Documentation:PATH417archive2020W2/Case 3

Path 417; Cases
Instructors
Nimah Kelly
Case Projects
	PATH4172020W2/Case_1; PATH417:2020W2/Case_2; Course:PATH4172020W2/Case_3; Course:PATH4172020W2/Case_4;

Case 3

A Red Rash

Robert is a 41-year-old man who came down with a high fever, headache, abdominal pain and muscle aches. He decides to tough it out at home but after 2 days, he also develops a red rash on his arms and legs. His wife drives him to the hospital where he is assessed by the emergency physician. The doctor asks about any insect bites and Robert tells him about getting tick bites during a camping trip one week prior to developing the first symptoms. Upon examining Robert he notes the macular rash on the arms and legs. Initial bloodwork shows that Robert has a low platelet count, low sodium levels and abnormal liver function tests.

The doctor orders a microbiological serology test and starts him on intravenous antibiotics. The acute serologic test for Rickettsia rickettsii shows a low level of IgM antibodies and Rick is diagnosed with Rocky Mountain Spotted Fever. The diagnosis is subsequently confirmed when convalescent serology shows a 16-fold rise in antibody titers to R. rickettsia.

1. The Body System

(i) Describe the signs (objective characteristics usually detected by a healthcare professional) and symptoms (subjective characteristics experienced by the patient). Also make note of any key history findings, what samples are taken (and why) and the meaning of any laboratory results reported. (There is no need to describe the laboratory testing as this is the basis of another question).

(ii) Which body system is affected. In what way has the normal physiological functioning of this area of the body been disturbed by the infection (without going into detail on the cause of this disturbance as this will be dealt with in questions 3 and/or 4). Representing this diagrammatically is always helpful.

(iii) What antibiotics might have been in the intravenous drip i.e. what is the antibacterial treatment of choice and how do the(se) antibiotic(s) work to rid the body of the organism?

(iv) What is the significance of the change in antibody titers with time? Is this a reportable communicable disease?

2. The Microbiology Laboratory

(i) Including the stated bacterial cause, what are the most common bacterial pathogens associated with this type of infectious scenario.

(ii) What samples are taken for laboratory testing and how important is the Microbiology Laboratory in the diagnosis of this particular infectious disease?

(iii) Explain the tests that will be performed on the samples in order to detect any of the potential bacterial pathogens causing this disease.

(iv) What are the results expected from these tests allowing for the identification of the bacteria named in this case.

3. Bacterial Pathogenesis

Using the following pathogenic steps outline the pathogenesis of the bacteria named as being responsible for this infection.

i. Encounter: where does the organism normally reside, geographically and host wise, and what are the bacterial characteristics that leave it suited to these places of residence. How would our patient have come in contact with this bacteria

ii. Entry: what facilitates the entry of the bacteria into the human host? What are the molecular, cellular and/or physiological factors at play in the initial entry/adherence step from the point of view of the organism and the host.

iii. Multiplication and Spread: does the organism remain extracellular or do they enter into cells and what are the molecular and cellular determinants of these events. Do the bacteria remain at the entry site or do they spread beyond the initial site i.e. are there secondary sites of infection and why do the bacteria hone in on these particular secondary sites.

iv. Bacterial Damage: do the bacteria cause any direct damage to the host (or is the damage fully attributable to the host response, as indicated below) and, if so, what is the nature of the bacterial damage. Can it be linked to any of the signs and symptoms in this case?

4. The Immune Response

i. Host response: what elements of the innate and adaptive (humoral and cellular) immune response are involved in this infection.

ii. Host damage: what damage ensues to the host from the immune response?

iii. Bacterial evasion: how does the bacteria attempt to evade these host response elements.

iv. Outcome: is the bacteria completely removed, does the patient recover fully and is there immunity to future infections from this particular bacteria ?

Responses

Q 1. The Body System

(i) Describe the signs (objective characteristics usually detected by a healthcare professional) and symptoms (subjective characteristics experienced by the patient). Also make note of any key history findings, what samples are taken (and why) and the meaning of any laboratory results reported. (There is no need to describe the laboratory testing as this is the basis of another question).

Rickettsia rickettsii is a gram-negative, obligate intracellular, coccobacillus bacterium that causes Rocky Mountain spotted fever (RMSF), an illness characterized by the infection of the vascular endothelial cells of the blood vessels (Snowden & Simonsen, 2020). Rocky Mountain spotted fever is among the most severe human infectious diseases, citing a mortality rate of 20-25% in the absence of appropriate antibiotic treatments, especially in male and elderly individuals (Walker, 1996). R. rickettsii is primarily transmitted through transovarial transmission from infected female ticks to infected ova that hatch into infected larval offspring, spreading via the bloodstream to infect the endothelium and vascular smooth muscle cells (Walker, 1996). The infection of endothelial cells leads to disseminated inflammation, loss of barrier function, and altered vascular permeability throughout the body, potentially causing fever, myalgias, central nervous symptoms such as headaches, rash, and cardiovascular instability (Snowden & Simonsen, 2020). The severity of Rocky Mountain spotted fever can be attributed to the severe damage that is caused to blood vessels; this damage leads to visible hemorrhages that can be visualized in the characteristic rash that patients exhibit (Walker, 1996). The pathologic effects of this disease originate from the areas of endothelial injury, with loss of intravascular fluid into tissue spaces, resultant lowered blood volume, the loss of perfusion of the organs, and disordered function of the damaged tissues (Walker, 1996). Advanced stages of RMSF are characterized by septic shock and systemic injury to the microvasculature, resulting in increased capillary permeability of organ systems (Paddock & Alvarez-Hernández, 2017).

Common Signs and Symptoms of RMSF
Signs	Symptoms
Macular or Petechial Rash	Macular or Petechial Rash
Fever	Headache
Lymphadenopathy	Fever
Hepatitis	Myalgias
Cardiovascular Instability	Vomiting

Signs:

The most obvious manifestation of Rocky Mountain spotted fever observed by physicians likely is the macular or petechial rash that develops following the infection of vascular endothelial cells (Snowden & Simonsen, 2020). Additional signs that a physician may note include fever or possible tick bites. In Robert’s case, the physician noted that he displayed a macular rash on his arms and legs. Rarer signs a physican may observe include lymphadenopathy, hepatitis, and cardiovascular instability (Snowden & Simonsen, 2020).

Figure 1. Typical manifestations of macular and petechial rashes in Rocky Mountain Spotted Fever. (Paddock & Alvarez-Hernández, 2017).

Symptoms:

Typical symptoms of Rocky Mountain spotted fever present themselves in the patient four to ten days following exposure (Snowden & Simonsen, 2020). Symptoms generally include fever, headache, and a petechial or maculopapular rash, but may include other symptoms such as myalgias and vomiting (Snowden & Simonsen, 2020). The rash typically begins as a maculopapular rash around the wrists and ankles, before progressing to petechia. RMSF rashes can present in 2 ways: maculopapular and petechiae (NORD, 2021). Maculopapular rashes present as a flat, red/pink are covered by confluent bumps. As RMSF progresses, the rash may evolve to papules, which are raised bumps with darker pigmentation (NORD, 2021). Petechiae is recognizable as pin-point red-purple spots that appear due to localized hemorrhaging due to broken capillaries (CDC, 2019). The pin-point petechiae may merge to form larger hemorrhagic patches, and in severe cases the lack of oxygen supply due to hemorrhaging may lead to tissue necrosis (NORD, 2021). Neurological symptoms are also frequently reported; headache, restlessness, insomnia, and back stiffness are common symptoms among patients (Sekeyová et al., 2019). Robert reports feeling a headache, abdominal pain, and muscle aches, all of which are within expectations for a Rocky Mountain spotted fever patient.

Laboratory Testing:

Robert, having reported tick bites on his camping trip one week prior the development of his symptoms, likely has Rocky Mountain spotted fever caused by R. rickettsii, especially given his reported fever, headache, abdominal pains, and rashes. His laboratory results collaborate with this conclusion. The physician ordered both blood tests and a microbiological serology test. Currently, most cases of Rocky Mountain spotted fever are diagnosed based on immunoglobulin M (IgM) and IgG serologic responses to R. rickettsii (Snowden & Simonsen, 2020). However, the hazardous nature of R. rickettsii prevents the timely completion of their diagnostic procedures in all but a few labs due to the difficult technique and high level of biosafety containment involved; thus, diagnosis of rickettsial infections often is reliant on serologic demonstration of the development of antibodies to rickettsial antigens in serum collected in the convalescent period following symptoms (Walker, 1996).

Robert’s initial serological tests do indicate low levels of IgM antibodies for R. rickettsii and is later confirmed by the rise in antibody titers to R. rickettsii during convalescence. Additional diagnostic markers can be thrombocytopenia (low platelet count), hyponatremia (low sodium levels), abnormal function of hepatic enzymes and cerebrospinal fluid pleocytosis, which can be determined using blood samples and CSF lumbar punctures (Snowden & Simonsen, 2020).

Low platelet counts can occur due to bacterial infection and usually result when the bacterium causes septic shock, which can alter coagulation pathways. Vascular injuries caused during RMSF infection often consume platelets, as microvascular bleeds from vessel damage recruit platelets to begin clotting (Dumler, 2019). RMSF infection often causes vascular permeability and edema, and the accumulation of fluid in circulation can directly dilute sodium levels (Traeger et al., 2015). Hyponatremia also could be caused by a secretion of antidiuretic hormone (ADH) in response to hypovolemia caused by RMSF infection (NORD, 2021). ADH can act on the kidneys directly to enhance reabsorption of water into the blood in an effort to maintain total body water, which can subsequently dilute the sodium content in the blood (NORD, 2021). Lastly, abnormal function of hepatic enzymes can be altered in bacterial infections as there is altered blood flow to the liver (Sahni et al., 2019). The blood tests indicate Robert has a low platelet count, low sodium levels, and abnormal liver function tests, consistent with a diagnosis of R. rickettsii infection.

History

While the physician was taking a patient history, it was noted that Robert was bitten by multiple ticks during a camping trip prior to the development of any symptoms. This is a key finding, as exposure to tick-infested environments is the primary risk factor for R. rickettsia transmission and subsequent RMSF diagnosis (Dumler, 2019). Additionally, the incubation period of infection following a tick bite is approximately one week, which aligns with Robert’s timeline (Dumler, 2019). An important detail to note would be the location and time of year of Robert's camping trip, as knowledge of where and when R. rickettsii is active assists with the early diagnosis and treatment of the illness, especially considering serological testing typically does not reveal antibodies against R. rickettsii in the first five days (CDC, 2020).

References:

CDC. (2019, February 19). Signs and symptoms of RMSF for healthcare providers | CDC. Centers for Disease Control and Prevention. https://www.cdc.gov/rmsf/healthcare-providers/signs-symptoms.html

CDC. (2020). Regions where ticks live | CDC. Centers for Disease Control and Prevention. https://www.cdc.gov/rmsf/healthcare-providers/signs-symptoms.htmlhttps://www.cdc.gov/ticks/geographic_distribution.html

Dumler, J. S. (2019, January 17). Rocky mountain spotted fever. Cancer Therapy Advisor. https://www.cancertherapyadvisor.com/home/decision-support-in-medicine/pediatrics/rocky-mountain-spotted-fever/

Mayo Clinic. (2021). Hyponatremia - Symptoms and causes. Mayo Clinic. https://www.mayoclinic.org/diseases-conditions/hyponatremia/symptoms-causes/syc-20373711

NORD. (2021). Rocky Mountain Spotted Fever. NORD (National Organization for Rare Disorders). https://rarediseases.org/rare-diseases/rocky-mountain-spotted-fever/

Paddock, C. D. & Alvarez-Hernández, G. (2017). 178 - Rickettsia rickettsii (Rocky Mountain Spotted Fever). In S. Long, C. Prober & M. Fischer. Principles and Practice of Pediatric Infectious Diseases (5^th ed.) (pp. 952-957). Elsevier. https://doi-org.ezproxy.library.ubc.ca/10.1016/B978-0-323-40181-4.00178-X

Sahni, A., Fang, R., Sahni, S. K., & Walker, D. H. (2019). Pathogenesis of Rickettsial Diseases: Pathogenic and Immune Mechanisms of an Endotheliotropic Infection. Annual review of pathology, 14, 127–152. https://doi.org/10.1146/annurev-pathmechdis-012418-012800

Sekeyová, Z., Danchenko, M., Filipčík, P., & Fournier, P. E. (2019). Rickettsial infections of the central nervous system. PLoS neglected tropical diseases, 13(8), e0007469. https://doi.org/10.1371/journal.pntd.0007469

Snowden, J. & Simonsen, K. A. (2020). Statpearls [Internet]. Statpearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK430881/

Traeger, M. S., Regan, J. J., Humpherys, D., Mahoney, D. L., Martinez, M., Emerson, G. L., Tack, D. M., Geissler, A., Yasmin, S., Lawson, R., Hamilton, C., Williams, V., Levy, C., Komatsu, K., McQuiston, J. H., & Yost, D. A. (2015). Rocky Mountain Spotted Fever Characterization and Comparison to Similar Illnesses in a Highly Endemic Area—Arizona, 2002–2011. Clinical Infectious Diseases : An Official Publication of the Infectious Diseases Society of America, 60(11), 1650–1658. https://doi.org/10.1093/cid/civ115

Walker, D. H. (1996). Medical Microbiology (4^th ed.). University of Texas Medical Branch at Galveston. https://www.ncbi.nlm.nih.gov/books/NBK7624/

(ii) Which body system is affected. In what way has the normal physiological functioning of this area of the body been disturbed by the infection (without going into detail on the cause of this disturbance as this will be dealt with in questions 3 and/or 4). Representing this diagrammatically is always helpful.

Integumentary System:

In order to enter the human body, the first point at which R. rickettsii disturb normal physiological functioning of the body is at the skin. The skin is part of the integumentary system, which serves as the anatomical boundary between the body and the environment. It protects the body from exterior damage by shielding internal organs and tissues and acts as a water barrier, maintaining temperature regulation, excretion of salts, and synthesis of Vitamin D (1).

The skin is composed of three layers:

1. Outer epidermis: stratified squamous cells (highly flattened) composed of keratinocytes

2. Deeper dermis: highly vascularized, fibrous connective tissue

3. Hypodermis/subcutaneous layer: Loose layer of subcutaneous connective tissue attaching the skin to underlying structures, thus allowing movement (1)

As mentioned in the case, Robert had been bitten by a tick. Ticks are blood-sucking arthropods which are able to attach to the human skin (2). While not caused by R. rickettsii itself, the attachment of ticks can cause lesions on the skin and thus disrupt the physiological barrier it provides. The penetration of the skin caused by tick bites can transfer toxic substances from the tick’s saliva to the skin. This can cause acute pruritic papular dermatitis, in which papules (raised areas of skin tissue that are less than 1 centimeter round) can manifest all over the skin (2). In addition to the direct damage caused by the arthropods, R. rickettsii is able to enter the body, as the protective layer of the skin is compromised (3). The bacteria can then penetrate through the dermis, resulting in inflammation causing host cell damage and death (3). Resident skin-associated immune cells including macrophages, dendritic cells, mast cells, dermal γδ T cells and innate lymphoid cells contribute to the immune response (4). As R. rickettsii target endothelial cells, the dermis endothelium is damaged by increased vascular permeability, and blood and fluid are able to leak out of the endothelial cell and into adjacent tissues causing a macular rash (3). Vasodilation of the skin endothelium also occurs, resulting in increased vascular permeability and fluid leakage which contributes to the rash seen in patients with RMSF (5).

Lymphatic System:

Once R. rickettsii has breached the skin, the initial target cells of infection are macrophages and dendritic cells (7). R. rickettsii can then spread to regional lymph nodes through lymphatic vessels, which has been studied to result in lymphangitis, thus disrupting the normal physiological function of the lymphatic system (7). The lymphatic system is a circulatory system that drains fluid from the blood vessels (8). The lymphatic system is involved in the filtration of lymph and blood, transportation of dietary lipids and immune system function and it consists of:

Lymph: a colourless fluid consisting of cellular debris and lymphocytes
Lymphatic vessels: the site of fluid drainage, pump lymph fluid using smooth muscle, and contain valves to prevent backflow
Lymph nodes: Small oval bodies of the lymphatic system distributed along the lymphatic vessels in the armpits, groin, neck, chest and abdomen. Lymph nodes filter lymph
Lymph organs: Structures dedicated to the circulation and production of lymphocytes including the spleen, thymus and bone marrow (8).

R. rickettsia may enter systemic circulation through the lymphatic system, as they are closely connected (9).

Circulatory System:

Once R. rickettsii gains access to the blood, the bacterium is able to spread hematogenously throughout the body and travel to many different body sites. R. rickettsii primarily infect endothelial cells lining the blood vessels of the vascular system (10). The vascular system is a network composed of the heart and vessels that circulate blood and lymph throughout the body (11). Under normal conditions, the arteries and veins carry blood, nutrients, and oxygen to tissues, and remove waste. The endothelium is composed of a single layer of endothelial cells that compose the inner layer of all the blood vessels in the body (12). There are numerous physiological functions of the endothelium in the human body. The endothelium provides a semi-permeable structural barrier between the blood and surrounding tissues, allowing small molecules like ions and oxygen, as well as small lipid- and water-soluble substances to pass through, but controlling the transport of larger molecules via vesicle and receptor mediated entry (13,14). Endothelial cells also produce vasoactive factors, chemical mediators that regulate blood pressure and flow and control the underlying vascular smooth muscle cells, controlling the exchange of gasses and nutrients between the blood and tissues (13,15). They can also contribute to blood fluidity and immune responses by expressing adhesion receptors like selectins and integrins, controlling the activation of platelets and maintaining coagulation and fibrinolysis (15).

R. rickettsii are able to attach to receptors on endothelial cell membranes and induce their own phagocytosis (16). Once inside host cells, the bacterium is able to escape the phagosome and enter the cytoplasm, which is mediated by the R. rickettsii enzyme phospholipase A2. Once inside the cytoplasm, R. rickettsii are able to divide inside the cell (17). R. rickettsii are then able to escape from host cell through their own filopodia, as well as by taking over host cell machinery, causing polymerization of host actin tails (16,17). This allows R. rickettsii to move through the cytoplasm and reach the membrane, enabling the bacterium to emerge through focal lysis. There are relatively few R. rickettsii that accumulate inside any particular cell; thus, the infection tends to spread quickly in order to infect other cells and continue the bacterium’s life in the host (17). The bacteria can then be spread to adjacent cells, allowing many endothelial cells in close proximity to become infected and subsequently damaged (18).

Figure 1: The changes in the blood vessel structure due to increased vascular permeability (Pollard-Smith, 2017).

R. rickettsii produces free radicals, proteases and phospholipases which damage the host cell membranes, resulting in injury of the endothelium and potentially cell death (9). There is increased vascular permeability and vasodilation in response, as the damaged endothelial cells begin an inflammatory response, activating cyclooxygenase and heme oxygenase and producing prostaglandins (9). Vascular endothelial cadherin, an adhesion molecule found between endothelial cells that is important for maintaining cellular contact, is also destabilized, further increasing the permeability of the vasculature (9). Immune cells enter the vasculature to respond to the bacterial infection, phagocytosing bacteria and killing infected host cells, resulting in lymphohistiocytic vasculitis, inflammation of the blood vessels (19). This mechanism is pictured in Figure 1.

With the increased permeability, fluid is able to escape from the blood vessels and enter the tissues, resulting in edema, decrease of the volume of fluid in the blood, decreased blood pressure, and decrease in the concentration of albumen in the blood (9). The hyponatremia seen in patients with Rocky Mountain Spotted Fever is due to the loss of fluid volume in the blood, as the body secretes antidiuretic hormone to try to maintain the proper osmolarity in the blood (9). R. rickettsia also induces a procoagulant state in the vasculature, with increased amounts of clotting compared to normal, due to the high levels of endothelial damage (9). The coagulation cascade is activated, resulting in production of thrombin and the activation of platelets, and other factors maintaining the procoagulant state, like the production of antifibrinolytic factors and secretion of cytokines promoting coagulation, are induced (9). The consistent use of platelets in coagulation results in the thrombocytopenia seen during Rocky Mountain Spotted Fever (9).

Once R. rickettsii is a part of systemic circulation, it is able to reach and infect various body sites. While inflammation can ensue following endothelial invasion of the blood vessels causing generalized vasculitis, vasculitis can also be specific to various organs, leading to compromises in the heart, lungs, liver, gastrointestinal tract, kidneys, muscles and brain (20). The loss of the integrity of the endothelial lining in the microvasculature in these body sites can result in edema, low blood volume, hypoalbuminemia, decreased osmotic pressure, hypotension and decreased tissue/organ perfusion.

Cardiovascular System:

The involvement of the heart with this infection is not common but possible and can create certain clinical conditions. Congestive heart failure, myocarditis and arrhythmias are all possible secondary to the damage R. rickettsii inflicts on the vascular system (21). As the heart is responsible for pumping highly oxygenated and nutrient rich blood to the organs and tissues, impairments to the heart function can result in decreased tissue perfusion.

Pulmonary System:

Increased concentration of R. rickettsii in the pulmonary circulation can create increased permeability allowing for more fluid and pathogens to enter, leading to manifestations such as interstitial pneumonia, pulmonary edema and alveolar infiltrates (22). Pulmonary involvement is less common; however, these conditions can be seen in those who present with a cough and R. rickettsii infection (22). The pulmonary system ensures the oxygen-waste exchange occurs and this requires specific conditions aided by its own microcirculatory system, therefore, the introduction of R. rickettsii can compromise ventilation and gas exchange (23).

Hepatic System:

In the liver, vasculitis can occur in portal triads. In addition, small foci of hepatocellular death and hyperbilirubinemia can be observed, but massive hepatic necrosis and failure do not occur. Lastly, elevated hepatic enzyme activity can occur, which we saw in Robert’s case (20). Damage to the hepatic cells disrupts the functions of the liver which are removing toxins and the production of certain enzymes and compounds. Since these functions are compromised, conditions like jaundice, hepatocellular necrosis and lesions can arise (22).

Gastrointestinal System

Reduced tissue perfusion to the abdominal cavity can cause focal lesions in the walls of the gastrointestinal tract, gallbladder and pancreas. The bacteria invading the endothelial cells initiate these symptoms and disturb the functions of the digestive system such as the breaking down and absorption of nutrients. In addition, loss of fluids due to hypovolemia can result in dehydration. This can lead to abdominal tenderness and abdominal pain, which is what we observed in Robert’s case (20).

Renal System:

In some cases of Rocky Mountain Spotted Fever, acute renal failure can occur as a consequence of the endothelial damage as well as hypovolemia (20, 24). This damage allows toxins and compounds to escape filtration and overall impairs the function of the kidneys which is filtering the blood and removing wastes from the body (20). The normal process of kidney filtration requires specific vascular conditions in terms of blood pressure, blood volume and vessel permeability which are altered with R. rickettsii infection (24).

Figure 2: Summary of infection and resulting symptoms caused.

Muscular System:

R. rickettsii can invade skeletal muscle and disturb the control and initiation of voluntary movement. Manifestations include rhabdomyolysis and ocular conditions such as papilledema (21). In addition, leaky capillaries result in decreased osmolality due to loss of electrolytes, such as sodium. Low levels of sodium in the blood can cause muscle pain, as seen in Robert’s case (25).

Central Nervous system

RMSF caused by R. rickettsii frequently is associated with neurological symptoms and the CNS appears to be one of the major systems involved in the last stage of infection (26). R. rickettsii can enter the CNS at the level of the brain microvasculature. R. rickettsii can enter the CNS either transcellular (through the endothelial cells of the BBB), or disrupt the integrity of the BBB and enter the CNS via the paracellular route (in between cells). Once in the CNS, the onset of symptoms due to R. rickettsii infection occurs relatively quickly (26). A few functions of the central nervous system (CNS) include controlled movements, the ability to form thoughts and memory and regulation of body homeostasis (27.). The vascular system within the brain is very sensitive and important in carrying out these functions. Once in the CNS, the onset of symptoms due to R. rickettsii infection occurs relatively quickly. Common symptoms associated with R. rickettsii in the CNS include headache, fever, chills, myalgias, and arthralgias (26). Injury to the vasculature by R. rickettsii can initiate inflammation in the brain or encephalitis, which may account for some of the neurological symptoms patients present such as confusion and drowsiness (24).

As this inflammation continues, symptoms become more severe. For instance, more aggressive inflammation can lead to seizures and ataxia (24). Further, vasculitis in the brain microvasculature can cause edema, thus resulting in perivascular hemorrhages. In addition, microinfarcts of the blood vessels can result in severe degeneration of the vasculature. As mentioned earlier, as endothelial cells become swollen due to infection, and this can occlude the lumen of blood vessels. This can lead to thrombosis in cerebral blood vessels, causing focal necrosis in regions of the brain (26). Other CNS manifestations, as a result of R. rickettsii infection, are loss of hearing, vertigo, increased reflex response, speech impairment, muscle weakness or paralysis and loss of bowel or bladder control (24). These conditions may or may not resolve after clearing of the infection (28).

A summary of R. rickettsii infections and the resulting damages to normal host function are outlined in Figure 2.

References

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Paddock, C. D., & Alvarez-Hernández, G. (2018). 178—Rickettsia rickettsii (Rocky Mountain Spotted Fever). In S. S. Long, C. G. Prober, & M. Fischer (Eds.), Principles and Practice of Pediatric Infectious Diseases (Fifth Edition) (pp. 952-957.e2). Elsevier. https://doi.org/10.1016/B978-0-323-40181-4.00178-X
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Chen, L. F., & Sexton, D. J. (2008). What’s New in Rocky Mountain Spotted Fever? Infectious Disease Clinics of North America, 22(3), 415–432. https://doi.org/10.1016/j.idc.2008.03.008
Sahni, A., Fang, R., Sahni, S. K., & Walker, D. H. (2019). Pathogenesis of Rickettsial Diseases: Pathogenic and Immune Mechanisms of an Endotheliotropic Infection. Annual review of pathology, 14, 127–152. https://doi.org/10.1146/annurev-pathmechdis-012418-012800
Gottlieb, M., Long, B., Koyfman, A. (2018). The Evaluation and Management of Rocky Mountain Spotted Fever in the Emergency Department: a Review of the Literature. The Journal of Emergency Medicine, 55(1), 42–50.
Blanton, L. S., Walker, D. H. (2020). Rickettsia rickettsii and Other Spotted Fever Group Rickettsiae (Rocky Mountain Spotted Fever and Other Spotted Fevers). In: Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases [Internet]. 9th ed. Philadelphia, PA: Elsevier, 2349-2357. Available from: https://www.clinicalkey.com/#!/content/book/3-s2.0-B9780323482554001867
4.4: Pulmonary Microcirculation [Internet] (2019). Medicine LibreTexts. [cited 2021 Mar 14]. Available from:
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Sekeyová, Z., Danchenko, M., Filipčík, P., & Fournier, P. E. (2019). Rickettsial infections of the central nervous system. PLoS neglected tropical diseases, 13(8), e0007469. https://doi.org/10.1371/journal.pntd.0007469
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Todar, K. (2020). Rickettsial Diseases, including Typhus and Rocky Mountain Spotted Fever [Internet]. Available from: http://textbookofbacteriology.net/Rickettsia_5.html
Pollard-Smith T. The Greatest Debate of Them All - part 1: Ice [Internet]. Open Health Clinic. 2017 [cited 2021 Mar 12]. Available from: https://openhealthclinic.com/the-greatest-debate-of-them-all-part-1-ice/

(iii) What antibiotics might have been in the intravenous drip i.e. what is the antibacterial treatment of choice and how do the(se) antibiotic(s) work to rid the body of the organism?

The prognosis of RMSF is closely related to the speed at which appropriate therapy is administered (Blanton & Walker, 2015), thus antibiotic treatment should be started immediately if there is any suspicion of RMSF based on clinical presentation, and should not be delayed pending laboratory results (Paddock & Alvarez-Hernández, 2018). It has been indicated that treatment should be provided within 5 days of symptom onset, as a delay in treatment has been associated with an increased risk of mortality (Holman et al., 2001).

Figure 1: Doxycycline Structure (Drugbank, 2021b)

Doxycycline

Doxycycline, administered orally or intravenously remains the antibiotic of choice for RMSF infection (Blanton & Walker, 2015), and thus is likely in Robert’s intravenous drip. Doxycycline belongs to the tetracycline class of antibiotics, which are derived from streptomyces, and are considered to be a broad-spectrum treatment (Shutter & Akhondi, 2021). Doxycycline is a tetracycline antibiotic that can be used to kill a wide range of bacteria, both gram-negative and gram-positive. It is commonly used to treat cholera, mycoplasma and other rickettsia infections, as well as some sexually transmitted infections, like chlamydia, and skin infections (Patel & Parmar, 2021). This antibiotic can also be used following exposure to anthrax and as prophylactic treatment for malaria (FDA, 2008). Doxycycline can also be used to treat some inflammatory diseases, like rosacea and rheumatoid arthritis, as it has been shown to have anti-inflammatory and immunosuppressive properties as well as its bactericidal functions (Patel & Parmar, 2021). Doxycycline is able to pass through the outer membrane of gram-negative bacteria via porin channels and it relies on active transport dependent on pH to move through the inner membrane of gram-negative bacteria and the cell membrane of gram-positive bacteria (Patel & Parmar, 2021). Tetracycline antibiotics are considered bacteriostatic, as they are capable of inhibiting the growth or reproduction of bacteria (Shutter & Akhondi, 2021). Doxycycline is a second-generation tetracycline, as it exhibits led toxicity than first-generation tetracyclines (Drugbank, 2021b).

Mechanism of Action

Figure 2: Cellular targets for Doxycycline and Chloramphenicol (Mechanisms of Antibacterial Drugs | Microbiology, 2021)

Tetracycline antibiotics inhibit protein synthesis through interaction with ribosomes (Shutter & Akhondi, 2021). In Prokaryotes such as bacteria, protein synthesis utilizes the 30S and 50S ribosomal subunits, and it is at these sites that ribosome transfer RNA (tRNA) binds to the mRNA template (Shutter & Akhondi, 2021). The binding of tRNA requires a charged amino acid, and the binding of each tRNA subsequently leads to the formation and elongation of a cellular protein (Shutter & Akhondi, 2021). Tetracycline antibiotics are able to reversibly bind the 16s rRNA found on the 30S ribosomal subunit (Drugbank, 2021b), and possibly the 50S subunit, thereby hindering the binding of the aminoacyl-tRNA to the receptor site on the MRNA-ribosome complex (Shutter & Akhondi, 2021). The binding of tetracycline to the 30S/50S ribosomal subunit inhibits the process of amino acid addition to the growing peptide chain, and thus the cell can no longer maintain proper functioning, grow, or replicate genetic material (Shutter & Akhondi, 2021). The bacteria eventually dies as essential proteins are not produced. The antibiotic can also prevent microtubular assembly and proliferation of immune cells and inhibits the function of proteases that are produced by immune cells, resulting in an anti-inflammatory response (Patel & Parmar, 2021).

Drug Administration

As mentioned above, it is possible to administer doxycycline either orally or intravenously (Shutter & Akhondi, 2021). The recommended dosage (oral or IV) for adults is 100mg twice daily, for 5 to 7 days or at least 3 days after fever subsides, whichever is longer (Biggs, 2016). For children and adolescents, an oral or IV dose of 2.2mg/kg/dose every 12 hours, to a maximum dose of 100mg/dose is recommended (Biggs, 2016). Similarly to adults, it is recommended that treatment is continued for a minimum of 5 to 7 days, and for treatment to continue for at least 3 days after clinical improvement is observed (Biggs, 2016). In severe or complicated disease, longer treatment lasting 10 days or longer is often recommended (Shutter & Akhondi, 2021).

Special Populations:

It has been found that doxycycline may cause tissue hyperpigmentation, and could disrupt bone and tooth development, however doxycycline has been approved for use in children ≤8 years of age with RMSF, as there are no adequate alternatives (Biggs, 2016). The use of tetracycline drugs in pregnant woman has traditionally been contraindicated due to concerns about risk related to musculoskeletal development of the fetus, cosmetic staining of primary dentition in fetuses exposed in the second or third trimester, and the development of acute fatty liver of pregnant mothers (Biggs, 2016). These concerns are related to the use of older tetracycline derivatives, and therefore newer derivatives such as doxycycline may be permitted for use in pregnant women (Biggs, 2016). A systematic review conducted in 2016 showed no correlation between the use of doxycycline and teratogenic effects during pregnancy (Cross et al., 2016), thus most pregnant women with RMSF will in fact be administered doxycycline, as it is the preferred treatment, as the benefits generally outweigh the risks (Biggs, 2016).

Expected Benefits

There are many benefits to the use of tetracycline drugs, specifically:

1. Tetracycline drugs have a lipophilic structure that readily allows concentration in host tissue

2. The bacterial transport system for tetracycline allows the drug to accumulate in bacterial cells at concentrations 50 times greater than that of the medium in vitro

(Todar, 2020)

The above factors vastly increase antibacterial effectiveness and promote the specificity of action of tetracycline. Doxycycline specifically is frequently chosen over other tetracyclines as it is less phototoxic, safer for patients with comorbid renal insufficiency, and has a longer plasma half-life (Paddock & Alvarez-Hernández, 2018).

Figure 3: Chloramphenicol Structure (PubChem, 2021)

Chloramphenicol

Chloramphenicol is a semi-synthetic broad-spectrum, bacteriostatic antibiotic derived from Streptomyces venequelae (PubChem, 2021). Chloramphenicol is the only known alternative agent for the treatment of RMSF, however epidemiological studies have indicated that patients with RMSF who are administered chloramphenicol have a higher risk of mortality than patients treated with tetracycline (DuPont et al., 1973). In the United States, chloramphenicol is only available in IV form, and is not as readily available as doxycycline (Biggs, 2016). The bioavailability following intramuscular administration is 70%, and oral bioavailability is 80%, although oral preparations of the medication are not used in practice (Drugbank, 2021a).

Mechanism of Action

Chloramphenicol inhibits protein synthesis by preventing protein chain elongation through the inhibition of peptidyl transferase on the bacterial ribosome (Biggs, 2016). Chloramphenicol is lipid soluble, and thus diffuses through the bacterial cell membrane (Drugbank, 2021a). Chloramphenocol is then able to reversibly bind to the L16 protein of the 50S bacterial ribosome subunit, thereby preventing the transfer of amino acids to growing peptide chains through suppression of peptidyl transferase activity (Drugbank, 2021a). Peptide bond formation and subsequent protein synthesis are inhibited within the bacterial cell (Biggs, 2016).

Administration

For serious infections such as RMSF in adults, an IV dose of 50 to 100 mg/kg/day in divided doses every 6 hours is recommended, with a maximum daily dose of 4g/day (Moffa & Brook, 2015). It should be noted that significant drug interactions exist (Drugbank, 2021b). For infants children, and adolescents, an IV dose of 12.5 to 25 mg/kg/day evert 6 hours, with a maximum daily dose of 4,000 mg/day is recommended (Mayo Clinic, 2021).

Special Populations

Chloramphenicol was traditionally the preferred treatment for RMSF during pregnancy due to perceived risks associated with doxycycline and teratogenicity, however new literature has indicated that doxycycline is likely safe for pregnant women (Biggs, 2016). There is some concern over the use of chloramphenicol during the third trimester of pregnancy due to risks associated with “gray baby syndrome” (Biggs, 2016). Gray baby syndrome is a circulatory collapse that can occur in newborn infants and is associated with high serum levels of chloramphenicol (Moffa & Brook, 2015). Signs associated with gray baby syndrome include ashen-gray colour, abdominal distention, vomiting, flaccidity, cyanosis, circulatory collapse and death (Moffa & Brook, 2015). Chloramphenicol and its inactive metabolites are also present in breast milk, and can cause serious adverse reactions in breastfed infants of mothers taking chloramphenicol, thus it is recommended for new mothers to either discontinue the drug or discontinue breastfeeding (Biggs, 2016).

Risks.

It has been indicated that chloramphenicol is not effective in the treatment of human ehrlichiosis or anaplasmosis, and thus should be used with caution in the empiric treatment of tickborne rickettsial disease (Biggs, 2016). Additionally, chloramphenicol has been associated with numerous adverse hematologic effects including aplastic anemia, hypoplastic anemia, thrombocytopenia, and granulocytopenia (Biggs, 2016). It is therefore advised that individuals using this medication have regular blood monitoring to detect adverse hematological effects (Biggs, 2016).

Intravenous Therapy and Fluid Replacement

In severe cases of RMSF, it is possible that patients may require intravascular fluid replacement alongside careful management of fluid status (Minniear & Buckingham, 2009). It is likely that severely ill patients will receive doxycycline intravenously (Biggs, 2016). In the case of patients demonstrating significant pulmonary and neurological compromise, mechanical ventilation may be warranted (Minniear & Buckingham, 2009). Robert is seen 2 days after the onset of his symptoms, which is within the 5 day window recommended to start treatment. It has been indicated that his antibiotics are being delivered via an IV drip intravenously.

References

Biggs, H. M. (2016). Diagnosis and Management of Tickborne Rickettsial Diseases: Rocky Mountain Spotted Fever and Other Spotted Fever Group Rickettsioses, Ehrlichioses, and Anaplasmosis — United States. MMWR. Recommendations and Reports, 65. https://doi.org/10.15585/mmwr.rr6502a1

Blanton, L., & Walker, D. (2015). Rickettsia rickettsii and Other Spotted Fever Group Rickettsiae (Rocky Mountain Spotted Fever and Other Spotted Fevers. In Principles and Practice of Infectious Disease. (8th ed., pp. 2198–2205).

Cross, R., Ling, C., Day, N. P. J., McGready, R., & Paris, D. H. (2016). Revisiting doxycycline in pregnancy and early childhood—Time to rebuild its reputation? Expert Opinion on Drug Safety, 15(3), 367–382. https://doi.org/10.1517/14740338.2016.1133584

Drugbank. (2021a). Chloramphenicol. https://go.drugbank.com/drugs/DB00446

Drugbank. (2021b). Doxycycline. https://go.drugbank.com/drugs/DB00254

DuPont, H. L., Hornick, R. B., Dawkins, A. T., Heiner, G. G., Fabrikant, I. B., Wisseman, C. L., & Woodward, T. E. (1973). Rocky Mountain spotted fever: A comparative study of the active immunity induced by inactivated and viable pathogenic Rickettsia rickettsii. The Journal of Infectious Diseases, 128(3), 340–344. https://doi.org/10.1093/infdis/128.3.340

FDA. (2008). DOXTERIC (doxycycline hyclate) Label. https://www.accessdata.fda.gov/drugsatfda_docs/label/2008/050795s005lbl.pdf

Holman, R. C., Paddock, C. D., Curns, A. T., Krebs, J. W., McQuiston, J. H., & Childs, J. E. (2001). Analysis of risk factors for fatal Rocky Mountain Spotted Fever: Evidence for superiority of tetracyclines for therapy. The Journal of Infectious Diseases, 184(11), 1437–1444. https://doi.org/10.1086/324372

Mayo Clinic. (2021). Chloramphenicol (Oral Route, Intravenous Route, Injection Route) Proper Use. https://www.mayoclinic.org/drugs-supplements/chloramphenicol-oral-route-intravenous-route-injection-route/proper-use/drg-20062754

Mechanisms of Antibacterial Drugs | Microbiology. (2021). Retrieved March 17, 2021, from https://courses.lumenlearning.com/microbiology/chapter/mechanisms-of-antibacterial-drugs/

Minniear, T. D., & Buckingham, S. C. (2009). Managing Rocky Mountain spotted fever. Expert Review of Anti-Infective Therapy, 7(9), 1131–1138. https://doi.org/10.1586/eri.09.94

Moffa, M., & Brook, I. (2015). Tetracyclines, Glycylcyclines, and Chloramphenicol. In Bennett JE, Dolin R, and Blaser MJ, eds. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases (8th ed.). Elselvier. https://www.elsevier.com/books/mandell-douglas-and-bennetts-principles-and-practice-of-infectious-diseases/bennett/978-1-4557-4801-3

Paddock, C. D., & Alvarez-Hernández, G. (2018). 178—Rickettsia rickettsii (Rocky Mountain Spotted Fever). In S. S. Long, C. G. Prober, & M. Fischer (Eds.), Principles and Practice of Pediatric Infectious Diseases (Fifth Edition) (pp. 952-957.e2). Elsevier. https://doi.org/10.1016/B978-0-323-40181-4.00178-X

Patel, R. S., & Parmar, M. (2021). Doxycycline Hyclate. In StatPearls. StatPearls Publishing. http://www.ncbi.nlm.nih.gov/books/NBK555888/

PubChem. (2021). Chloramphenicol. https://pubchem.ncbi.nlm.nih.gov/compound/5959

Shutter, M. C., & Akhondi, H. (2021). Tetracycline. In StatPearls. StatPearls Publishing. http://www.ncbi.nlm.nih.gov/books/NBK549905/

Todar, K. (2020). Rickettsial Diseases, including Typhus and Rocky Mountain Spotted Fever. http://textbookofbacteriology.net/Rickettsia_5.html

(iv) What is the significance of the change in antibody titers with time? Is this a reportable communicable disease?

Serological testing is typically helpful after the first 5 days of Rocky Mountain Spotted Fever (RMSF) given the production of antibodies is not detectable before (1). This is evident in Robert’s acute serological test as it showcases low levels of IgM antibodies therefore, it is necessary to conduct a convalescent sera serological test later. Antibodies take about 7-10 days to be produced and to accumulate to levels which are detectable through serology for first time RMSF patients (2). This is because the adaptive immune response takes a while to become activated when an individual is infected with a bacterium that is new to them. In addition, the antibody response in RMSF has been known to be late, with immunogenic antibodies appearing as late as day 12 (3).

The early production of IgM antibodies is attributed to the early stages of the adaptive immune response where a small amount of poorly immunogenic antibodies are made without the help of CD4 T cells (3). IgM is known to be the first type of antibody produced, typically within 5 to 7 days of infection, and can aid in fighting the infection through activating complement before more specific antibodies can be produced (4)(5). As the adaptive immune response develops, antigen-activated naïve T cells present antigens to B cells (6). B cells become activated and differentiate into plasma cells which then can generate antibodies including IgM, IgG, IgA and IgE (7)(6). To create more specific antibodies such as IgG, it takes about 10 days after the onset of symptoms (5). Once these steps are carried out and repeated, a detectable presence of antibodies in the patient’s sera is possible. These antibodies differ from those generated earlier as they are specific to the OmpA and OmpB proteins found on the Rickettsia rickettsii outer membrane and they are able to rid bacteria from tissues in 24 hours (3). At this time, about 2 to 3 weeks into infection, a convalescent antibody test is done and will show a spike in antibody titers compared to that of the acute test, confirming the presence of a Rickettsia rickettsii infection (8). If there is not a large difference in the convalescent and acute tests, this may suggest that the circulating antibodies are due to a prior R. rickettsii infection since these can survive in the blood for months or years (2). These persistent antibodies are usually of the IgG variety and because of their longevity, a low IgG antibody titer early in infection does not allow for RMSF diagnosis (8). Therefore, regulation indicates there must be at least a 4-fold rise in antibody titers between the acute and convalescent sera tests (9). Robert showcased a 16-fold increase which meets the diagnostic criteria, implying RMSF infection.

R. rickettsii infection is considered a communicable reportable disease because it fits the definition outlined by the BC Public Health Act stating that a communicable disease is an illness which manifests due to an infectious agent being transmitted from an infected human, animal, a vector or due to environmental exposure (10). R. rickettsii is transmitted to humans through the vector of infected ticks, thus deeming it a communicable disease. A reportable or notifiable disease is an illness which requires prevention and control through timely and frequent reporting of cases (11). Reporting cases is vital to measure disease trends, monitor the success of prevention and control strategies as well as analyze which populations are most at risk (12). Prevention measures for RMSF include bug repellent, wearing light coloured clothing, tucking in pant legs and checking and removing any ticks (5). There has been an increase in R. rickettsii cases in the past 2 decades and other bacterial infections under the general category of Spotted Fever Rickettsiosis (13). This category was created because of the lack of a serological test that can distinguish between the different spotted fever group Rickettsia species and includes Rickettsia parkeri rickettsiosis, rickettsialpox, Pacific Coast tick fever and RMSF (13). Given the timely manner in which R. rickettsii infection must be treated, the severity of disease and the mode of transmission, it is important to monitor this illness on a population level.

References

Kaplan JE, Schonberger LB. The Sensitivity of Various Serologic Tests in the Diagnosis of Rocky Mountain Spotted Fever. The American Journal of Tropical Medicine and Hygiene. 1986 Jul 1;35(4):840–4.
Trexeler Hessen M. Rocky Mountain spotted fever- ClinicalKey [Internet]. 2021 [cited 2021 Mar 17]. Available from: https://www.clinicalkey.com/#!/content/clinical_overview/67-s2.0-533d1714-64e0-4c32-b6cf-1f7b8cf24e07
Osterloh A. Immune response against rickettsiae: lessons from murine infection models. Med Microbiol Immunol. 2017 Dec 1;206(6):403–17.
Charles A Janeway J, Travers P, Walport M, Shlomchik MJ. The distribution and functions of immunoglobulin isotypes. Immunobiology: The Immune System in Health and Disease 5th edition [Internet]. 2001 [cited 2021 Mar 17]; Available from: https://www.ncbi.nlm.nih.gov/books/NBK27162/
Todar K. Rickettsial Diseases, including Typhus and Rocky Mountain Spotted Fever [Internet]. 2020. Available from: http://textbookofbacteriology.net/Rickettsia_5.html
Williams JC, Walker DH, Peacock MG, Stewart ST. Humoral immune response to Rocky Mountain spotted fever in experimentally infected guinea pigs: immunoprecipitation of lactoperoxidase 125I-labeled proteins and detection of soluble antigens of Rickettsia rickettsii. Infect Immun. 1986 Apr;52(1):120–7.
Charles A Janeway J, Travers P, Walport M, Shlomchik MJ. The course of the adaptive response to infection. Immunobiology: The Immune System in Health and Disease 5th edition [Internet]. 2001 [cited 2021 Mar 17]; Available from: https://www.ncbi.nlm.nih.gov/books/NBK27125/
Straily A, Stuck S, Singleton J Jr, Brennan S, Marcum S, Condit M, et al. Antibody Titers Reactive With Rickettsia rickettsii in Blood Donors and Implications for Surveillance of Spotted Fever Rickettsiosis in the United States. The Journal of Infectious Diseases. 2020 Mar 28;221(8):1371–8.
CDC. Clinical and laboratory diagnosis of RMSF | CDC [Internet]. Centers for Disease Control and Prevention. 2018 [cited 2021 Mar 17]. Available from: https://www.cdc.gov/rmsf/healthcare-providers/ClinLab-Diagnosis.html
Electronic Health Information System (Re) [Internet]. 2010 [cited 2021 Mar 17]. Available from: https://canlii.ca/t/28z42
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CDC. Epidemiology and statistics of spotted fever rickettsioses | CDC [Internet]. Centers for Disease Control and Prevention. 2020 [cited 2021 Mar 17]. Available from: https://www.cdc.gov/rmsf/stats/index.html

2. The Microbiology Laboratory

(i) Including the stated bacterial cause, what are the most common bacterial pathogens associated with this type of infectious scenario?

A table of tick-borne diseases.

Robert presents high fever, headache, abdominal pain and muscle aches, and later develops a red rash on arms and legs. These symptoms are non-specific and can be indicative of other viral or bacterial infections or drug allergic reactions (1). Upon seeing the doctor, Robert reveals that he got tick bites during a camping trip a week prior to symptom development. This infectious scenario is commonly associated with bacterial pathogens that cause similar non-specific symptoms and are transmitted by tick bites, some of which include: Rickettsia rickettsii, Ehrlichia species, Borrelia burgdorferi, Rickettsia parkeri, and Francisella tularensis.

Rickettsia rickettsii

R. rickettsii causes an acute, febrile tick-borne illness known as Rocky Mountain spotted fever (RMSF) (2). RMSF occurs after an infected tick bite results in the rapid entry of R. rickettsii into endothelial cells of human hosts, although humans are not the natural reservoir of this bacteria (2). Infection of vascular endothelial cells leads to disseminated inflammation, loss of barrier functional and change in vascular permeability (2). Symptoms that are presented with RMSF include fever, myalgias, headache, confusion, rash, and cardiovascular instability (2). Infected individuals are initially presented with maculopapular rashes around the wrists and ankles which further progress into petechiae (2). Influenza-like symptoms during the warmer months of summer are typically good indicators of rickettsial infection (2). Late stage (5 or more days into disease) signs and symptoms can include altered mental status, cerebral edema, necrosis and multiorgan system damage (3). Patients with RMSF often also present with thrombocytopenia, elevated hepatic transaminases and hyponatremia, although this usually appears later on in the disease manifestation (3). Established laboratory testing for R. rickettsii involves an indirect immunofluorescence assay (IFA) against IgG or IgM antibodies in the bloodstream (4). Alternatively, another test for RMSF is a PCR to detect the presence of rickettsiae DNA (4). Immunohistochemical staining of a skin biopsy can also be undertaken, but the accuracy rate of this method is lower than serological methods (4). Additional testing for metabolite levels and liver function may also be performed (4).

R. rickettsii bacteria are obligate, intracellular organisms shaped like coccobacilli (2). As one of its virulence factors, R. rickettsii contain the outer-membrane proteins OmpA and OmpB which allow them to interact with host cell receptors such as α2β1, Ku70, and FGFR1 to gain entrance to host endothelial cells through a process known as endocytosis (5,6). Once entering the host cell, these bacteria are able to avoid macrophage induced killing by releasing toxic chemicals through their Type IV secretion system (T4SS) (5). In doing so, they reach the cytoplasm to acquire host nutrients through the use of host proteins such as actin (5). Actin allows these intracellular bacteria to spread throughout host endothelial cells in a process called actin-based motility (6). This actin-based motility mediated by the host enables R. rickettsii to move throughout endothelial cells whilst avoiding the host immune system (6).

Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia muris-like Species

Ehrlichiosis disease is caused by the tick-borne bacterial species E. chaffeensis (the causative agent of Human monocytotropic ehrlichiosis or HME), E. ewingii and E. murin-like (7,8). These three members of the Ehrlichia genus are all gram-negative, obligate intracellular bacteria and are all transmitted to humans by ticks (7). Both E. chaffeensis and E. ewingii are primarily transmitted by the lone star tick (A. Americanum) whereas E. muris-like species is effectively transmitted by the blacklegged tick (Ixodes scapularis) (9). Both E. chaffeensis and E. muris-like species infect the monocytes and macrophages of the human host whereas E. ewingii infects neutrophils (8). Morphologically, these bacteria are coccoid and coccobacillary in shape (10). They are also quite small, pleomorphic and enveloped within a thin outer membrane and inner membrane where a periplasmic space lies between (11). The presence envelope proteins provide the interface between bacteria and hosts due to their lack of capsules, pili, and LPS (11). Therefore, these bacteria bind to and infect vertebrate host mononuclear cells via its outer membrane (10). Survival within the host cell requires iron acquisition due to complex molecules and biochemical pathways which require iron for cytochromes and other iron-containing enzymes (10).

Infection and the development of Ehrlichiosis generally requires an infected tick to feed on the human host (and thus remain attached) for at least 24 hours to be sufficiently transmitted (7). Once infected, the incubation period to the first appearance of symptoms is usually 7-14 days, with a median of 9 days (7,8). In the early stage of illness (generally the first 5 days of illness), the signs and symptoms may appear mild and include fever, chills, myalgia, nausea/vomiting and diarrhea, headache and appearance of a rash that may be maculopapular or petechial in nature or may even present as widespread erythema (7,12). This rash, although more commonly observed in children, is normally diffuse around the body and may appear on the face, trunk, extremities and potentially the palms and soles of the patient (7,12). If not treated with antibiotics in a timely manner, late stage illness of ehrlichiosis can develop with symptoms and signs including meningoencephalitis (or inflammation of the central nervous system and brain), respiratory and/or organ failure, uncontrollable bleeding and potentially death (12). Leukopenia, thrombocytopenia and elevated liver enzymes can also commonly be seen in Human monocytotropic ehrlichiosis or HME, which is one of the most prevalent fatal tick-borne diseases in the US (8). Those at higher risk for more severe cases of disease include those who have a weakened immune system (such as cancer patients receiving chemo-therapy, HIV patients and patients taking specific medications) and those who are either very young or elderly (12).

Borrelia burgdorferi

Borrelia burgdorferi is also a bacterial species associated with another tick-borne illness known as Lyme disease (13). This gram-negative “like” obligate parasite infects but does not cause disease to small mammals, lizards, and birds (13,14). Ticks most frequently acquire spirochetes from infected rodents during their larval feeding. However if transmitted to humans will result in infectious illness (13). Morphologically speaking, these bacteria have a spiral, wavelike body and motile flagella belonging to the family of Spirochaetaceae(13,14). The disease manifestation of Lyme disease normally presents in stages (15). In stage 1 or the early stage, which usually begins 3-30 days after the tick attachment, common signs and symptoms include erythemia migrans rash at the location of the initial tick bite (usually appears as a “bulls-eye”), fever, headache, general malaise, muscle and joint pain and swollen lymph nodes (15,16).In Stage 2 or the later stage, signs and symptoms can include severe headaches and neck stiffness, secondary erythema migrans on areas other than initial bite location, neurological issues like facial palsy, nerve pain/tingling and inflammation of central nervous system (16). Chronic Lyme disease, sometimes referred to as Stage 3, can involve symptoms such as dermatitis, neurological abnormalities including Bell’s-like palsy and potentially myocardial abnormalities such as pericarditis (15). When transmitted to human hosts, B. burgdorferi feeds on the blood which allows them to colonize in dermal tissue causing a localized infection (17). While B. burgdorferi is Gram-negative, its outer membrane lacks lipopolysaccharide which is a key component in many Gram-negative bacteria (18). Outer surface proteins such as OspC enable initial host colonization however the expression of other proteins such as VlsE enable the bacteria to survive attack from the host immune system, disseminate to other sites within the host, and acquire host nutrients (13). Since this bacterium does not secrete any sort of toxins, its main form of virulence arises from exploiting the host immune system (17). Inflammation in the host as a response to the infection will result in this disease (17). Laboratory testing for B. burgdorferi includes performing an ELISA and Western blot for antibodies against B. burgdorferi antigens (19). If required, testing for cardiac function and nervous system function may also be conducted. After 2-4 weeks after infection onset, testing for IgM and IgG antibodies against B. burgdorferi is used to confirm a positive diagnosis through IgG test results are preferred as IgG antibodies last far longer than IgM (19).

Rickettsia parkeri

R. parkeri is a gram-negative member of the bacterial Rickettsia genus which widely infect ticks, and subsequently humans through tick biting and attachment (20,21). The Rickettsia genus members are all obligate intracellular bacterial species that can be subdivided into five different groups including: the spotted fever group (or SFG), the typhus group (or TG), bellii group (or TRG), the canadensis group and the ancestral groups (20). R. parkeri is a member of the SFG species group (20). Additionally, as members of the Rickettsia bacterial genus, R. parkeri is also non-motile and lacks the ability to form spores (22). Infection by the pathogenic R. parkeri through tick bites, especially by the Amblyomma maculatum species (also known as the Gulf Coast tick), can result in R. parkeri rickettsiosis (10). Gulf Coast ticks primarily inhabit grassland and costal upland areas and can be found largely in the southeaster and mid-Atlantic states of the US (9,23). Clinical manifestation, including signs and symptoms, of R. parkeri rickettsiosis can closely mimic those of Rocky Mountain Spotted Fever (RMSF) caused by R. rickettsii (a closely related Rickettsia species) (23) R. parkeri rickettsiosis can also closely resemble disease caused by rickettsialpox, which is an eschar-associated illness caused by the bacterial species R. akari (10). With an incubation period between 2-10 days, patients with R. parkeri rickettsiosis develop fever, headache, muscle aches and a rash that can appear on the patient’s trunk and extremities and can include spare maculopapular or papulovesicular eruptions (23). Conversely to RMSF, R. parkeri rickettsiosis often also presents with an inoculation eschar at the location of the tick bite and initial attachment (23). Eschar is not commonly observed in patients with RMSF (23). R. parkeri rickettsiosis can also present with several clinical signs such as mildly elevated hepatic transaminases, mild leukopenia, and mild thrombocytopenia, although this is not as common as the other two (23). In general, R. parkeri rickettsiosis often mimics the signs and symptoms associated with RMSF, however, these symptoms and disease manifestation are usually milder than in patients diagnosed with RMSF (23).

Francisella tularensis

Tularemia resembles the above infectious scenario as well and is associated with the bacteria Francisella tularensis (24). Morphologically, this facultative intracellular pathogen is a small, gram-negative, non- spore forming, nonmotile aerobic bacillus (25). Transmission via tick bites to humans occurs through the skin in reported situations including hunting or skinning infected rabbits, and other rodents (24). Household, domestic cats are also susceptible to infection and may transmit these bacteria to humans (24). Exposure to humans may also occur when inhaling dust or aerosols that may be contained with F. tularensis (24). When spread to humans through insect bites or direct exposure to an infected animal, these bacteria can invade and rapidly multiply in a range of cell types, resulting in a widespread inflammatory response (26). In particular, it can invade cells of the immune system such as macrophages and arrest maturation of the phagosome, downregulating the host immune response (27). Signs and symptoms also vary depending on bacteria-host contact however flu-like symptoms such as fever, chills, headaches, myalgia, arthralgia and loss of appetite typically occur at the onset of infection (24,28). The ulceroglandular form of this disease is caused by tick and deer fly bites which eventually leads to the formation of a skin ulcer at the site of infection (24). Swelling of regional lymph glands also accompany this ulcer in the armpit or groin region, this can be referred to as glandular tularemia (24,28). Both ulceroglandular and glandular tularemia occur in 75 percent of diagnosed cases (28). Pneumonic tularemia and conjunctiva may also occur (28).

References

Biggs HM, Behravesh CB, Bradley KK, Dahlgren FS, Drexler NA, Dumler JS, et al. Diagnosis and Management of Tickborne Rickettsial Diseases: Rocky Mountain Spotted Fever and Other Spotted Fever Group Rickettsioses, Ehrlichioses, and Anaplasmosis — United States. MMWR.Morbidity and Mortality Weekly Report.Recommendations and Reports 2016;65(2):1-1–44.
Snowden J, Simonsen KA. Rickettsia Rickettsiae. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2020.
Rocky Mountain Spotted Fever (RMSF) | Tick-borne Diseases | Ticks | CDC [Internet]. 2020 [cited 2021 Mar 12]. Available from: https://www.cdc.gov/ticks/tickbornediseases/rmsf.html
Clinical and Laboratory Diagnosis - RMSF [Internet]. Centers for Disease Control and Prevention. Centers for Disease Control and Prevention; 2018 [cited 2021Mar13]. Available from: https://www.cdc.gov/rmsf/healthcare-providers/ClinLab-Diagnosis.html#:~:text=The%20standard%20serologic%20test%20for,rickettsii%20antigen
Proietti R. Rickettsia rickettsii [Internet]. Mechpath.com. 2017 [cited 2021 Mar 13]. Available from: https://mechpath.com/2017/12/11/rickettsia-rickettsii/
Sahni A, Fang R, Sahni SK, Walker DH. Pathogenesis of rickettsial diseases: Pathogenic and immune mechanisms of an endotheliotropic infection. Annu Rev Pathol. 2019;14(1):127–52.
Muzumdar S, Rothe MJ, Grant-Kels JM. The rash with maculopapules and fever in adults. Clinics in Dermatology. 2019 Mar 1;37(2):109–18.
Ismail N, Bloch KC, McBride JW. Human Ehrlichiosis and Anaplasmosis. Clinics in Laboratory Medicine. 2010 Mar 1;30(1):261–92.
Biggs HM, Behravesh CB, Bradley KK, Dahlgren FS, Drexler NA, Dumler JS, et al. Diagnosis and Management of Tickborne Rickettsial Diseases: Rocky Mountain Spotted Fever and Other Spotted Fever Group Rickettsioses, Ehrlichioses, and Anaplasmosis — United States: A Practical Guide for Health Care and Public Health Professionals. MMWR Recomm Rep. 2016 May 13;65(2):1–44.
Paddock CD, Childs JE. Ehrlichia chaffeensis: a Prototypical Emerging Pathogen. Clin Microbiol Rev. 2003;16(2):355–355.
Rikihisa Y. Molecular pathogenesis of Ehrlichia chaffeensis infection. Annu Rev Microbiol. 2015;69(1):283–304.
Signs and symptoms of ehrlichiosis | CDC [Internet]. Centers for Disease Control and Prevention. 2019 [cited 2021 Mar 12]. Available from: https://www.cdc.gov/ehrlichiosis/symptoms/index.html
Tilly K, Rosa PA, Stewart PE. Biology of infection with Borrelia burgdorferi. Infect Dis Clin North Am. 2008;22(2):217–34.
Shapiro E. Borrelia burgdorferi (Lyme Disease). Pediatrics in Review. 2014;35(12):500-509.
Bratton RL, Corey GR. Tick-Borne Disease. AFP. 2005 Jun 15;71(12):2323–30.
Signs and symptoms of Lyme disease | CDC [Internet]. Centers for Disease Control and Prevention. 2021 [cited 2021 Mar 12]. Available from: https://www.cdc.gov/lyme/signs_symptoms/index.html
Hyde JA. Borrelia burgdorferi keeps moving and carries on: A review of borrelial dissemination and invasion. Front Immunol. 2017;8:114.
Motaleb MDA, Liu J, Wooten RM. Spirochetal motility and chemotaxis in the natural enzootic cycle and development of Lyme disease. Current Opinion in Microbiology. 2015;28:106–13.
Diagnosis and Testing - Lyme Disease [Internet]. Centers for Disease Control and Prevention. Centers for Disease Control and Prevention; 2019 [cited 2021Mar13]. Available from: https://www.cdc.gov/lyme/diagnosistesting/index.html
Sebastian PS, Tarragona EL, Bottero MNS, Mangold AJ, Mackenstedt U, Nava S. Bacteria of the genera Ehrlichia and Rickettsia in ticks of the family Ixodidae with medical importance in Argentina. Exp Appl Acarol. 2017 Jan;71(1):87–96.
Rickettsia parkeri. In: Wikipedia [Internet]. 2021 [cited 2021 Mar 12]. Available from: https://en.wikipedia.org/w/index.php?title=Rickettsia_parkeri&oldid=1008688775
Rickettsia. In: Wikipedia [Internet]. 2021 [cited 2021 Mar 12]. Available from: https://en.wikipedia.org/w/index.php?title=Rickettsia&oldid=1008683571
Rickettsia parkeri Rickettsiosis | Tick-borne Diseases | Ticks | CDC [Internet]. Centers for Disease Control and Prevention. 2019 [cited 2021 Mar 12]. Available from:https://www.cdc.gov/ticks/tickbornediseases/rickettsiosis.html
CDC. Signs & Symptoms [Internet]. Cdc.gov. 2019 [cited 2021 Mar 13]. Available from: https://www.cdc.gov/tularemia/signssymptoms/index.html
Mansfield KG, Fox JG. Bacterial Diseases. In: The Common Marmoset in Captivity and Biomedical Research. Elsevier; 2019. p. 265–87.
Tularemia - Symptoms and causes [Internet]. Mayo Clinic. 2020 [cited 12 March 2021]. Available from: https://www.mayoclinic.org/diseases-conditions/tularemia/symptoms-causes/syc-20378635
Collins FM. Pasteurella, Yersinia, and Francisella. In: Baron's Medical Microbiology (Baron S et al., eds.) (4th ed.). Univ of Texas Medical Branch. 1996. ISBN 0-9631172-1-1.
Tularemia - NORD (national organization for rare disorders) [Internet]. Rarediseases.org. 2015 [cited 2021 Mar 13]. Available from: https://rarediseases.org/rare-diseases/tularemia/

(ii) What samples are taken for laboratory testing and how important is the Microbiology Laboratory in the diagnosis of this particular infectious disease?

Rocky Mountain Spotted Fever (RMSF) can be difficult to diagnose solely through clinical examination due to non-specific signs and symptoms in the early stages of illness, which makes the laboratory results crucial to the diagnosis of this disease (1). Blood samples, tissue samples and anthropod samples should be taken for laboratory testing to diagnose RMSF (2, 3). All specimens should be accompanied by relevant clinical information, including patient information, date of illness onset, date of specimen collection, brief clinical summary and exposure history and tests requested (4).

Blood Samples

Blood samples should be taken before antimicrobial therapy, and will be used for laboratory testing, including polymerase chain reaction (PCR), cell culture and serological testing (3, 5). Antimicrobial therapy can inhibit growth of bacteria in cell cultures, and decrease chance of identification in other laboratory testing (3, 5).

The whole blood sample will be used to obtain a complete blood cell count (CBC) to count the number of red blood cells, white blood cells, and platelets that are circulating in Robert’s blood (6, 7). It will also be used for a comprehensive metabolic panel to test for sodium, other metabolites and liver enzymes (6, 7). These laboratory findings from blood samples are often general and not specific to R. rickettsii infection, so cannot be used for diagnosis (5). However, abnormal laboratory findings such as low circulating blood platelets, elevated levels of liver enzymes, and low sodium, which are found in Robert’s case, in the blood may be suggestive of RMSF (5).

A whole blood sample can also be used for PCR and cell culture (8). For PCR, 3-5 mL of whole blood is required and it is collected in a citrate tube or an ethylenediaminetetraacetic acid (EDTA) tube, which are used to prevent coagulation of the sample (8). If collected in a citrate or EDTA tube, blood samples should be transported to the lab in less than 24 hours, and kept at 2-8 degrees Celsius, otherwise, it must be stored at -20 degrees Celsius for preservation (8). For cell culture, a whole blood sample of 3-5 mL should be collected in a heparin tube (8). This sample can be transported for longer than 24 hours, but should be stored and transported on dry ice (8). These handling considerations are important for microbiology laboratory testing because proper collection and transportation practices are important to maintain integrity of the sample and limit contamination in order to ensure accuracy of testing (9). Microbiology samples deteriorate with time and proper handling and timely transport can minimize loss of viability of the pathogen and overgrowth of contaminating organisms (9).

A blood serum sample can be collected for serological testing; indirect immunofluorescent assay is the most common test for RMSF diagnosis (3). Blood serum, when collected, is a clear fluid that remains from blood plasma when clotting factors are removed (5). Therefore, this blood serum contains no blood cells or proteins that function in blood clotting (5). However, it does contain glucose, antibodies and other proteins that are of interest in indirect immunofluorescence assay (IFA) (5). 12 mL of whole blood should be collected for each 5 mL of serum needed (10). These serum samples can be collected in a serum separator or anticoagulant tube (8). Samples should be transported to the lab in less than 24 hours, and kept at 2-8 degrees Celsius (8). Typically, these blood samples are collected 1 week after the appearance of symptoms and again 2 to 4 weeks later (5). These samples represent the acute phase and convalescent phase of the disease for “paired” serological testing to demonstrate evidence of a fourfold IgG titre seroconversion, which can confirm R. rickettsii infection (3, 11).

Tissue Samples

Tissue biopsy specimens from the rash should be collected because they can be used for cell culture, immunohistochemical staining and PCR (2, 3). The optimal tissue sample is a punch biopsy of skin approximately 4mm in size that includes the central aspect of the lesion (4, 8) To perform a punch biopsy, a circular blade is used and rotated down through the epidermis and dermis, and into the subcutaneous fat, yielding a 3- to 4-mm cylindrical core of tissue sample (12). The sample should be taken before or within 24 hours of antibiotic treatment (4).

Tissue biopsy may be fresh, frozen or formalin-fixed and can be submitted to laboratories for testing (4). A fresh tissue is preferred because it allows for cell culture isolation, PCR assay and immunostaining (4). After collection, place the specimen on sterile gauze pad and package in a sterile specimen collection cup to be refrigerated and sent to the lab by overnight delivery (4). Formalin-fixed tissue can be evaluated by PCR and immunostaining and may be stored and transported at room temperature (4, 8). A frozen sample can be put on dry ice, but can only be used for PCR testing (4).

Other Samples

If possible, an eschar sample from the site of the tickbite should also be taken (4). This can be used for PCR or culture tests (4 8). To take the sample, cleanse the eschar with a disinfectant, then use sterile tweezers to lift the scab and place it into a sterile specimen container (4). Use a dry, sterile cotton swab to sample the ulcerated area and place the swab in a sterile specimen container (4).

If possible, ticks or arthropods can also be taken as samples that can be used to test for R. rickettsii via cell culture or polymerase chain reaction (3). Identification of a particular bacterial species that is infecting the isolated tick may indicate the species that is responsible for the disease in the human host and may help narrow down testing procedure for confirmation and definite diagnosis (13).

Other samples like organ tissues, cerebrospinal fluid or pleural fluid can also be used for molecular diagnosis via PCR or cell culture, but these are not preferred specimens because R. rickettsii will only be present in severe disease cases (8). These samples can be collected in a sterile tube (8). Samples should be transported to the lab in less than 24 hours, and kept at 2-8 degrees Celsius; if more than 24 hours, the sample should be transported at -20 degrees Celsius (8). These handling considerations are important for similar reasons as stated above, to ensure pathogen viability and accuracy of laboratory diagnostic testing (9).

Why is the Microbiology Laboratory Important for Diagnosis?

Microbiology laboratory is key in diagnosis of RMSF because clinical symptoms are non-specific, and clinical symptoms and patient history are not sufficient for diagnosis of R. rickettsii (2). Therapeutic interventions are started based on clinical suspicion and epidemiological clues because treatment cannot be delayed (2). When a patient presents with the symptoms of a rickettsial disease and reports experiencing a tick bite, treatment with doxycycline will be administered as soon as possible, even before all samples and diagnostic tests are conducted (14). The findings from microbiology laboratory testing for RMSF commonly include data from PCR assays and indirect immunofluorescence assays to confirm suspicions of a particular rickettsial disease (14). Microbiology laboratory allows for diagnosis of R. rickettsii from the other potential bacterial pathogens to help select a specific and effective course of treatment (2).

Furthermore, according to the CDC, spotted fever rickettsioses (SFR), including RMSF, are classified as nationally notifiable diseases, so proper laboratory testing and diagnosis is important to ensure confirmed cases are reported to public heath officers (1) This can help with epidemiological studies to monitor and control the infectious disease (1).

Summary Table: Samples and Laboratory Tests for RMSF Diagnosis

SAMPLE	LABORATORY TEST
Whole Blood Sample	Complete Blood Cell Count Comprehensive Metabolic Panel Polymerase Chain Reaction Cell Culture
Blood Serum Sample	Indirect Immunofluorescence Assay
Tissue Biopsy Sample	Immunohistochemical staining Polymerase Chain Reaction Cell Culture
Arthropod Sample	Polymerase Chain Reaction Cell Culture
Cerebrospinal fluid or pleural fluid or organ tissue	Polymerase Chain Reaction Cell Culture

References

Clinical and Laboratory Diagnosis - RMSF [Internet]. Centers for Disease Control and Prevention. 2018 [cited 2021Mar13]. Available from: https://www.cdc.gov/rmsf/healthcare-providers/ClinLab-Diagnosis.html#:~:text=The%20standard%20serologic%20test%20for,rickettsii%20antigen
Biggs HM, Behravesh CB, Bradley KK, Dahlgren FS, Drexler NA, Dumler JS, et al. Diagnosis and Management of Tickborne Rickettsial Diseases: Rocky Mountain Spotted Fever and Other Spotted Fever Group Rickettsioses, Ehrlichioses, and Anaplasmosis — United States. MMWR.Morbidity and Mortality Weekly Report.Recommendations and Reports 2016;65(2):1-1–44.
Brouqui P, Bacellar F, Baranton G, Birtles RJ, Bjoërsdorff A, Blanco JR, et al. Guidelines for the diagnosis of tick-borne bacterial diseases in Europe. Clinical Microbiology and Infection 2004;10(12):1108-1132.
Instructions for Submitting Diagnostic Specimens for Testing by the Rickettsial Reference Diagnostic Laboratory. 2019; Available at: https://www.cdc.gov/ncezid/dvbd/specimensub/rickettsial-shipping.html. Accessed Mar 10, 2021.
Rocky Mountain spotted fever - NORD (national organization for rare disorders) [Internet]. Rarediseases.org. 2015 [cited 2021 Mar 13]. Available from: https://rarediseases.org/rare-diseases/rocky-mountain-spotted-fever/
Chapman AS. Diagnosis and Management of Tickborne Rickettsial Diseases: Rocky Mountain Spotted Fever, Ehrlichioses, and Anaplasmosis [Internet]. Centers for Disease Control and Prevention. 2006 [cited 2021 Mar 12]. Available from: https://www.cdc.gov/mmwr/preview/mmwrhtml/rr5504a1.htm
Shen N, Saumoy M, Wan DW. Rocky Mountain Spotted Fever: An Unusual Cause of Elevated Liver Function Tests. American Journal of Gastroenterology. 2015;110.
Portillo A, de Sousa R, Santibáñez S, Duarte A, Edouard S, Fonseca IP, et al. Guidelines for the Detection of Rickettsia spp. Vector borne and zoonotic diseases (Larchmont, N.Y.) 2017;17(1):23.
Specimen Collection and Handling [Internet]. St. James Healthcare. [cited 2021 Mar 19]. Available from: https://www.sclhealth.org/locations/st-james-healthcare/services/laboratory/specimen-collection-and-handling/
Guzman N, Yarrarapu SNS, Beidas SO. Anaplasma Phagocytophilum. [Updated 2021 Jan 5]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK513341/
Centers for Disease Control and Prevention. Diagnosis and Management of Tickborne Rickettsial Diseases: Rocky Mountain Spotted Fever and Other Spotted Fever Group Rickettsioses, Ehrlichioses, and Anaplasmosis — United States A Practical Guide for Health Care and Public Health Professionals. 2016.
Zuber T. Punch Biopsy of the Skin [Internet]. Aafp.org. 2021 [cited 12 March 2021]. Available from: https://www.aafp.org/afp/2002/0315/p1155.html
Sebastian PS, Tarragona EL, Bottero MNS, Mangold AJ, Mackenstedt U, Nava S. Bacteria of the genera Ehrlichia and Rickettsia in ticks of the family Ixodidae with medical importance in Argentina. Exp Appl Acarol. 2017 Jan;71(1):87–96.
Rocky Mountain Spotted Fever (RMSF) | Tick-borne Diseases | Ticks | CDC [Internet]. 2020 [cited 2021 Mar 12]. Available from: https://www.cdc.gov/ticks/tickbornediseases/rmsf.html

(iii) Explain the tests that will be performed on the samples in order to detect any of the potential bacterial pathogens causing this disease.

Serological Tests

Peripheral Blood Smear

Atypical lymphocytes were more commonly seen in Ehrlichia-infected cases. They were typically large, with hyperchromatic nuclei, abundant basophilic cytoplasm, and contained prominent cytoplasmic granules (Wright's-stained blood film, patient 6, original magnification × 300).

In a blood smear microscopy test, a drop of blood is spread thinly onto a glass slide and is then treated with eosin-azure type dyes, such as the Wright-Giemsa stain, so that blood cells can be examined and evaluated using light microscopy (1,2). Examinations of these smears may help laboratory technicians identify morulae located within the cytoplasm of infected leukocytes and monocytes in the circulation (2,3). Morulae can usually be observed in 3% of patients with HME; however, this testing method for HME is insensitive and should only be performed by experienced lab technicians (2,3). These trained technicians will also examine the blood smear’s shape, size and other characteristics manually with a light microscope (1).

Blood smear examination is useful for the diagnosis of E. chaffeensis ehrlichiosis or anaplasmosis as manual observation of morulae in the cytoplasm of infected leukocytes is suggestive of Ehrlichia or Anaplasma infection (4). However, positive identification of morulae is conducive to, but not sufficient to form a diagnosis of an Ehrlichia infection (7). Negative blood smear results also should not be used to rule out HME (2). When compared to HME, blood-smear microscopy is known to be more sensitive for detection of HGA (2). Additionally, this technique is not useful for B. burgdorferi testing because it does not cause bacteremia, so it cannot be visualized on a blood smear (5). Furthermore, these examinations are not useful for diagnosis of RMSF, or other Spotted Fever Group (SFG) rickettsioses as Rickettsia species are most often located inside cells within the tissues and thus not available in sufficient amounts in the blood to be visible in blood smears (4,6).

Immunofluorescence Assay (IFA)

Serological assays can help to distinguish between infection and disease caused by R. parkeri, R. rickettsia, E. chaffeensis, E. ewingii and E. murin-like (2). The gold standard, with the highest specificity, for serological testing for these species involves the use of indirect immunofluorescence antibody (IFA) assays for IgG antibodies in paired serum samples using select antigens unique to each species (8,9). These IFA or secondary immunofluorescence assays are used to detect the levels of circulating IgG antibodies to different bacterial pathogens in patient serum (10). To perform an indirect IFA, the lab technician fixes cells with R. rickettsii antigen onto a glass slide and exposes it to a sample of the patient’s serum (which has been allowed to clot and centrifuged to remove this clot) (11). Any antibodies against R. rickettsii present in the patient’s blood should bind to the fixed antigen on the slide (11). A control slide without R. rickettsii antigen is also exposed to the same serum to control for non-specific binding (11). After an incubation period, the serum is washed off and only the bound antibodies remain on the slide. The slide is then exposed to a secondary antibody specific to the primary antibody (the anti-R. Rickettsii IgG, in this case) and is labelled with a fluorescent marker (11). IFA assays for IgG antibodies reactive against many types of tickborne rickettsial pathogens are commercially available and are the recommended serologic method for confirming this type of disease in the United States (11). Upon visualization under a fluorescent microscope, the lab technician should be able to detect the presence of the antibody of interest determined by the presence or absence of fluorescent labelling in the sample (11). Additionally, the titre level of the patient’s IgG to the specific bacteria (its antigen) can be quantified through relative levels of fluorescence (10).

Although IFA is the principal diagnostic test that is used for the diagnosis of Rickettsia and Ehrlichia infections, there are currently no standard antigens used for identification of these pathogens in laboratories (2). For diagnosis of both HME caused by E. chaffeensis and Rickettsia, it is important to conduct IFA in paired serum samples, which requires the collection of one acute blood sample taken during the first week of illness and a second convalescent sample 2-4 weeks later (12). This paired sampling technique is required as antibodies can remain detectable for months or years after infection; additionally, antibody titers usually return a negative result within the 7-10 days of the disease manifestation (4,12). As a result, unpaired acute test results cannot be used by themselves to form a diagnosis (12).

At 14 days into disease manifestation, the IFA diagnostic test has been shown to be 94-100% sensitive, although this sensitivity increases if paired samples are examined (2). While some laboratories may test for IgM antibodies in IFAs (as are the first to appear in infection), IgM antibodies have lower specificity, can be cross-reactive with multiple antigens, can persist after acute illness and have been shown to be much more likely to return false positives; therefore they cannot be used to form a laboratory diagnosis on their own (4,12,13). Thus, diagnosis is supported but not confirmed by one or more samples of IgM antibody titer >64 in patients with a compatible acute illness (4). One limitation of IFAs is that false positives may also arise due to the fact that approximately 3-20% of healthy individuals have an elevated titre level at any time due to past disease or from related or unrelated cross-reactivity between antibodies reactive to different species and their antigens (9). Antibodies detected by IFA are group-specific, but not always species-specific, such that antibodies reactive with R. rickettsii detected in a serologic test may be from infection with another SFG rickettsii (4,6). Enzyme-linked immunosorbent assays (ELISAs) are more useful for B. burgdorferi (see below) (5).

Polymerase Chain Reaction (PCR)

An overview of a Polymerase Chain Reaction (PCR)

PCR allows for amplification of specific DNA sequences and can be performed on DNA that is extracted from the patient’s whole blood during the acute phase or from a skin biopsy sample of a rash lesion (12,14). It is useful for early detection of the infection before the development of detectable antibodies (15). Specific PCR primers can be used to confirm the presence of microorganisms or specific features or virulence factors (4,6).To perform a PCR, the patient’s blood is blended into a slurry and the solids are filtered out to leave behind a semi-translucent fluid called the supernatant (16). The supernatant contains DNA whose concentration can be amplified. RNA primers are added into the supernatant, along with DNA polymerase (such as taq) and deoxynucleoside triphosphates (dNTPs) are added to begin the reaction (16).

PCR cycles:

Denaturation: The sample DNA strands are separated by heating to 90-96 ℃ (16).
Annealing: The mixture is then cooled to 55-65 ℃ to enable the primers to bind to their complementary bases on the single-stranded DNA and act as origins of replication (16).
- Some target DNA sequences for Rickettsia PCR testing include the citrate synthase encoding gene (gltA), the 17 kDa antigen gene, the outer membrane proteins genes, ompA and ompB, and surface cell antigens genes, sca1 and sca4 (6).
- These primers are complementary to sequences upstream and downstream of the gene of interest (16).
Elongation: The temperature is then raised to the optimal functional temperature for the DNA polymerase (68-72 ℃) resulting in the synthesis of new DNA starting from the primers and attaching new dNTPs to the template DNA (16)

Each cycle of PCR duplicates the number of copies of the gene of interest that are specific to the bacteria species (16). This cyclic process is completed for nearly 30-40 rounds resulting in roughly one billion copies of the original DNA sample (17).

Identification and detection of bacterial DNA by PCR provides a rapid technique for diagnosing tick-borne diseases (2). PCR tests can be acquired from the CDC, some state health labs, and a small number of commercial or research labs (2). At the CDC, nested PCR assays are the standard molecular diagnostic test for blood and fresh tissue samples (18). The nested assays target two genes found in spotted fever group species which include the 17-kDa-protein-encoding gene and the ompA outer membrane protein (18). The ompA is later sequenced for species identification (18). However, this method is time-consuming and not sensitive enough for acute cases (18). To increase sensitivity for acute cases, real-time PCR assays have been developed such as R. rickettsii-specific assays which target 16S rRNA, sca4, ompB, gltA, and ompA genes (18). The specific primer used for R. rickettsii real-time assays is RRi6 (18). Real-time PCR does not rely on downstream analysis such as electrophoresis which reduces the time and effort needed for detection (19). Detection of E. chaffeensis also relies on real-time TaqMan PCR testing through the use of single-copy 16S rRNA gene and primers that amplify an 81-bp region of the gene (20). Positive results of the real-time assays imply the presence of pathogens. Moreover, F. tularensis detection may also be detected through the use of real-time TaqMan PCR testing (21).

Conventional PCR tests for whole blood samples that are more readily available lack a specified standard and the diagnostic sensitivity and selectivity may differ between different assays (2). Additionally, doxycycline treatment can also reduce the sensitivity of PCR assays and positive results usually only appear in patients with severe illness (2,9). However, due to its rapid turnover time, high specificity (60-85%) and sensitivity (60-85%) for E. chaffeensis, PCR has emerged as the diagnostic test of choice for HME confirmation (3). Additionally, PCR is the only confirmatory test available for E. ewingii infection as this bacterial species cannot be cultured in typical hospital labs (3). Various conserved genes between different Ehrlichia species are used as PCR targets which may include the rrs gene (for 16S rRNA) and the groESL heat shock operon (3). For diagnosis of RMSF, PCR is more useful when performed on a skin biopsy specimen than on an acute blood sample as R. rickettsii infects the endothelial cells that line blood vessels and may not circulate in large numbers until an advanced stage of the disease (2,14). Even when performed on skin biopsy specimens, PCR assays lack high sensitivity and negative results cannot exclude an RMSF diagnosis (2). Laboratory diagnosis of RMSF from samples collected during the acute phase of the disease can be improved by performing both PCR in conjunction with IHC staining (2). There are currently some new emerging techniques including real-time PCR and panrickettsia/R. rickettsii-specific assays that have been shown to reduce the occurrence of false negatives and improve the detection of R. rickettsii and other Rickettsia species while allowing for rapid, reproducible results (2,18). While the PCR test is very precise in detecting DNA, it is not as useful in this scenario compared to the IFA assay for RMSF as negative results from a PCR do not rule out a diagnosis (14).

Metabolite & Enzyme Panels

To quantify serum levels of sodium and other metabolites, Robert’s physician ordered a comprehensive metabolic panel (CMP) performed on Robert’s blood. Two results of interest are Robert’s sodium levels and bilirubin. Bilirubin is created when the liver naturally breaks down red blood cells (22). In addition to the CMP, Robert’s physician may have also ordered for a hepatic panel to be done on Robert’s blood. A hepatic panel is a group of blood tests that provide comprehensive information of the state of a patient’s liver which includes tests for biomolecules such as albumin, bilirubin, and various transaminases (23). In the case of RMSF, albumin and bilirubin levels are normal but levels of key liver enzymes such as alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) are elevated which suggest some injury or damage to the liver (23).

Histological Tests

Immunohistochemistry (IHC)

Positive staining pattern of a tissue sample with RMSF.

Stain positivity in immunohistochemistry (IHC)

Several tick-borne diseases, including F. tularensis infection, RMSF and Ehrlichiosis, can also be diagnosed through immunohistochemical staining of collected skin or tissue biopsy samples (2,14). Immunohistochemistry (IHC) is a microscopy-based technique for visualizing cellular components to identify discrete tissue components by targeting antigens of interest with labelled antibodies (24). These immunostaining methods are able to detect the presence of organisms in patients before or within the first 48 hours of using antibacterial therapy (14). The tissue collected is preserved to prevent breakdown of protein and degradation of tissue architecture (25). The tissue is rinsed free of blood before fixation/preservation to remove blood-derived antigens (25). The tissue is then fixed in formaldehyde and embedded in paraffin to maintain tissue shape during long-term storage (25). These fixed tissues are cut into small sections and mounted onto glass slides coated with tissue adhesive where they will be dried in an oven for deparaffinization (25). Antigen retrieval is performed before staining to improve antigen expression (25). Blocking of endogenous materials before staining also occurs to minimize the false-positive staining (25). The antibody-mediated antigen detection step can be done using direct and indirect methods (24).

Immunostaining agents against spotted fever group rickettsiae and E. chaffeensis can be obtained from the CDC and different university-based hospitals or commercial laboratories (2). An example of an immunostaining agent for R. rickettsia includes an anti-Rickettsia rickettsii polyclonal antibody from rabbit serum (26). The tissue sample is usually also stained with other dyes such as hematoxylin-eosin, which differentially stains different histological structures within the tissue sample and allows for observation of any morphological and anatomical changes (8). In an indirect assay, these agents are added to the tissue sample, incubated, washed and then incubated with secondary antibodies against the primary antibodies bound to the tissue sample (26). These secondary antibodies are either conjugated to an enzyme which can catalyze a colour-producing reaction (visualized by light microscopy) or are conjugated to a fluorophore that can be visualized with fluorescence microscopy (27).

For patients with a rash or eschar, immunostaining of skin biopsy specimen is a useful diagnostic tool for SFG rickettsioses (4). Sensitivity may be higher for tests on eschars compared to rash lesions due to the higher concentration of organisms in eschars (4). Accordingly, itt has been reported that immunostaining of skin specimens from biopsies of a rash lesion is 100% specific and 70% sensitive for the diagnosis of RMSF (2). However, because the causative bacteria may be focally distributed in the tissue, IHC performed on a small portion of collected tissue may not always detect the bacteria (2). Additionally, immunostaining of bone marrow biopsy species can be used for diagnosis of ehrlichiosis and anaplasmosis (4).

Diagnostic testing to detect B. burgdorferi infection and the diagnosis of Lyme disease (LD)

The most common tests for Lyme disease (LD) caused by B. burgdorferi involve the testing for antibodies against B. burgdorferi in the blood (28). The official recommended protocol from the CDC for testing for Lyme Disease includes a 2-tier methodology including a sensitive screening test which usually consists of an enzyme-linked immunosorbent assay (ELISA) performed first followed by a Western Immunoblot if the ELISA result is either positive or equivocal (29). The ELISA test provides quantitative information on the estimated concentration of antibodies against B. burgdorferi within the patient’s serum whilst the immunoblot presents qualitative information and reveals the specificity of the detected antibodies (29).

Enzyme-linked Immunosorbent Assay (ELISA)

An overview of an Enzyme-linked Immunosorbent Assay

An indirect ELISA follows a similar procedure to that of IFA. It allows for the detection and quantitative estimation of the levels of antibodies to specific antigens in a patient’s serum (30).

This technique is used to identify the presence of antigens or antibodies by triggering colour changes on particular enzymes by immobilization of antigens/antibodies on solid surfaces such as plastic beads (31).

The following steps are required for an ELISA (31):

A specific capture antibody is immobilized on high protein-binding plates by overnight incubation.
Samples taken from the patient and standard dilutions are added to the wells and will be captured by the bound antibodies.
Specific biotinylated detection antibody is added to the wells to enable the detection of the captured protein.
Streptavidin conjugated with alkaline phosphatase or horseradish peroxidase is added to the wells and will bind to the biotinylated antibody.
Colorimetric substrate is added to the wells and will form a coloured solution when catalyzed by the enzyme.
Absorbance is measured in an ELISA reader and the amount of protein (based on relative colour production) in the samples is determined.

Some rickettsial serologic testing is available in the ELISA format (11). Commercial laboratories might offer ELISA because of the ease in reading (11). For a positive ELISA test, the Western blot test will be used to further confirm the diagnosis of B. burgdorferi by identifying proteins based on molecular weight using gel electrophoresis (32,33).

Western Blot

A readout of a Western Blot indicating a positive result for Lyme disease.

If the ELISA returns positive or inconclusive (non-negative), then the more specific Western blot is performed to detect a series of antibodies against 10 proteins in B. burgdorferi (28,34). In a Western blot, proteins from the sample are separated by size using gel electrophoresis (36).

Steps to a Western blot (35):

Protein samples of a known concentration are loaded into an acrylamide gel along with a ladder
A 2-stage electric current is applied to pull the protein samples through the gel.
Once separated, proteins are then transferred to a solid support (usually membrane-like nitrocellulose) and marked using primary and secondary antibodies.
After incubation with primary and secondary, the membrane is then developed onto filmed or scanned into the computer.

The Western blot produces a strip with several dark bands, with each band representing the presence of an antibody directed against the pathogen in question (36). Each band thus represents a patient’s antibodies that display different specificity to different bacterial antigens (37). Oftentimes, a combination of many antibodies is used to confirm the presence of a particular bacterium (36). CDC guidelines require that at least 5 out of the 10 proteins return as positive for an overall positive diagnosis of Lyme disease (28,34). It is recommended that testing not be done prior to 2 weeks post-infection as IgM and IgG levels cannot reliably be detected during that time (28). IgG antibody tests are more reliable, but it takes upwards of six weeks for the body to produce a significant quantity that can be detected to confirm a diagnosis (36). Conversely, IgM antibodies are produced more quickly but are more likely to provide results that are false positives (36). There are currently tests being developed to identify and quantify the presence of OspA antigens shed by live B. burgdorferi into the urine, but the CDC does not recommend the use of such tests at the moment (34).

**Summary Table of the Different Tests Discussed in this Section**
Diagnostic Test	Goals of the test	Bacteria this test is relevant for
Immunofluorescence Assay (IFA)	Uses fluorescent-labelled antibodies to test for the presence of antibodies against bacterial antigens.	R. rickettsii, R. parkeri, E. chaffeensis, E. ewingii and E. murin-like
Immunohistochemical staining (IHC)	Uses reporter-antibody conjugates to detect the presence of specific antigens of interest.	R. rickettsii, R. parkeri, E. chaffeensis, E. ewingii, E. murin-like, and F. tularensis
Polymerase Chain Reaction (PCR)	Uses amplification amount to determine the presence of and quantify the amount of a target piece of DNA.	R. rickettsii, R. parkeri, E. chaffeensis, E. ewingii and E. murin-like
Enzyme-linked Immunosorbent Assay (ELISA)	Uses antibodies to determine the presence of and quantify the amount of a target protein.	B. burgdorferi (most useful for)
Western Blot	Uses fluorescence to determine the size and quantity of a particular target protein.	B. burgdorferi
Complete Metabolic Panel (CMP)	Assays for various biomolecules such as sodium ions and bilirubin that are important for metabolic processes.	R. rickettsii, R. parkeri
Hepatic Panel	Detects and quantifies the amount of specific key liver enzymes.	R. rickettsii, R. parkeri

References

Blood Smear. 2018; Available at: https://labtestsonline.org/tests/blood-smear. Accessed Mar 12, 2021.
Chapman AS. Diagnosis and Management of Tickborne Rickettsial Diseases: Rocky Mountain Spotted Fever, Ehrlichioses, and Anaplasmosis [Internet]. Centers for Disease Control and Prevention. 2006 [cited 2021 Mar 12]. Available from: https://www.cdc.gov/mmwr/preview/mmwrhtml/rr5504a1.htm
Ismail N, Bloch KC, McBride JW. Human Ehrlichiosis and Anaplasmosis. Clinics in Laboratory Medicine. 2010 Mar 1;30(1):261–92.
Biggs HM, Behravesh CB, Bradley KK, Dahlgren FS, Drexler NA, Dumler JS, et al. Diagnosis and Management of Tickborne Rickettsial Diseases: Rocky Mountain Spotted Fever and Other Spotted Fever Group Rickettsioses, Ehrlichioses, and Anaplasmosis — United States. MMWR.Morbidity and Mortality Weekly Report.Recommendations and Reports 2016;65(2):1-1–44.
Laboratory Methods for the Detection of Lyme Disease and Other Tick-Transmitted Diseases. Marshfield Labs 2013 Mar:1-3.
Portillo A, de Sousa R, Santibáñez S, Duarte A, Edouard S, Fonseca IP, et al. Guidelines for the Detection of Rickettsia spp. Vector borne and zoonotic diseases (Larchmont, N.Y.) 2017;17(1):23.
Clinical and Laboratory Diagnosis - Ehrlichiosis [Internet]. Centers for Disease Control and Prevention. Centers for Disease Control and Prevention; 2019 [cited 2021Mar13]. Available from: https://www.cdc.gov/ehrlichiosis/healthcare-providers/diagnosis.html
Paddock CD, Finley RW, Wright CS, Robinson HN, Schrodt BJ, Lane CC, et al. Rickettsia parkeri Rickettsiosis and Its Clinical Distinction from Rocky Mountain Spotted Fever. Clinical Infectious Diseases. 2008 Nov 1;47(9):1188–96.
Weis E. Rocky Mountain Spotted Fever (RMSF): Laboratory Testing.
Chhabra S, Minz R, Saikia B. Immunofluorescence in dermatology. Indian J Dermatol Venereol Leprol. 2012;78(6):677.
Centers for Disease Control and Prevention. Diagnosis and Management of Tickborne Rickettsial Diseases: Rocky Mountain Spotted Fever and Other Spotted Fever Group Rickettsioses, Ehrlichioses, and Anaplasmosis — United States A Practical Guide for Health Care and Public Health Professionals. 2016.
Rocky Mountain Spotted Fever (RMSF) | Tick-borne Diseases | Ticks | CDC [Internet]. 2020 [cited 2021 Mar 12]. Available from: https://www.cdc.gov/ticks/tickbornediseases/rmsf.html
Abdad MY, Abou Abdallah R, Fournier P, Stenos J, Vasoo S. A Concise Review of the Epidemiology and Diagnostics of Rickettsioses: Rickettsia and Orientia spp. J Clin Microbiol 2018;56(8):1728.
Clinical and laboratory diagnosis of RMSF | CDC [Internet]. Centers for Disease Control and Prevention. 2018 [cited 2021 Mar 12]. Available from: https://www.cdc.gov/rmsf/healthcare-providers/ClinLab-Diagnosis.html
Brouqui P, Bacellar F, Baranton G, Birtles RJ, Bjoërsdorff A, Blanco JR, et al. Guidelines for the diagnosis of tick-borne bacterial diseases in Europe. Clinical Microbiology and Infection 2004;10(12):1108-1132.
Walker, K. Identification of Bacterial Species Using Colony PCR. Ouachita Baptist University; 2015.
What is PCR (polymerase chain reaction)? [Internet]. Yourgenome.org. 2021 [cited 12 March 2021]. Available from: https://www.yourgenome.org/facts/what-is-pcr-polymerase-chain-reaction
Kato CY, Chung IH, Robinson LK, Austin AL, Dasch GA, Massung RF. Assessment of Real-Time PCR Assay for Detection of Rickettsia spp. and Rickettsia rickettsii in Banked Clinical Samples. J Clin Microbiol. 2013 Jan;51(1):314–7.
Maddocks S, Jenkins R. Quantitative PCR. In: Maddocks S, Jenkins R, editors. Understanding PCR. San Diego, CA: Elsevier; 2017. p. 45–52.
Loftis AD, Massung RF, Levin ML. Quantitative real-time PCR assay for detection of Ehrlichia chaffeensis. J Clin Microbiol. 2003;41(8):3870–2.
Emanuel PA, Bell R, Dang JL, McClanahan R, David JC, Burgess RJ, et al. Detection of Francisella tularensis within infected mouse tissues by using a hand-held PCR thermocycler. J Clin Microbiol. 2003;41(2):689–93.
Jewell T. What’s the Difference Between a CMP and BMP, the Two Common Blood Tests Ordered by Doctor? [Internet]. Luo EK, editor. Healthine. Healthine Media; 2018 [cited 2021Mar12]. Available from: https://www.healthline.com/health/cmp-vs-bmp
Shen N, Saumoy M, Wan DW. Rocky Mountain Spotted Fever: An Unusual Cause of Elevated Liver Function Tests. American Journal of Gastroenterology. 2015;110.
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Explanation of Lyme Disease Laboratory Testing. Utah Department of Health 2010 Apr:1-2.
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(iv) What are the results expected from these tests allowing for the identification of the bacteria named in this case?

Robert’s initial bloodwork shows low platelet count, low sodium levels and abnormal liver function. This is consistent with the expected results for R. rickettsii RMSF, which include thrombocytopenia (low platelet levels), and elevation in hepatic transaminases (increased liver enzymes) and hyponatremia (low sodium) (1). However, this initial bloodwork is not sufficient for identification of R. rickettsii because these laboratory values are non-specific and are often slightly deviated from the reference range early in the course of illness (1). Furthermore, other Rickettsia species, Ehrlichia species and A. phagocytophilum, which are also possible causes of the infectious scenario, show similar laboratory findings of thrombocytopenia and elevated hepatic transaminases (1).

Polymerase Chain Reaction (PCR)

DNA amplification is done to achieve serovar-level identification of the bacterium from samples isolated from the skin biopsy. PCR products are then visualized on an agarose gel, using gel electrophoresis. PCR methods for detection of R. rickettsii in clinical samples include nested assays for two SFG target genes, the 17-kDa-protein-encoding gene and the ompA outer membrane protein gene (2). The ompA nested amplicon may then be sequenced for species identification (2). However, this methodology takes 1-2 days and has not proven highly sensitive for the detection of Rickettsia spp. in blood samples during acute disease (2). Thus, it is likely these tests would be negative if conducted in the first week of illness, but positive in weeks 2-4.

Several real-time PCR assays have been developed for rickettsial agent detection, including Rickettsia genus-specific (panrickettsia) and R. rickettsii-specific assays (2). Panrickettsia assays have utilized conserved sites in the 17-kDa, outer membrane protein (ompB), 16S rRNA, and citrate synthase (gltA) genes, while species-discriminating assays have targeted the 16S rRNA, sca4, ompB, gltA, and ompA genes. These assays can significantly improve the detection of R. rickettsii and other Rickettsia spp, as they are rapid and more specific than traditional nested assays, so they are more likely to be positive early on in the illness (2).

Immunofluorescence Assay (IFA)

Representative indirect immunofluorescence assay staining of susceptible cells against either spotted fever group rickettsiae (A and B)

A positive result is seen when the target antigen associated with the disease can be visualized with the use of a fluorescent microscope. However, IFAs are insensitive during the first week of rickettsial infection, so a negative result early in the infection should not rule the disease out, and treatment should be initiated upon clinical suspicion (3).

In the majority of RMSF cases, the first IgG IFA titer is low or negative and the second shows a fourfold increase in IgG antibody levels (4). IgM antibodies usually rise at the same time as IgG (4). Also, IgM antibodies are less specific than IgG antibodies and are likely to result in a false positive. This is because IgM antibodies reactive with R. rickettsii are frequently detected in patients for whom no other supportive evidence of a recent rickettsiosis exists (4). IgM is associated with a high false-positive rate, including in patients with no history of any Rickettsia infection, but IgM may be falsely positive in patients with various other conditions. For these reasons, an IgG test is used in conjunction with IgM.

Immunohistochemical (IHC) Staining

Anti- Rickettsia immunohistochemistry demonstrating numerous organisms in the skin of animals inoculated with R . parkeri at 4 dpi as opposed to rare Rickettsia in the tick-only animal.

About 70% of IHS tests for R. rickettsii are positive when performed during the first 5 days of symptoms. (3). A positive result can be detected when using light or fluorescent microscopy to visualize the antibodies bound to the target antigen (5).

Immunostaining of skin biopsy specimens is 100% specific and 70% sensitive in diagnosing RMSF (4). Immunostaining can be particularly useful for diagnosing fatal tickborne rickettsial diseases in tissue specimens from patients who had not developed diagnostic levels of antibodies before death (4).

Enzyme Linked Immunoassay (ELISA)

Similar to the results with IFA, ELISAs used in the first week of infection may not be as sensitive as those used later on in the illness (4). Furthermore, the currently marketed ELISA kits for this bacterium offer only qualitative results (antibody presence or absence relative to a threshold value) and do not provide a quantitative method of demonstrating increases or decreases in antibody levels (4). We would expect to see a positive ELISA, detected through fluorescence.

Western Blot

As Rickettsia rickettsia is typically confirmed through IFA on paired samples 2-4 weeks apart, a western blot is usually not performed unless primary testing was positive for Lyme Disease (6).

References

Biggs HM, Behravesh CB, Bradley KK, Dahlgren FS, Drexler NA, Dumler JS, et al. Diagnosis and Management of Tickborne Rickettsial Diseases: Rocky Mountain Spotted Fever and Other Spotted Fever Group Rickettsioses, Ehrlichioses, and Anaplasmosis — United States. MMWR.Morbidity and Mortality Weekly Report.Recommendations and Reports 2016;65(2):1-1–4
Kato C, Chung IH, Robinson LK, Austin AL, Dasch GA, Massung RF. Assessment of Real-Time PCR Assay for Detection of Rickettsia spp. and Rickettsia rickettsii in Banked Clinical Samples. Chlamydiology and Rickettsiology 2012, 51 (1) 314-3174.
Rocky Mountain Spotted Fever [Internet]. Centers For Disease Control and Prevention. 2019 [cited 12 March 2021]. Available from: <http://www.cdc.gov/rmsf/index.html>
Centers for Disease Control and Prevention. Diagnosis and Management of Tickborne Rickettsial Diseases: Rocky Mountain Spotted Fever and Other Spotted Fever Group Rickettsioses, Ehrlichioses, and Anaplasmosis — United States A Practical Guide for Health Care and Public Health Professionals. 2016.
Direct vs indirect immunofluorescence | Abcam [Internet]. Abcam.com. 2021 [cited 13 March 2021]. Available from: https://www.abcam.com/secondary-antibodies/direct-vs-indirect-immunofluorescence
Lyme disease home | CDC [Internet]. Centers for Disease Control and Prevention. 2021 [cited 12 March 2021]. Available from: http://www.cdc.gov/lyme/index.html

3. Bacterial Pathogenesis

Using the following pathogenic steps outline the pathogenesis of the bacteria named as being responsible for this infection.

i. Encounter: where does the organism normally reside, geographically and host wise, and what are the bacterial characteristics that leave it suited to these places of residence. How would our patient have come in contact with this bacteria

Geography

Transstadial and Transovarian Transmission of R. rickettsii (Eremeeva & Dasch, 2015)

Rickettsia rickettsii is a small, Gram-negative bacilli that causes Rocky Mountain spotted fever (RMSF) (1). R. rickettsia is transmitted through tick bites, and there are three ticks that transmit this intracellular bacterium: the American dog tick, the Rocky Mountain wood tick, or the brown dog tick (1). The American dog tick is also known as Dermacentor variabilis and is found along the east of the Rocky Mountains and in portions of the Pacific Coast. The Rocky Mountain wood tick is also known as Dermacentor andersoni and is located in the Rocky Mountain region. Lastly, brown dog ticks known as Rhipicephalus sanguineus are found worldwide, and are the main vector for R. rickettsii in the southwest United States and in Mexico, where the diagnosis is most common (2). Globally, it is also restricted to the Americas, most commonly found in the USA, Western Canada, Mexico, Panama, Costa Rica, Argentina, Brazil, Columbia and Bolivia (3). Most reported cases occur between May and August as this coincides with the season when adult Mountain wood tick (Dermacentor andersoni) which are the primary arthropod in North America that transmits infection, are most active (4). Due to transmission by tick bite, the diagnosis is more common in the summer months when there is increased outdoor exposure (1). Ticks can live in grassy, bushy, or wooded areas and on animals, meaning you may be in close contact to ticks if you spend time camping outdoors, gardening or hunting (5). This is consistent with Robert’s case, as he had recalled getting a tick bite during a camping trip a week before his symptoms. This organism is suited for these places of residence because those are the locations for their tick hosts. Ticks become colonized by the R. rickettsii by feeding on bacteremic animals or by transstadial and transovarial transmission. Transovarial is when an adult female tick passes on the bacteria to the egg, and transstadial passage is when the egg retains the bacterium as it matures (6). Since R. rickettsii is transmitted both transstadially and transovarially in the respective tick species, those ticks are the reservoir for the bacteria and distribution of R. rickettsii will be identical to the distribution of the tick hosts (6). Ticks are a suitable place of residence for R. rickettsii because R. rickettsii can multiply in almost all organs and fluids of the ticks. They particularly invade via the salivary glands, allowing for transmission to vertebrate hosts during feeding (6). They can also only grow within host cells, meaning they can only reside within the ticks or a vertebrate, with small mammals often serving as hosts for the bacterium to amplify (7).

See figures for more information.

Host (human)

R. rickettsii is only stable in tick tissues, feces or blood and does not survive long outside its host (3). Within a human host, the bacteria infects the vascular endothelial cells lining the small and medium vessels in the body, leading to inflammation, loss of barrier function and altered vascular permeability (1). They can grow in the nucleus or the cytoplasm of the host cells, multiplying rapidly (8). The bacteria is able to leave the host cell because they have long, thin cell projections called filopodia, allowing the infection to spread rapidly to other cells (4). R. rickettsii is able to attach to the endothelial cell membrane which leads to its subsequent phagocytosis and escape into the cytosol (4). The bacterium induces phagocytosis through several surface proteins, OmpA, OmpB, Sca1 and Sca2 (9). Once phagocytosed, the bacteria is able to produce phospholipases to dissolve the phagocytic vesicle by destroying the phagosomal membrane that the lysosomes would fuse to (7). Since R. rickettsii is an obligate intracellular organism, it relies on the host endothelial cells to metabolize glucose and to synthesize lipids and nucleotides for use (10).

Certain individuals are at a higher risk of disease caused by R. rickettsia (11). The frequency of RMSF is highest among males and children (11). Two-thirds of the cases occur in children under the age of 15 (11). Individuals at a higher risk of severe RMSF are immunocompromised individuals, the elderly, and people with glucose-6-phosphare dehydrogenase deficiency (12). Repeated exposure to dogs who live near wooded areas, or locations with high grass may also increase your risk of infection with R. rickettsii (11).

Given that the geographic location of R. rickettsii is determined by the infected tick reservoir, it is most likely that Robert came into contact with the bacterium on his camping trip. Depending on the location of his camping trip, the corresponding tick will have been the reservoir host for the bacterium. The bacteria will infect the vascular endothelial cells in Robert, leading to vascular leakage that would explain his rash. Typically symptoms do not appear until a week after however, as seen in Robert (13).

References (3.i)

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Rickettsial Diseases, including Typhus and Rocky Mountain Spotted Fever [Internet]. [cited 2021 Mar 9]. Available from: http://textbookofbacteriology.net/Rickettsia_2.html
Rickettsia rickettsii - Infectious Disease and Antimicrobial Agents [Internet]. [cited 2021 Mar 10]. Available from: http://www.antimicrobe.org/r05.asp#t5
Minniear TD, Buckingham SC. Managing Rocky Mountain spotted fever. Expert Review of Anti-infective Therapy. 2009 Nov 1;7(9):1131–8.
Kenneth Todar M. Rickettsial diseases, including Typhus and Rocky Mountain fever [Internet]. Online Textbook of Bacteriology. [cited 12 March 2021]. Available from: http://textbookofbacteriology.net
Rocky Mountain spotted fever home | CDC [Internet]. Centers for Disease Control and Prevention. 2021 [cited 12 March 2021]. Available from: https://www.cdc.gov/rmsf/index.html
Nicholsan W, Paddock, Christopher. Rickettsial Diseases (Including Spotted Fever & Typhus Fever Rickettsioses, Scrub Typhus, Anaplasmosis, and Ehrlichioses) [Internet]. Centers for Disease Control and Prevention.

ii. Entry: what facilitates the entry of the bacteria into the human host? What are the molecular, cellular and/or physiological factors at play in the initial entry/adherence step from the point of view of the organism and the host.

As stated previously, R. rickettsii enters the human host via tick salivary secretions during tick feeding (1). Approximately six to ten hours of tick feeding on the human host is required for the bacterium to be transmitted into the individual, however, even longer periods of feeding may be required (1). Less commonly, humans can be infected by R. rickettsii during the removal of ticks from an infected human or animal, as the tick tissue may be crushed between their fingers (1). Another infrequent route of transmission is human exposure to R. rickettsii-infected tick feces in close proximity to abraded skin (2). The mean infectious dose of R. rickettsii is 23 organisms, but as few as one bacteria can cause infection (1).

Tick saliva may play an important role in the dissemination of R. rickettsii infection and the initial survival of the bacterium within the human host (3). Murine studies indicate that specific molecules in tick saliva support local infection of R. rickettsii in the skin (3). Ixodid (tick) saliva has been shown to alter the immune response to lipoteichoic acid which is an activator of toll-like receptor 2 (3). The saliva decreased the downstream signalling of the toll-like receptors while increasing the production of immunosuppressive cytokine interleukin 10 (3). Also, the saliva of ticks has been shown to inhibit neutrophil function, disrupt the complement system, natural killer cell activity, macrophage activity and decreases the production of cytokines (ex. interleukin-12, interferon-gamma) (4). In regards to RMSF, the effects of tick saliva may decrease the influx of inflammatory cells at the site of tick feeding, therefore, suppressing the recognition of R. rickettsii and the clearance of the pathogen (3).

After inoculation with R. rickettsii, the bacterium spreads throughout the body via the bloodstream (1). Adherence to and eventual invasion of target cells is necessary for the successful establishment of a R. rickettsii infection in the human body (4). R. rickettsii uses various proteins, such as OmpA, OmpB, Sca1, Sca2, and fibroblast growth factor receptor-1, to attach to and/or induce phagocytosis by their target cells, which are non-phagocytic vascular endothelial cells (1). OmpA, OmpB, Sca1 and Sca2 are R. rickettsii autotransporter proteins (5). Autotransporters comprise of an N-terminal signal sequence, a central passenger peptide, and a C-terminal “translocation module” (5). After translation, the peptide is transported across the inner membrane of the bacteria using the N-terminus sequence, then the C-terminus inserts into the outer membrane to form a transmembrane pore (5). The transmembrane pore is used to transport the passenger peptide into the extracellular environment (5).

OmpA is an outer-membrane protein of R. rickettsii that acts as an adhesin to aid in the attachment of the bacterium to the host cell, such as endothelial cells (4). The OmpB Rickettsia protein has been shown to bind to the receptor Ku70, a DNA-dependent protein kinase found in the plasma membrane of endothelial cells (6). The binding of OmpB to Ku70 recruits additional Ku70 proteins to the host membrane, which allows for more binding (4). Ubiquitin ligase is also recruited to the site of Rickettsia infection where Ku70 is ubiquinated, which allows for a signal transduction event that results in the recruitment of the Arp2/3 complex (4). Multiple molecules, such as cdc42, protein tyrosine kinase, phosphoinositide 3-kinase, and Src-family kinases, stimulate Arp2/3, which results in the phagocytosis of Rickettsia via the reorganization of the host cytoskeletal actin (4).

Sca1 (surface cell antigen) is present in the outer membrane of Rickettsia and may help with attachment of the bacterium to the host cell, however, this protein does not play a role in internalization (6). Sca2 can also be found on the surface of Rickettsia and may be involved in adherence and engulfment of the bacterium, however not much is known about the mechanism of function of Sca2 (7). One study demonstrated that expression of Sca2 on nonadherent, non-invasive E. coli was sufficient to facilitate adherence and invasion of mammalian cells, including endothelial cells (7). Sca2 has been shown to stimulate Arp2/3, which can result in reorganization of host cell actin and potentially phagocytosis of the bacterium (1). These findings suggest that along with other bacterial factors, Sca2 may help mediate bacterial attachment and engulfment into the host cell (7).

Heparan sulfate proteoglycans (HSPGs) induce the interaction between fibroblast growth factors (FGFRs) and their tyrosine kinase receptors, which results in receptor dimerization and activation, and has been involved in bacterial adhesion to host cells (8). Fibroblast growth factor-1 (FGFR-1) is commonly expressed on endothelial cells and has been associated with R. rickettsii adhesion and entry (8). A recent study proposes that the FGR1/HSPG complex on the host cell interacts with Rickettsia OmpA protein, FGR1 then binds to caveolin-1 and initiates bacterial entry via caveolin-1 dependent endocytosis (8).

After phagocytosis, the bacteria will escape the phagosome by producing phospholipases which can dissolve the phagocytic vesicle before it can fuse to the lysosome, allowing the bacteria to escape into the cytosol (2). Specifically, R. rickettsii will secrete phospholipase D and hemolysin C to disrupt the phagosomal membrane (9).

Therefore, to summarize entry for R. rickettsii is completed by four distinct steps (5,18):

OmpB on the bacteria attaches to Ku70 on the surface of human target endothelial cells while OmpA attaches to FGFR1 on the human target endothelial cell to increase binding (10,8).
CBL unbiquinates Ku70 and signal transduction events involving Cdc42, protein tyrosine kinase, phosphatidylinositol and 3’kinase (PI3-K) and Src-family kinases activate the Arp 2/3 complex to induce cytoskeleton actin remodelling and phagocytosis of the bacterium (10).
Phosplipase D, phospholipase A2 and haemolysin C mediate bacterial escape from the phagosome (10).
RickA on the bacterium stimulates Arp2/3 to mediate actin remodelling to propel the bacterium through the cytosol and into filopodia where they are either released extracellularly or infect adjacent endothelial cells (10). Sca4 is thought to assist with this process as well.

References (3.ii)

Blanton LS, Walker DH. Mandell, Douglas, and Bennett's principles and practice of infectious diseases. 9th ed. Philadelphia, PA: Elsevier; 2020. Rickettsia rickettsii and other spotted fever group rickettsia (Rocky Mountain spotted fever and other spotted fevers).
Walker DH. Medical microbiology 4th edition. Texas: University of Texas Medical Branch at Galveston; 1996. 21, Rickettsiae.
Grasperge B, Morgan T, Paddock C, Peterson K, Macaluso K. Feeding by Amblyomma maculatum(Acari: Ixodidae) Enhances Rickettsia parkeri (Rickettsiales: Rickettsiaceae) Infection in the Skin. J Med Entomol. 2014;51(4):855-863.
Walker D, Valbuena G, Olano J. Pathogenic Mechanisms of Diseases Caused by Rickettsia. Ann NY Acad Sci. 2003;990(1):1-11.
Riley S, Goh K, Hermanas T, Cardwell M, Chan Y, Martinez J. The Rickettsia conorii Autotransporter Protein Sca1 Promotes Adherence to Nonphagocytic Mammalian Cells. Infection and Immunity. 2010;78(5):1895-1904.
Chan Y, Riley S, Martinez J. Adherence to and Invasion of Host Cells by Spotted Fever Group Rickettsia Species. Front Microbiol. 2010;1.
Cardwell M, Martinez J. The Sca2 Autotransporter Protein from Rickettsia conorii Is Sufficient To Mediate Adherence to and Invasion of Cultured Mammalian Cells. Infection and Immunity. 2009;77(12):5272-5280.
Sahni A, Patel J, Narra H, Schroeder C, Walker D, Sahni S. Fibroblast growth factor receptor-1 mediates internalization of pathogenic spotted fever rickettsiae into host endothelium. PLOS ONE. 2017;12(8):e0183181.
Walker DH. Rickettsiae and Rickettsial Infections: The Current State of Knowledge. Clinical Infectious Diseases. 2007 Jul 15;45(Supplement_1):S39–44.
Walker DH, Ismail N. Emerging and re-emerging rickettsioses: endothelial cell infection and early disease events. Nat Rev Microbiol. 2008 May;6(5):375–86.

iii. Multiplication and Spread: does the organism remain extracellular or do they enter into cells and what are the molecular and cellular determinants of these events. Do the bacteria remain at the entry site or do they spread beyond the initial site i.e. are there secondary sites of infection and why do the bacteria hone in on these particular secondary sites.

R. rickettsii is an obligate intracellular pathogen, meaning it requires an intracellular lifestyle to replicate and propagate itself in host tissues. This is because R. rickettsii has undergone an extraordinary amount of genome reduction through evolution, now dependent on the host for its metabolic and motility-related functions. Possessing a genome of approximately 1.27Mb, this pathogen has evolved to take advantage of the host cells’ cytosolic environment and nutrients for its growth and maintenance (1). R. rickettsii is the most pathogenic species of Rickettsiae (1). It is intriguing that through the depletion of most non-essential genetic elements, R. rickettsii has acquired many important virulent elements: namely OmpA, OmpB, Sca1, and Sca2 adhesion molecules; phospholipase D and hemolysin C for persistence in the cytosol; followed by RickA, Sca2, and Sca4 to infect neighbouring cells through an actin-based motility (ABM) mechanism (2). Please see the graphically summary of R. rickettsii interactions with the host cell presented in Figure 2 (2).

Entry into host cells

Figure 2 from David H. Walker and Nahed Ismail. 2008. Nat Reviews Microbiology, p379

R. rickettsii is known to preferentially invade the vascular endothelium-- with less frequency they are observed to infect the underlying smooth muscle cells and macrophages (1). This is no small feat, as the bacterium must securely attach to the host cell membrane despite present antibacterial components (complement, antimicrobial peptides) and high velocity of circulating blood. OmpB is highly expressed on the surface of R. rickettsii and is used to interact specifically with Ku70 on the host endothelium (2, 3). Additional surface molecules assist the pathogen in adhering to the endothelial cell membrane, such as OmpA's interaction with fibroblast growth factor receptor (FGFR1). As many as 17 unique ‘surface cell antigens’, like Sca 1 and 2, may participate in facilitating R. rickettsii’s adherence to host cells (3). Once bound by OmpB, Ku70 is ubiquitinylated on its cytosolic side resulting in the activation of actin-related proteins (Arp2/3 complex). Specifically, Ku70 is ubiquitinylated by Cbl (E3 ubiquitin-protein ligase), triggering a signal transduction involving Cdc42-tyrosine kinase, PI3 and src-family kinases, resulting in the activation of the Arp2/3 complex. This signals a cytoskeletal rearrangement and phagocytic uptake of the bacterium, representing one of many methods R. rickettsii attempts to hijack the host actin assembly and remodelling pathways (2, 3).

Persistence in the cytosol

Once in the cytosol, R. rickettsii must evade destruction by the phagosome, while also preventing host cell death. This is achieved primarily through the production of phospholipase A2, phospholipase D, and hemolysin C (2). These virulence factors have membrane disruptive properties and prevent neutralization by the phagosome. The specific mechanism action is poorly defined, but it is speculated that these phospholipases oligomerize and form pores in the phagosome membrane (4).

By evading the host phagosome the bacterium persists within the cytosol, sequestering host nutrients, ATP, amino acids, and nucleotides for its own growth and division (5). They rely on host endothelial cells to metabolize glucose into useable substrates and to synthesize lipids and nucleotides for use (6,7). Through this mechanism, R. rickettsii is able to actively deplete available nutrients and ATP from the host, depleting the available energy to the cell (1, 6, 7).

To survive intracellularly for an extended period, R. rickettsii can manipulate host cell death mechanisms through the stimulation of NF-kB (nuclear factor kappa light chain enhancer of activated B cells). NF-kb is a regulator of inflammatory genes and prevents host cell death during infection (8). The activation of NF-kb is mediated by upstream activation of IkappaB kinase (IKK)- therefore, selective inhibition of IKK may provide a potential target for enhanced clearance of R. rickettsii and an effective strategy to reduce inflammatory damage (8).

Dissemination to neighbouring cells and distal tissues

R. rickettsii appears to be a “professional disseminator”. Unlike other intracellular pathogens, R. rickettsii replicates relatively few times by binary fission, before exiting the cell to infect a neighbouring endothelial or smooth muscle cell. This allows the pathogen to spread and infect a large number of cells, rather than accumulate in high numbers in any one given endothelial cell (1,2). This achieved through an actin-based motility (ABM) mechanism; the major virulence factors involved are RickA, Sca2, and Sca4 (7).

The ABM strategy employed by R. rickettsii can be described in five major stages (7). As previously described, R. rickettsii must first evade phagosomal destruction and prevent cell apoptosis before it may successfully gain access to the host actin machinery. Second, R. rickettsii attempts to hijack the host actin remodelling complexes to acquire actin-based motility (ABM). The bacteria manipulate this machinery through the production of RickA, Sca2, and Sca4 virulence factors. In uninfected cells, actin polymerization is mediated by actin nucleators and their cognate regulators (9). In cells infected by R. rickettsii, ABM is established by manipulating these host cell actin nucleators or by utilizing unique bacterial nucleators (9). These actin nucleators can be observed at the bacterial pole, causing polar actin polymerization allowing bacterial transport through the cytosol (9). Specifically, R. rickettsii encodes RickA, a protein with similar structure to N-WASP (a protein that induces actin polymerization and filopodia formation), that recruits Apr2/3 to the bacterial surface and initiates ABM (9). R. rickettsii also produces Sca2 which is necessary for cell-to-cell spread and functions similarly to actin nucleators. It is proposed that RickA mediates the Apr2/3 nucleation of actin filaments and Sca2 catalyzes the progressive nucleation at the barbed end, creating long actin filaments, (7, 9) with 'early motility' events being driven by RickA and Apr2/3 activation while late motility is more dependant on Sca2 (9).

In the third stage, the bacterium forms the membrane protrusion in the adjacent cell. Formation of the membrane protrusion, also called the filopodia, requires a release of tension at the neighbouring cell membrane and then elongation of the protrusion by an undefined mechanism involving Sca2 and Sca4 (7). The fourth step in cell-to-cell spread is the transformation of the membrane protrusions into vacuoles within the adjacent cells (7). These protrusions become double membrane vacuoles (DMVs), comprising an outer membrane of the infected cell and an inner membrane of the neighbouring cell (7). The formation of the DMV requires disassembly of the actin network; Sca4 is proposed to play a role in protrusion resolution (7, 8). Lastly, the bacteria must escape from the DMV once inside the neighbouring cell. In order for Rickettsia to infect and multiply within the adjacent cell, it has to escape the DMV, however, this process is more complicated than phagosomal escape as two membranes need to be disrupted. The mechanism of R. rickettsii escape from the DMV is poorly characterized, however Sca4 is thought to be involved (7). The process of cell-to-cell spread results in a network of cells infected with R. rickettsii, resulting in the rapid dissemination of infection (1, 7). R. rickettsii also uses the process of ABM to escape and exit the apical surface of the infected endothelial cell and return to circulation (1,2). In contrast with other intracellular bacteria, which typically accumulate in large numbers before exiting the cell, R. rickettsii leaves the host cell after only a few rounds of binary fission in the cytosol, allowing the pathogen to rapidly propagate and disseminate to many host cells and tissues (1, 2, 7).

R. rickettsii rapidly invades multiple cells and can disseminate to distal tissues. Cell-to-cell spreading represents a strong strategy to rapidly infect many host cells and spread into circulation before the host immune system can respond (2, 7). As a result, the infection can accumulate in distal organs and cause secondary infections. As the vascular endothelium is R. rickettsii’s preferred cell to invade, secondary infection can arise in organs with increased vasculature such as the skin, brain, lungs, heart, kidneys, liver, and gastrointestinal tract (10). In severe cases, R. rickettsii causes systemic infection resulting in toxic shock of the blood stream (1).

References (3.iii)

1. LS Blanton, DH Walker. Rickettsia rickettsii (Rocky Mountain Spotted Fever). Available from:http://www.antimicrobe.org/r05.asp#t5

2. Walker DH, Ismail N. 2008. Emerging and re-emerging rickettsioses: endothelial cell infection and early disease events. Nat Rev Microbiol. 6(5):375-86. doi: 10.1038/nrmicro1866. PMID: 18414502.

3. G Blanc, M Ngwamidiba, H Ogata, PE Fournier, JM Claverie, D Raoult. 2005. Molecular Evolution of Rickettsia Surface Antigens: Evidence of Positive Selection, Molecular Biology and Evolution, (22):10 p2073–2083, https://doi.org/10.1093/molbev/msi199

4. Whitworth T, Popov VL, Yu XJ, Walker DH, Bouyer DH. 2005. Expression of the Rickettsia prowazekii pld or tlyC gene in Salmonella enterica serovar Typhimurium mediates phagosomal escape. Infect Immun 73(10):6668-73. doi: 10.1128/IAI.73.10.6668-6673.2005. PMID: 16177343

5 - Rydkina E, Silverman DJ, Sahni SK. 2005. Activation of p38 stress-activated protein kinase during Rickettsia rickettsii infection of human endothelial cells: role in the induction of chemokine response: Rickettsia rickettsii-induced activation of p38 MAP kinase. Cellular Microbiology. 8;7(10):1519–30.

6 - Sahni SK, Rydkina E. Host-cell interactions with pathogenic Rickettsia species. Future Microbiol. 2009 Apr;4(3):323–39.

7. Weddle E, Agaisse H. 2018. Principles of intracellular bacterial pathogen spread from cell to cell. PLoS Pathog 14(12): e1007380. https://doi.org/10.1371/journal.ppat.1007380

8. DH Walker, GA. Valbuena, JO. Olano. 2003. Pathogenic Mechanisms of Diseases Caused by Rickettsia. Ann. N.Y. Acad. Sci. 990: 1–11.

9. Reed SCO, Lamason RL, Risca VI, Abernathy E, Welch MD. Rickettsia actin-based motility occurs in distinct phases mediated by different actin nucleators. Curr Biol. 2014;24(1):98-103. doi:10.1016/j.cub.2013.11.025

10. Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7624

R. rickettsii Cell-to-Cell Spread (Weddle & Agaisse, 2018)

iv. Bacterial Damage: do the bacteria cause any direct damage to the host (or is the damage fully attributable to the host response, as indicated below) and, if so, what is the nature of the bacterial damage. Can it be linked to any of the signs and symptoms in this case?

Infection by R rickettsii causes insults to endothelium and vascular smooth muscle, possibly through the activity of a rickettsial phospholipase, rickettsial protease or through free-radical peroxidation of host cell membranes (1). The pathologic effects of these rickettsial diseases originate from endothelial injury with loss of intravascular fluid into tissue spaces resulting in edema, resultant low blood volume, reduced perfusion of the organs, and disordered function of the tissues with damaged and inflamed blood vessels leading to rashes (1).

The major pathophysiologic effect of infection is increased microvascular permeability due to disruption of junctions between endothelial cells, inter-endothelial gap formation, stress fiber formation, and morphological endothelial cell shape change from polygons to large spindles resulting in damage (2). It is hypothesized that this is due to reactive oxygen species produced by infected cells that cause lipid peroxidative damage to the host cell membrane (2). This may be exacerbated by inflammatory molecules such as by IFNγ + TNFα. The altered vascular permeability that leads to fluid imbalance within the cell and this leads to oedema within the infected organ.

Oxidative stress-induced damage of host endothelial cells can also result in the reduction of host factors such as glutathione and increased concentrations of catalase, which increases the level of hydrogen peroxide and finally, a large reduction in enzymes such as glucose-6-phosphate dehydrogenase (G6PD), which is a major host defense against ROS-mediated cell damage (3). This is why individuals with a G6PD deficiency are at a higher risk for RMSF (4). In combination with ROS-mediated endothelial cell damage, R. rickettsii phospholipases and proteases contribute to host cell injury (5). The onset of RMSF symptoms usually occurs 2-14 days after the tick bite infected with R. rickettsia (5). The first signs and symptoms experienced by Robert are a headache, high fever and muscle aches, which can be attributed to the host immune system attempting to eradicate the R. rickettsii infection with the production of pro-inflammatory cytokines and interferons (2).

As R. rickettsii disseminates throughout the body via the bloodstream and cell-to-cell spread, the bacterium infects and damages endothelial cells, creating a network of infected blood-vessel lining cells (6) . Injury to the vascular endothelium results in blood leaking through holes in the vessel walls into neighbouring tissue; this results in increased vascular permeability (5,6). Increased vascular permeability and lesions in the blood-vessel lining can result in the characteristic rash associated with RMSF, as seen on Robert, which is typically observed 3-5 days after the occurrence of fever (5,6).

Prostaglandins and leukotrienes which are vasoactive modulators of tone and permeability are potential mediators of microvascular injury and vasculitis (7). They are generated by an inducible isoenzyme cooxygenase 2(COX2) (7). COX2 activity can be decreased by heme oxygenase, an antioxidant and anti-inflammatory that mediates activity (8). The production of this seems to be dependant on several factors such as dose and infection kinetics, protein synthesis by host cells, bacterial replication and patient immune status among others (8). This may dictate extent of autoimmune host damage as a result of bacterial infection. In addition to antioxidant and anti-inflammatory capabilities, heme oxygenase also plays an important role in the vascular endothelium by the diminution of vascular constriction, decrease in smooth muscle cell proliferation and inhibition of apoptosis (9). Increase COX2 activity results in prostaglandin secretion, specifically PGE2 and PGI2 (9). They are known to cause increased vascular permeability and edema which result in acute inflammation during infection (9). Therapeutic approaches targeting heme oxygenase or COX2 may serve as potential therapeutic modalities (9).

Increased vascular permeability can result in edema, resultant loss of blood volume, hypoalbuminemia, decreased osmotic pressure, hypovolemia and hypotension (5,6). Edema is the result of damage to the vascular endothelium, allowing for loss of intravascular fluid into the underlying tissue (1). These effects can be fatal, causing pulmonary edema and adult respiratory distress syndrome, shock, or acute tubular necrosis (6). Hyponatremia is a result of the inappropriate secretion of antidiuretic hormone (ADH) as a response to hypovolemia (5). As stated above, increased vascular permeability results in edema and decreased blood volume (hypovolemia); in response, an increased amount of ADH is secreted which acts on kidneys to increase the reabsorption of water in the blood, in order to uphold optimal water levels in the body (10). Reabsorption of water in the blood by ADH results in a decreased concentration of sodium levels (hyponatremia), which is a sign observed in the patient (10).

Damage to endothelial cells of the lining of the inside of small blood vessels causing increased vascular permeability can also increase the amount of blood cells involved in blood clotting (ex. platelets), in an attempt by the host to repair the damage (10). This results in a procoagulant state causing the release of procoagulant components, activation of the coagulation cascade with thrombin production and platelet activation (5). An increased amount of platelets recruited to the site of infection results in a depletion in the number of circulating platelets within the bloodstream, causing thrombocytopenia which is observed in the patient Robert (10).

There is also generalized vascular inflammation, oedema, and release of powerful vasoactive mediators that promote coagulation such as thrombomodulin plasminogen-activator inhibitor 1 and pro-inflammatory cytokines such as, IL-1, IL-6, IL-8 and E-selectin (11). This is exacerbated by by IFNγ + TNFα released by the host. Activation of genes encoding proinflammatory molecules occurs through signalling mechanisms requiring activation of NF-κB, a ubiquitous nuclear transcription factor involved in the control of apoptosis, inflammation and stress response. (9,12,13). The release of procoagulant factors, and subsequent coagulation cascade activation with generation of thrombin, platelet activation, increased fibrinolytic factors, and consumption of natural anticoagulants may lead to hemorrhages and infarcts but this is rare (2).

The cell-to-cell spread of R. rickettsii also creates a focal network of hundreds of contiguous infected endothelial cells that corresponds to lesions that cause rashes on the skin (5). Endothelial cell induction of heme oxidase and cyclooxygenase, production of prostaglandins and phosphorylation-induced destabilization of vascular endothelial cadherin is a contributor to vasodilation and increased vascular permeability (5). Platelets are consumed locally in various locations of infection, and thrombocytopenia is observed in 32 to 52% of patients, which leads to a procoagulant state. Procoagulant components are released and the coagulation cascade is activated with thrombin generation, platelet activation, increased antifibrinolytic factors, consumption of natural anticoagulants, activation of the kallikrein-kinin system, and secretion of coagulation-promoting cytokines (5).

The infection of endothelial cells by R. rickettsii results in injury to blood vessel-lining cells, therefore, damage can be done to any vascularized tissue within the body which may cause rash, interstitial pneumonia, encephalitis, interstitial nephritis, interstitial myocarditis and lesions in the liver, gastrointestinal wall, pancreas, brain, lungs and heart (5,6). Specifically, injury to liver cells by R. rickettsii infection can result in the disruption of liver function, which is observed in Robert (10). Also, damage to the gastrointestinal tract by R. rickettsii can result in Robert’s symptom of abdominal pain (5).

To summarize, R. rickettsii causes damage to the host by infecting endothelial cells, utilizing resources causing conformational change and death. Then, R. rickettsii infect neighbouring cells, or systematically spread to other organs. The systematic spread may explain the abnormal liver test observed in Robert. The damage to the vascular network can lead to rashes as seen in Robert. To fight infection, the host causes damage due to increased permeability that accompanies inflammation and oxidative stress. Autoimmune damage can occur resulting in further damage to the vasculature and contributing to the rash. The fluid imbalance and oedema resulting from altered permeability may explain the altered sodium levels, which is a key osmotic regulator. There is increased clotting due to inflammation which may explain the low platelet count observed in Robert. Thus, damage to the host is due to inflammatory insults as well as R. rickettsii damage.

References (3.iv)

References

1. Walker D. Chapter 38 Rickettsiae. In: Medical Microbiology. 4th Edition. Galveston (Tx) University of Texas Medical Branch; 1996.

2. Walker DH. Rickettsiae and Rickettsial Infections: The Current State of Knowledge. Clinical Infectious Diseases. 2007 Jul 15;45(Supplement_1):S39–44.

3. Walker DH, Valbuena GA, Olano JP. Pathogenic Mechanisms of Diseases Caused by Rickettsia. Annals of the New York Academy of Sciences. 2003 Jun;990(1):1–11.

4. CDC Centers for Disease Control and Prevention. Rocky Mountain spotted fever [Internet]. 2021. Available from: https://www.cdc.gov/rmsf/index.html

5. LS Blanton, Walker D. Rickettsia rickettsii and other spotted fever group rickettsia (Rocky Mountain spotted fever and other spotted fevers). In: Mandell, Douglas, and Bennett’s principles and practice of infectious diseases. 9th edition. Philadelphia: Elsevier;

6. Todar K. Rickettsial diseases including Typhus and Rocky Mountain fever. [Internet]. Online Textbook of Bacteriology; Available from: http://textbookofbacteriology.net/

7. Rydkina E, Sahni A, Baggs RB, Silverman DJ, Sahni SK. Infection of Human Endothelial Cells with Spotted Fever Group Rickettsiae Stimulates Cyclooxygenase 2 Expression and Release of Vasoactive Prostaglandins. IAI. 2006 Sep;74(9):5067–74.

8. Rydkina E, Sahni A, Silverman DJ, Sahni SK. Rickettsia rickettsii Infection of Cultured Human Endothelial Cells Induces Heme Oxygenase 1 Expression. IAI. 2002 Aug;70(8):4045–52.

9. Sahni SK, Rydkina E. Host-cell interactions with pathogenic Rickettsia species. Future Microbiol. 2009 Apr;4(3):323–39.

10. National Organization for Rare Disorders (NORD). Rocky Mountain Spotted Fever [Internet]. 2021. Available from: https://rarediseases.org/rare-diseases/rocky-mountain-spotted-fever/

11. Walker DH, Ismail N. Emerging and re-emerging rickettsioses: endothelial cell infection and early disease events. Nat Rev Microbiol. 2008 May;6(5):375–86.

12. Clifton D. Expression and secretion of chemotactic cytokines IL-8 and MCP-1 by human endothelial cells after infection: Regulation by nuclear transcription factor NF-B. International Journal of Medical Microbiology. 2005 Aug 22;295(4):267–78.

13. Rydkina E, Silverman DJ, Sahni SK. Activation of p38 stress-activated protein kinase during Rickettsia rickettsii infection of human endothelial cells: role in the induction of chemokine response: Rickettsia rickettsii-induced activation of p38 MAP kinase. Cellular Microbiology. 2005 Jul 8;7(10):1519–30.

4. The Immune Response

i. Host response: what elements of the innate and adaptive (humoral and cellular) immune response are involved in this infection.

Morphology of R. rickettsii

The Rickettsia genus belongs to the Rickettsiaceae family and are a group of small, aerobic, coccobacilli (rod shaped) that have a typical gram-negative cell wall and lack flagellum (1). These bacteria are obligate intracellular parasites of eukaryotic cells meaning they are able to reside in the cytoplasm or within the nucleus of the cells that they invade (1). This genus is then divided into three main groups of species: spotted fever group, typhus group, and other (1). We will be focusing on the spotted fever group because the bacteria Robert is diagnosed with, Rickettsia rickettsii, belongs to this group. The spotted fever group consists of a large group of tick-, mite-, and flea-borne zoonotic infections (2). R. rickettsia is one of the strains associated with human infection and alongside R. prowazekii is considered the most virulent rickettsia (1).

Innate Immune Response:

The host innate immune response is the first line of defense; it includes physical and chemical barriers, mucus, antimicrobial peptides (AMPs), the complement cascade, and innate cells such as macrophages, dendritic cells, granulocytes, and natural killer cells (3). Inflammation-induced by an invading pathogen is also an essential mechanism of the innate immune response and is characterized by redness, swelling, heat, pain, and loss of tissue function. Important microcirculatory events that occur during the inflammatory process include vascular permeability changes, white blood cell recruitment and accumulation, and inflammatory mediator release (4). These are further explained below. The physical and chemical barriers that the innate host defense is comprised of the skin and linings of respiratory, urogenital, and gastrointestinal tracts which are made of thin layers of epithelial cells (3). The skin has a variety of antimicrobial properties that form a protective shield against pathogens, including AMP’s, CD8+T cells, and specialized dendritic cells (DCs), known as Langerhans cells, that sample microbes and migrate to lymph nodes eliciting the appropriate immune response (3). These cells are part of both the innate and adaptive immune response and play a strong role in controlling rickettsial infections. However, physical barriers part of the innate immune system do not play a big role in rickettsial infections. As mentioned above, R.rickettsii are tick-borne diseases that are found in the saliva of ticks. The ticks can bite the host and cause the laceration of the skin and mucosal surfaces (5). As mouthparts of the tick are thrust into the dermis and into blood vessels, its saliva comes into contact with the blood and allows for the R. rickettsii bacteria to get into the bloodstream and start infecting the endothelial cells lining the vessels (5). As a result, it is able to bypass the physical barriers by using the tick as a vector. Isolation of R. rickettsii from its arthropod vector leads to low pathogenicity in humans, as it cannot get through physical host barriers (6). In Robert’s case, we are aware he went camping a week prior to his symptoms and suffered through multiple tick bites. When Robert was bit by an infected tick, the bacteria would have entered through the skin and spread through the bloodstream leading to the infection of vascular endothelium in his skin, brain, lungs, heart, kidneys, liver, gastrointestinal tract, and other organs (7).

Antigens Specific to R. rickettsii:

OmpA and OmpB, also known as Sca0 and Sca5, respectively, as well as Sca1 and Sca2 are part of the Sca, or ‘surface cell antigen’, protein family of autotransporters (8, 9). These bacteria cell wall proteins are key factors for the adhesion of R. rickettsii to host cells and tissue, and therefore influence the pathogenicity of the bacteria (10). Specifically, OmpA is thought to contribute to adherence to host cells, while OmpB is involved in both bacterial adherence and invasion processes (11). OmpB binds to Ku70, which is a host-cell receptor located in the cytoplasm and on the plasma membrane, that is recruited to the site of bacterial entry (9,12).

Endothelial Cells:

Summary of factors released by infected endothelial cell to induce the immune

The first step of infection, the tick bite and entrance of bacteria through the skin, is a direct breach of the skin/ physical barrier of the innate immune defenses. The bacteria’s main targets when they enter hosts are vascular endothelial cells and medium blood vessels, so once they enter the bloodstream, they are able to infect the endothelial cells that coat the inner wall of the blood vessels (13). ECs have defense mechanisms that get activated upon binding of pathogens. They function to trap and kill pathogens and activate additional innate immune cells (3). ECs also generate a variety of inflammatory cytokines and chemokines through the binding of their PRRs which mediate the inflammatory cytokines through NF-kB (3) These chemokines include CXCL8, CCL2/MCP-1(monocyte chemoattractant protein 1), CXCL9, CXCL10, and CCL5. CXCL8 predominantly recruits neutrophils to the site of infection, CCL2/MCP-1 and CCL5 recruit monocytes to the site of the infection, and CXCL9 and CXCL10 attract activated natural killer (NK) to the site of the infection (14). Furthermore, NF-κB is a nuclear transcription factor within ECs that can help dictate the outcomes in innate immune responses because it regulates the expression of cytokines and chemokines that ultimately protect the host from bacterial infection (15). The transcription factor NF-κB that gets activated in rickettsia-infected ECs has been demonstrated to be involved in many of these events, including the expression of TF, CXCL8, and CCL2 (14). Additionally, the NF-κB induces the release of several inflammatory cytokines such as IL-6, IL-12, IL-1β, and TNFα, which are soluble mediators of inflammation (14). Additionally, rickettsia-infected ECs are involved in innate immunity because they produce vasoactive prostaglandins known as PGF1α, PGI2 and PGE2. rickettsial-infected ECs produce the enzyme haem oxygenase, which regulates the cyclooxygenase-2 (COX2) enzymes responsible for producing prostaglandins (16). Vasoactive prostaglandins contribute to the regulation of the acute inflammatory response by increasing vascular permeability during spotted fever rickettsioses (16,17).

PRR and PAMPs:

Specific cells in the body known as antigen-presenting cells (APCs), such as macrophages, dendritic cells, and B cells contain pattern recognition receptors ( PRRs) (3). There are many types of pattern recognition receptor (PRR) families; however, some of the best-characterized families are Toll-Like receptors (TLRs) and Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) (3). There are a variety of TLRs and they each function as a key element to the immune system because they have the ability to activate both the innate and adaptive immune system. PRRs function to recognize and respond to pathogens that are able to breach the immediate defenses through the recognition of pathogen-associated molecular patterns (PAMPs) (3). PAMPs are often components of the cell wall and include proteins, carbohydrates, nucleic acids, and lipids that are associated with the pathogen. Some examples of the PAMPs associated with R. rickettsii are the OmpA, OmpB and Adr2 surface proteins, peptidoglycan, and lipopolysaccharide (LPS) (3). It is important to note that for rickettsia infections a PRR such as TLR4 would be involved because TLR4’s are known to recognize LPS of gram-negative bacteria.

Macrophages and Neutrophils:

Macrophages and neutrophils are cells of the immune system that respond to many pathogens and are non-specific. They are early responders that quickly migrate towards the infection site. Both of these cells are effective killers, as they can phagocytose the pathogen and highly destructive substances such as reactive chemicals or enzymes to kill the pathogen [18]. Most macrophages and neutrophils express pattern-recognition receptors (PRRs) on their surfaces. Once the PRR’s are engaged, these professional phagocytes (also including monocytes, eosinophils, and DCs) phagocytose microbes in their vicinity and fuse with lysosome(s) to form phagolysosome(s) that become acidified and are able to kill the microbe (3). Though macrophages are similar to DCs they have a limited capacity to migrate to regional lymph nodes (3). Instead, when phagocytose killing a bacterium, their phagolysosomes are able to reach a much lower pH, meaning they have better microbe killing capabilities when phagocytosing (3). Subsequently, the recognition of rickettsial LPS by TLR4 on host macrophages provides the priming signal for the activation of the ASC–caspase-1–IL-1β inflammasome during rickettsial infection. Inflammasomes are cytosolic multiprotein complexes that control the inflammatory response and coordinate host antimicrobial immunity (19). The inflammasome detects danger signals and infectious pathogens in the cytosol by cytosolic receptors, such as the NLRs mentioned before, and subsequently activates caspase-1, which is an enzyme. As a result, the activated caspase-1 cleaves pro-IL-1β/IL-18 into functional molecules of IL-1β and IL-18, which are pro-inflammatory cytokines that are released extracellularly and help with the inflammation response (19). It has been demonstrated that the ASC inflammasome plays an important role in host control of rickettsiae, as it promotes host surveillance during cytosolic infection with Gram-negative bacteria, such as R. rickettsii. The inflammasomes are also responsible for recognizing intracellular R. rickettsii bacteria at a late stage of infection, so they might be ideal candidates for future vaccine development targeting inflammasome activation (20).

Post PRR Activation:

Following engagement of the PRRs different pathways are activated which all work to protect the body and clear the bacteria through the recruitment of an array of signaling complexes that mediate inflammation, autophagy, or cell death (3). More specifically, if TLR4 recognizes the LPS of R. rickettsii, it activates immune cell activation through the use of myeloid differentiation primary response 8 (MyD88) which results in the activation of dendritic cells (DCs) and natural killer (NK) cells (3). Additionally, MyD88 signaling leads to the secretion of pro-inflammatory cytokines such as IL-1β and IFN-γ by DCs (11, 21). These cytokines will further induce the local inflammatory response, and IFN-γ will contribute to the killing of intracellular R. rickettsii by infected endothelial cells. The importance of MyD88-signaling in the immune response against rickettsiae infection is seen in mouse models where mice with loss-of-function mutations in the MyD88 gene have higher severities of infection compared to those with normal MyD88 genes (21). Dendritic cells are important because they link the innate and adaptive immune systems together due to their ability to activate naïve T cells (3). DCs are divided into two categories, the myeloid (mDC) and plasmacytoid (pDC) types which originate from a common DC precursor. The mDC’s play a big role in R. rickettsii infections because of their PRR-rich cell surface, sensitivity to microbes and inflammation, and their ability to quickly alert and instruct the immune system, through proinflammatory cytokine production and their ability to migrate to lymph nodes (3) Dendritic cells phagocytose pathogens early in infection and present antigens on major histocompatibility complex class I (MHC I) and MHC II pathways to CD8+ cytotoxic T cells and CD4+ helper T cells, respectively (22). Furthermore, NK cells are innate cytotoxic cells that can directly kill infected target cells and are implicated in early innate defense against R. rickettsii infection (23). These cells respond to rickettsiae infection by secreting cytotoxic perforin and granzyme granules and inflammatory cytokines, IFN-γ and TNF-ɑ (11). Perforin and granzyme are cytotoxic granules that punch holes in target cell membranes to induce apoptosis (11). IFN-γ initiates the defense and clearance of the pathogen by activating macrophages (11). The production of nitric oxide by these activated macrophages helps restrict rickettsial growth (11). Additionally, IFN-γ assists in the Th-1 cell response (11). On the other hand, TNF-ɑ contributes to the local inflammatory response (23). IFN-γ and TNF-ɑ cytokines released by NK cells can also activate endothelial cells and enhance their ability to kill pathogens via nitric oxide and hydrogen peroxide production (24). Interestingly, IFN-γ and TNF-ɑ provided from cells of the adaptive immune system to NK cells, later on, can further enhance their role in defense (23).

Adaptive Immune Response:

The adaptive immune response is referred to as the second line of defense and it functions to further eliminate the infection that was not resolved by the innate immune response as well as provides protection in cases of reinfection through immunological “memory” (25). While innate immune responses are very important in early defense against rickettsial infections, adaptive immunity is also essential for protection (26). Unlike innate responses, the adaptive immune system responses are highly specific to the particular pathogen that induced them. The adaptive immune response is based on the antigen-specific responses of T and B cells (26), and is separated into two parts: the humoral immune response and cell-mediated immune response. As mentioned above, DCs play a bridging role between the innate and adaptive immune systems for they are able to activate the adaptive immune system. DCs digest the pathogen and present its antigen material on Major Histocompatibility Complex Class I and II (MHC I and II) proteins, which are found on the surface of dendritic cells. MHC I and II proteins play a pivotal role in the adaptive branch of the immune system because both classes of proteins share the task of presenting peptides on the cell surface for recognition by T cells (27). CD4+ T helper cells become activated once they bind to MHC II on dendritic cell surfaces via T cell receptors (TCR), and CD8+ T cells become activated once they bind to MHC I on DC via TCRs as well. The activation of T cells is needed for the activation of the adaptive immune system.

Cellular Response:

The cellular component of the adaptive immune response heavily involves T cells. T cells play a great role in defending the body from rickettsial infection both through helping B cells with antibody production and the ability they have to release effector molecules that help with the role of CD8+ T cells (13). Beginning with the induction phase, bacteria are ingested by cells such as dendritic cells, also known as antigen-presenting cells (APSc). The bacteria are then proteolytically processed as the APCs travel to the lymph nodes where they are presented to T cells through the use of MHC molecules (28). Although activated antigen-specific CD4+ and CD8+ T cells both proliferate and differentiate into antigen-specific cells that produce IFN-γ and TNF-α, the most important mechanism of defense against rickettsiae is ascribed to cytotoxic CD8+ T cells. This is because one of the roles of CD4+ T cells is driving the activation of B cells and the humoral immune response, to produce specific antibodies against bacteria. However, R. rickettsii are intracellular pathogens, so the direct killing of infected cells by cytotoxic CD8+ T cells helps them fight rickettsial infections more than CD4+ T cells (14).

Overview of T cell-mediated protection mechanisms

CD8 + T cells

CD8+ T cells that are activated during rickettsial infections work in a similar manner to NK cells, except they are specific to R. rickettsii infected host cells. Cytotoxic CD8+ T cells induce apoptosis (cell death) in rickettsial-infected cells via the release of perforin and granzymes, in a similar mechanism as mentioned above. In addition, as mentioned previously, CD8+ T cells produce IFNγ and TNFα that have host protective effects as described previously (14). These cytokines induce the production of nitric oxide (NO) by macrophages and neutrophils and thus enable bacterial killing. CD8 + CTLs are especially crucial in the host response against R. rickettsii infection due to the intracellular nature of this pathogen (29). CD8 + CTLs recognize peptides presented on MHC class I molecules, which are predominantly endogenous peptides (except in cases of cross-presentation). Therefore, peptides from intracellular R. rickettsii can be presented on MHC class I molecules during infection and activate CD8 + CTLs. CD8 + CTLs will eliminate the pathogen through direct killing as explained above.

CD4 + T cells

CD4+ T cells can also contribute to protection against rickettsial infections by the release of effector molecules that can activate phagocytes. The main effector molecules that are considered to be involved in CD4+ T cellular–mediated defense against intracellular pathogens are IFNγ and tumor necrosis factor-alpha (TNFα) (14). CD4+ T cells usually differentiate into T Helper 1 (TH1) cells that produce IFNγ and TNFα in the infection with rickettsiae, and the induction of TH1 cells is mediated by IL-12 production by DCs. In the absence of external IFNγ, CD4+ T cells develop into T Helper 17 (TH17) cells in the infection with rickettsiae (14). These cells release inflammatory cytokines such as IL-17A, IL- 22 and TNFα. IL-17A and TNFα can then work together to induce the production and release of NO and ROS by macrophages. IL-17A induces the production of chemokines, leading to the recruitment of neutrophils that also contribute to local inflammation. IL-22, on the other hand, does not act on immune cells but instead acts on various tissue cells to induce the release of antimicrobial peptides to contribute to bacterial elimination (11).

Comparing innate versus adaptive immune response

When examining the effectiveness of role of CD8+T and CD4+ T cells in rickettsial infections in mice it, was noted that CD8+ T cells play a great role in the clearance of the infection through their cytotoxic activity and release of IFN- γ while CD4+ T cells seem to more important for the control of the persisting bacteria (5). CD8+ Cytotoxic T cells (CTLs) mainly function to kill host cells that have been infected by a pathogen or have undergone deleterious changes (30). Furthermore, because rickettsiae are known to infect host cells, the cytotoxic role of CTLs is a critical defense mechanism (30).

Humoral Response:

The humoral response is centered around antibody production. B cells secrete immunoglobulins (or antibodies) in the extracellular spaces of the body in places where pathogens tend to spread through, to help fight infections (25). Antibodies then work to control infections through mechanisms such as neutralization, opsonization, and complement activation (25).

While the specific functions of antibodies in R.rickettsii infection and have not been fully characterized yet, it is speculated that opsonization is at least one of the mechanisms that are used by antibodies against this pathogen (39). However, antibodies are not formed until the later stages of infection and since R. rickettsii is an intracellular pathogen that can largely avoid detection by antibodies, it is likely that antibodies are not a significant defense during primary infection. They may instead contribute to defense mainly during secondary infections. OmpA and OmpB contain epitopes that are targets of humoral immunity by the host (8). IgG and IgM antibodies against these antigens are produced and can be used for the diagnosis of R. rickettsii infection (31). Although R. rickettsii contains LPS that can also be targeted by humoral immunity, antibodies against LPS alone do not provide protection against infection by this pathogen (8).

Better describes roles that the innate and adaptive immune cells play during a bacterial infection such as R. ricketsii infection.

References

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ii. Host damage: what damage ensues to the host from the immune response?

The target cells of R. rickettsii are the endothelial cells that line the vasculature, which serve regulatory functions in angiogenesis (formation of new blood vessels), hemostasis (cessation of bleeding), vascular tone, immunity, and inflammation; therefore, bacterial infection of these endothelial cells around the body affects all of these functions [1]. During R. rickettsii infection, the host immune inflammatory and coagulation system are activated to recruit immune cells and to repair vascular damage from infection [2]. Under-activation of the immune response can result in ineffective killing of the invading pathogen; however, when overstimulated, these immune responses can cause host damage.

Figure 1. R. rickettsii replication in endothelial cells and induction of local inflammation.

Host Damage from Inflammation

Although the endothelial cells play an active role in the immune response, they can also actively play a role in host damage that ensues from the immune response. The main mechanism through which the immune response against rickettsiae ensues damage to the host is through vasculitis, which is defined as the inflammation of the blood vessels [3]. When endothelial cells are infected by R. rickettsii bacteria, they release inflammatory cytokines and vasoactive prostaglandins, which increases vascular permeability. Eventually, due to the immune response, there will also be perivascular infiltration of CD4+ T cells, CD8+ T cells and macrophages [2,4] at the site of the infection. ROS and TNF-α produced by neutrophils and macrophages, and IFN-ɣ produced by NK cells and macrophages, can cause an overwhelming systemic release of inflammatory cytokines and an exaggerated inflammatory response [4]. The dendritic cells and macrophages will also recruit mast cells that are known to release pro-inflammatory cytokines and preformed mediators such as histamine, prostaglandins, and leukotrienes, which result in blood vessel dilation [5]. Additionally, there is another mechanism by which increased vascular permeability can occur in rickettsial infections, and it also involves the release of pro-inflammatory cytokines during the innate immune response. During infection, host endothelial cells will destabilize cadherins, which are cell adhesion molecules between vascular endothelial cells. When endothelial monolayers are infected by R. rickettsii in the presence of inflammatory cytokines, such as TNF-α and L1-1β, the redistribution of P120 and β-catenin proteins occurs [3]. P120 and β-catenin proteins are molecules that attach endothelial cells to the extracellular matrix and regulate interaction of vascular endothelial (VE) cadherin with the actin cytoskeleton [3]. Vascular endothelial (VE)-cadherin is a strictly endothelial specific adhesion molecule located at junctions between endothelial cells, and is essential for the maintenance of endothelial cell contacts [3,6]. Rickettsial activation of endothelial cells causes phosphorylation of the VE-cadherin, which results in decreased VE-cadherin interactions between the endothelial cell junctions and thus disruption of endothelial barrier function [7]. As a result, all of these molecules function to increase vascular permeability and vasodilation [8].

The result of increased vascular permeability is the leakage of fluid from the bloodstream into the interstitial space, which accumulates as interstitial fluid in the tissues surrounding rickettsiae-infected endothelial cells. The accumulation of fluid in the surrounding tissues leads to edema (swelling), hypotension (low blood pressure), hypoalbuminemia (deficit of blood albumin), poor blood delivery to tissues and organs [9], and hypovolemia, which is defined as a lower amount of fluid remaining in the circulation within blood vessels [3]. Moreover, an inflammatory response involves an enormous expenditure of metabolic energy and often results in damage and destruction of host tissues [5]. Additionally, many of the pro-inflammatory cytokines released during inflammation are the culprit for some of the symptoms seen in Robert, such as his fever and headache [10].

Although the activation of the innate and adaptive immune system is critical for the clearance of infection, the cytokines and other cells produced may result in increased vascular permeability or increased inflammation that ultimately harms the host [11]. For instance, the R.rickettsii bacteria can spread to the endothelial cells within the brain and lungs. The brain and lungs lack lymphatic vessels to drain any excess interstitial fluid that may arise due to vascular permeability from the host immune response. The resulting excess interstitial fluid can cause edema in the brain and prevent blood from entering it, leading to loss of neurologic function in the area of the brain involved and central nervous system implications [12]. The excess interstitial fluid within the lungs can fill the airspaces with fluid and reduce gas exchange, resulting in hypoxemia. The vasculitis within the lungs can also lead to non-cardiogenic pulmonary edema, which is excess fluid in the lung tissues due to non-cardiac mechanisms [12], thus leading to respiratory distress [8]. The combination of vascular damage and the host immune response can contribute to many other clinical manifestations as well, including encephalitis and myocarditis [8]. Encephalitis and myocarditis are inflammation of the brain and heart muscle, respectively. In addition to encephalitis, other neurological manifestations can arise if the pathogen reaches the central nervous system. These include meningitis and acute disseminated encephalomyelitis (ADEM). These conditions are associated with an inflammatory response in the brain and spinal cord, and can result in severe headaches, photophobia (fear of light), temporary deafness, and focal neurologic deficits [8].

The hypovolemia (reduced blood volume) resulting from immune responses to rickettsial infection may lead to the secretion of antidiuretic hormone (ADH), which can cause low sodium levels (hyponatremia), a sign that was present in Robert’s case upon investigation by the physician. Symptoms of hyponatremia range from mild to severe and can include headaches and nausea [8]. Hypovolemia can cause other issues as well, such as reduced perfusion to organs like the kidneys. The reduced perfusion in the kidneys can impair renal function [12] and cause a loss of sodium through the kidneys, further contributing to hyponatremia [13]. Decreased perfusion of organs and tissue can also lead to potential skin necrosis or gangrene (tissue death in the digits or limbs) [14]. Gangrene occurs in 4% of cases and may require amputation [6]. There is also typically an eschar, or slough of dead tissue, resulting from necrosis at the site of pathogenic entry into the host [4].

Host Damage from Oxidative Stress

In addition to injury due to the inflammatory response, another source of damage to the host from the immune response is oxidative stress. Oxidative stress occurs as a result of the reactive oxygen species (ROS), such as superoxide anion (O2-) and hydroxyl radical (OH-), and reactive nitrogen species (RNS), such as nitric oxide (NO), produced by neutrophils and macrophages [2,15]. This is induced by the combination of IFN-γ and TNF-ɑ in the immune response [2,15]. However, studies have demonstrated that ROS produced by endothelial cells infected by R. rickettsii can also contribute to damage via oxidative stress, with the accumulation of the ROS [3]. As a result, ROS and RNS induced by the combination of TNF-α and IFN- γ act as both host defense and infection-induced injury mechanisms [10]. These molecules result in changes to blood consistency, as well as cause damage to surrounding host cells in the process of infection dissemination [14].The paradox of ROS produced by endothelial cells appearing to play a role in both host defense and host damage is unresolved, but presumably involves different sub-cellular compartments within the cell [12].

Reactive oxygen species function to combat bacteria through denaturation, disruption of lipids, and DNA damage; however, in this process, they also inflict the same fate upon surrounding host cells [5]. Furthermore, when neutrophils are recruited to combat an infection, they phagocytose any microbes surrounding them and direct their granules towards phagosomes to ensure the destruction of the bacteria [5]. These granules contain proteases and function to degrade phagocytosed materials [5]. However, in cases where neutrophils have sensed the TNF-α but do not encounter microbes to phagocytose at the site of infection, they still release their granules and that can generate significant damage to host tissue and cells [5]. As a result, neutrophils contribute to the damage caused to hosts through their ability to produce ROS, reactive nitrogen species (RNS), and granules [5,16]. Therefore, although there is much damage caused to the endothelium and vascular smooth muscles by the R. rickettsii bacteria itself, it is likely that the free radical peroxidation of the endothelial cell membranes accounts for a large portion of this damage as well [14]. Damage to endothelial cells due to oxidative stress is exacerbated by decreased levels of antioxidant enzymes, which include superoxide dismutase, G6PD (glucose-6-phosphate dehydrogenase), and glutathione peroxidase [12]. When hydrogen peroxide is increased in endothelial cells, it depletes enzymes such as glucose‐6‐phosphate dehydrogenase, glutathione peroxidase, and catalase, which are host defenses against ROS‐induced damage [12]. This can be evidenced by the fact that G6PD-deficient individuals have poorer prognosis upon R. rickettsii infection due to increased oxidative damage, which can result in hemolysis and acute renal failure [10]. Therefore, while ROS and RNS can help kill R. rickettsii, they can also cause oxidative damage to DNA, proteins and lipids of host cells, which can lead to inflammation and cell death [2]. Overall, this manifests as oxidative stress-mediated endothelial injury due to immune response to infection [15], and this may eventually contribute to endothelial cell death [17], which damages host endothelial integrity.

Figure 2. Petechiae from Rocky Mountain Spotted Fever

Endothelial Damage Complications

The damage of endothelial cells (via oxidative stress) or disruption to endothelium integrity (via actin rearrangements) can lead to focal areas of hemorrhage, which is defined as the escaping of blood from a damaged blood vessel [18]. Therefore, extravasation of the blood occurs into the surrounding tissues, and causes a petechial rash [3], as seen in Robert’s case. As the pathogen disseminates throughout the bloodstream, it will induce inflammatory responses throughout the host organs and skin which contribute to a spotted skin rash in 60-70% of infected individuals, similar to what Robert displays on his arms and legs [14]. This is contributed by the fact that R. rickettsii has the ability to enter almost all organs and tissues in the human host [4].

When blood vessels are damaged by the immune response, nearby platelets are stimulated to come to the area of the lesion and form a hemostatic plug, to prevent the hemorrhage from occurring. This is because damaged endothelial cells allow a subendothelial collagen protein called thromboxane to be exposed to the platelets circulating in the blood. Thromboxane causes the platelets to clump together and adhere to the vessel wall, and this continues as more platelets congregate to form a platelet plug that seals the injured area [19]. Platelet adhesion to injured endothelium is the major mechanism of thrombocytopenia in the spotted fever group of diseases, since a decreased amount of platelets are circulating in the bloodstream [20]. Thrombocytopenia occurs in 32-52% of infected patients [8], and the overconsumption of platelets causing this condition occurs because of the need to repair the vascular wall destruction during R. rickettsii infection, which explains Robert’s low platelet count. Since platelets have blood clotting capabilities, without their presence, an individual’s body will have a difficult time plugging blood vessel injuries [21]. To overcome this, the body will enter a procoagulant state involving platelet activation, a decrease in anticoagulants and increase in procoagulant components, increased activation of the coagulation cascade, and an upregulation of cytokines that promote coagulation [2,8]. These conditions can result in excessive bruising or prolonged bleeding in the patient. This means an individual infected with R. rickettsii is susceptible to internal bleeds. Although rare, it may also lead to fatal hemorrhages and thrombotic complications [8].

Additionally, in cases of overwhelming and prolonged illness, T-regulatory cells may suppress the immune response, which can further contribute to cases of fatality. For instance, cytotoxic CD8+ T cells eliminate infected cells within the host by inducing apoptosis. Inducing apoptosis kills the intracellular bacteria within the infected cell and prevents spread to other uninfected host cells. However, if cytotoxic CD8+ T cells undergo increased clonal expansion and activation, there can be an extensive killing of infected endothelial cells, which causes an immune mediated pathologic effect [12]. The mode of death of rickettsia‐infected cells usually appears to be oxidant-mediated necrosis, although cytotoxic T‐lymphocytes can contribute to the removal of infected cells by inducing their apoptosis [12].

Macroscopic Damage to Host Organs

Furthermore, the vascular lesions because of damage to the endothelial cells from the immune response can be found throughout the body, and create macroscopic damage within the host. The bacteria can spread to many areas in the body, such as the lungs, brain, liver, gastrointestinal tract, and the kidneys [22]. When the bacteria reach these areas, they again will induce an inflammatory and oxidative-stress response within the organs that can lead to its damage within the host. In severe cases of vascular injury, hemorrhages can develop and are observed in approximately half of infected individuals [14]. For instance, vasculitis due to R.rickettsii can also be seen in the liver, once the bacteria has spread there. The vasculitis from the immune responses and the oxidative-stress injury in the endothelial cells within the portal triads can lead to small, focal lesions of hemorrhaging and hepatocellular death, and thus reduced liver function [3]. These findings correlate with the abnormal liver function test in Robert’s case. Decreased perfusion to the liver may also be a cause of the abnormal liver function test in Robert. Furthermore, vasculitis and vascular focal lesions in the wall of the gastrointestinal tract, gallbladder, and pancreas can lead to abdominal pain, a symptom that is experienced by Robert [3,23]. Thus, overall, the clinical manifestation is vasculitis in almost all organs, which leads to cutaneous vasculitis (skin), interstitial pneumonitis (lungs), hepatic portal triaditis (liver), interstitial nephritis (kidney), interstitial myocarditis (heart), and meningoencephalitis (brain) [24].

References:

1. Valbuena G, Walker DH. 2009. Infection of the endothelium by members of the order Rickettsiales. Thrombosis and haemostasis. 102(6): 1071-1079. https://doi.org/10.1160/TH09-03-0186

2. Walker DH, Ismail N. 2008. Emerging and re-emerging rickettsioses: endothelial cell infection and early disease events. Nature Rev. Microbiol. 6(5): 375-386. https://doi.org/10.1038/nrmicro1866

3. Carrillo JLM, Garcia FPC, Coronado OG, Garcia AM, Cordeo, JFC. 2017. Physiology and Pathology of Innate Immune Response Against Pathogens. https://doi.org/10.5772/intechopen.70556

4. Osterloh A. 2017. Immune response against rickettsiae: Lessons from murine infection models. Medical Microbiol. and Immun. 206(6): 403-417. https://doi.org/10.1007/s00430-017-0514-1

5. Barton GM. A calculated response: control of inflammation by the innate immune system. J Clin Invest. 2008 Feb 1;118(2):413–20.

6. Vestweber D. 2008. VE-cadherin: the major endothelial adhesion molecule controlling cellular junctions and blood vessel formation. Arteriosclerosis, Thrombosis, and Vascular Biology,28(2):223-232. https://doi.org/10.1161/ATVBAHA.107.158014

7. Iyer S, Ferreri DM, DeCocco NC, Minenear FL, Vincent PA. 2004. VE-cadherin-p120 interaction is required for maintenance of endothelial barrier function. American Journal of Physiology. Lung Cellular and Molecular Physiology. https://doi.org/10.1152/ajplung.00305.2003

8. Walker D, Blanton L. Rickettsia rickettsii and other spotted fever group rickettsiae (Rocky Mountain spotted fever and other spotted fevers). InMandell, Douglas, and Bennett's principles and practice of infectious diseases 2014 Aug 28 (pp. 2198-2205). Elsevier Inc..

9. Walker DH, Dumler JS. 2015. The role of CD8 T lymphocytes in rickettsial infections. Sem. in Immunopath. 37(3): 289-299. https://doi.org/10.1007/s00281-015-0480-x

10. Walker DH, Alcamo IE. Rocky Mountain Spotted Fever. Infobase Publishing; 2008. 96 p.

11. Rickettsia rickettsii and Other Spotted Fever Group Rickettsiae (Rocky Mountain Spotted Fever and Other Spotted Fevers)- ClinicalKey [Internet]. [cited 2021 Mar 8]. Available from: https://www.clinicalkey.com/#!/content/book/3-s2.0-B9780323482554001867?scrollTo=%23hl0000543

12. Walker DH, Valbuena GA, Olano JP. 2003. Pathogenic mechanisms of diseases caused by Rickettsia. Annals of the New York Academy of Sciences, 990:1-11. https://doi.org/10.1111/j.1749-6632.2003.tb07331.x

13. Razzaq S, Schutze GE. 2005. Rocky Mountain Spotted Fever: A Physician's Challenge. Pediatrics in Review. An Official Journal of the American Academy of Pediatrics,26(4):125-130. https://doi.org/10.1542/pir.26-4-125

14. Walker DH. Rickettsiae. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 38. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7624/

15. Schroeder C, Chowdhury I, Narra H, Patel J, Sahni A, Sahni S. 2016. In S. Thomas (ed.), Rickettsiales. Springer International Publishing, Galveston, Texas. https://doi.org/10.1007/978-3-319-46859-4_19

16. Hong JE, Santucci LA, Tian X, Silverman DJ. Superoxide Dismutase-Dependent, Catalase-Sensitive Peroxides in Human Endothelial Cells Infected by Rickettsia rickettsii. Infect Immun. 1998 Apr;66(4):1293–8.

17. Santucci LA, Gutierrez PL, Silverman DJ. 1992. Rickettsia rickettsii induces superoxide radical and superoxide dismutase in human endothelial cells. Infection and Immunity, 60(12):5113-5118. https://doi.org/10.1128/IAI.60.12.5113-5118.1992

18. Petri, WA. 2020. Overview of Rickettsial and Other Infections. Merck Manual Professional Version. https://www.merckmanuals.com/professional/infectious-diseases/rickettsiae-and-related-organisms/overview-of-rickettsial-and-related-infections

19. Hemostasis. Boundless Anatomy and Physiology. https://courses.lumenlearning.com/boundless-ap/chapter/hemostasis/

20. Pantanowitz, L. 2002. Mechanisms of Thrombocytopenia in Tick-Borne Diseases. The Internet Journal of Infectious Diseases, 2:1-6. https://print.ispub.com/api/0/ispub-article/3023

21. Thrombocytopenia (low platelet count) - Symptoms and causes [Internet]. Mayo Clinic. [cited 2021 Mar 10]. Available from: https://www.mayoclinic.org/diseases-conditions/thrombocytopenia/symptoms-causes/syc-20378293

22. Paddock CD, Alvarez-Hernandez G. 2018. 178 - Rickettsia rickettsii (Rocky Mountain Spotted Fever). Principles and Practice of Pediatric Infectious Diseases(5th Edition), Pages 952-957. https://doi.org/10.1016/B978-0-323-40181-4.00178-X

23. Zaidi SA, Singer C. 2002. Gastrointestinal and Hepatic Manifestations of Tickborne Diseases in the United States. Clinical Infectious Diseases,34:1206–1212. https://doi.org/10.1086/339871

24. Khayat A, Rathore MH. 2008. Rickettsia rickettsii. In L.L, Barton & N.R. Friedman (eds.). The Neurological Manifestations of Pediatric Infectious Diseases and Immunodeficiency Syndromes. Humana Press, Totowa, New Jersey

iii. Bacterial evasion: how does the bacteria attempt to evade these host response elements.

The infection of endothelial cells activates the endothelium, which results in the upregulation and secretion of a variety of cytokines and chemokines and the activation of the host immune response (1). However, Rickettsia has developed different strategies to evade the innate and adaptive immune response to facilitate its intracellular niche (1). It has been shown that such evasion mechanisms could be operating in rickettsial infections, but there is still a lot more research that needs to be done in this area (2).

Phagosome Escape

One of the major evasion mechanisms that R. rickettsii bacteria possess is the ability to escape the phagosomes found within the cytosol of the host cells that they have infected (2). Investigations with electron microscopy indicate that rickettsial entry into cells occurs within minutes of bacterium-host cell contact (3). This internalization allows bacteria to gain access to nutrients, adenosine triphosphate, amino acids and nucleotides required for growth and replication (4). Once the bacteria have been internalized by the host cells, they will be situated within a vesicle that has formed around the pathogen, called a phagosome (5). Usually, the host cell will fuse the phagosome, containing the pathogen, with a lysosome to form a phagolysosome (6). The phagolysosome will then contain various bactericidal components, such as hydrolytic enzymes and reactive oxygen species, to facilitate the killing of the foreign pathogen (6).

Figure 2: Rickettsia phagosome escape during multiplication and R. rickettsii filopodia formation during release stages of infection (14).

However, once internalized, the bacteria is able to quickly escape into the cytoplasm before the fusion of the phagosome with the lysosome via its own phospholipase activity (3). The mechanisms governing the quick escape of R. rickettsii from the phagosome into host cytosol remain to be fully elucidated; however, it likely involves membranolytic activities of hemolysin, phospholipase D and phospholipase A2 (7). This activity is most likely mediated by the rickettsial genes pld, which encodes an enzyme with phospholipase D activity, and tlyc, which encodes hemolysin (3). Phospholipase D and hemolysin C are two pore forming proteins that are able to lyse the phagosomal membrane to facilitate the escape of R. rickettsii from the phagosome to the cytoplasm (3, 9). Phospholipase D is an enzyme that hydrolyzes the phosphodiester bond of glycerophospholipids to remove the polar head and destabilize the membrane (10). Hemolysin C forms transmembrane channels to facilitate uncontrolled permeation of water and ions and discharge of important ion gradients to disrupt cell membranes (10). Forming pores to lyse the phagosome membrane prevents the killing of intracellular bacteria in phagolysosomes, allowing R. rickettsii to escape degradation by lysosomal contents (3). Additionally, this releases the bacterium into the host cell cytosol, where nutrients and energy are available to support its growth and intracellular replication (3). By escaping, this obligate intracellular pathogen is able to grow and replicate freely in the nutrient rich host cytoplasm (3).

Anti-Apoptotic Pathway

Host cells are known to induce apoptosis (programmed cell death) as a method to limit the spread of infection (4). However, this host defense mechanism can be manipulated by intracellular pathogens to promote their own persistence and survival (9). This is because apoptosis of infected endothelial cells early in the course of infection would remove the pathogen’s intracellular niche and interfere with its replication (9). Some studies have explored how R. rickettsii may have evolved strategies to overcome host cell apoptosis, as an evasion mechanism (11). Studies have shown that when R. rickettsii-induced activation of nuclear factor kappa B (NF-κB) was inhibited, apoptosis of infected, but not uninfected, endothelial cells rapidly ensued (11). These studies support the idea that R.rickettsii can exert an anti-apoptotic effect and modulate the host cell’s apoptotic response to its own advantage to allow the host cell to remain as a site of infection (11).

Upon infection, Rickettsia directly triggers activation of NF-κB, which can alter the expression of several genes under its control (9, 11). Normally, NF-κB is a major transcription factor that when activated, upregulates important cytokine and chemokine genes responsible for mediating the immune response (8). However, R. rickettsii activates the NF-κB signalling pathway in the infected host cell to suppress apoptosis (1). More specifically, NF-kB will repress upstream apical caspase-8 and caspase-9, and the executioner enzyme caspase -3, which are all cysteine proteases involved in the induction of apoptosis (4, 12). This allows maintenance of mitochondrial integrity and delays apoptosis (4, 12). Additionally, Rickettsia activation of NF-κB will regulate the expression of pro-apoptotic and antiapoptotic proteins, belonging to the Bcl-2 protein family, which also regulate the function and integrity of the host mitochondria to favour apoptosis inhibition (9, 12). It is also known that R. rickettsii-infected endothelial cells produce IL-8, which has an anti-apoptotic effect (13). These mechanisms via R. rickettsii NF-κB activation all contribute to the delay of apoptosis of infected endothelial cells, which gives the infecting R. rickettsii time to grow and proliferate intracellularly, protected from extracellular host defenses (further discussed in the next section) (1, 12).

Overall evasion of the host apoptotic defence prevents early killing of the infected host cell to allow sufficient time for R. rickettsii to replicate for a few cycles in the host cell before spreading to adjacent cells (1). Thus, this facilitates R. rickettsii survival and persistence in the host (1). Interestingly, eventually, this pathway results in the eventual death of the host cell, which contributes to damage to the host endothelium by this pathogen and disease progression (1).

Cell to Cell Movement Via Actin

Figure 1: Rickettsia actin polymerization and motility using RickA protein, Arp2/3 complex and actin monomers to create filopodia to spread from cell to cell (14)

After invasion into the host, bacterial movement between endothelial cells is a critical step in the spread of R. rickettsii and involves a mechanism called actin-based motility (4). Rickettsial pathogens belonging to the Rocky Mountain Spotted Fever Group, like R. rickettsii, use actin-polymerization machinery in the cytosol of the infected cell to facilitate intracellular and intercellular movement (4). After a few cycles of binary fission in a host cell, R. rickettsii leaves via long, thin cell projections called filopodia made of actin cytoskeleton to infect a new host cell (14).

In the cytosol, rickettsiae express a surface protein called Sca2, which recruits the Arp2/3 complex, which is a central actin nucleator (4,15). Activation of the Arp2/3 complex, via the RickA protein found in R. rickettsii, results in nucleation of host actin polymerization and induction of the formation of a network of long, unbranched filaments in R. rickettsii actin tails (4,15). Actin polymerization derived from the host cytoskeleton allows for the formation of the characteristic F-actin ‘comet tail’ (9). This actin polymerization propels the bacteria through the cytoplasm to the cell membrane and filopodia projections are induced when the bacteria collide with the host plasma membrane to create protrusions and outward deformations in the cell membrane (4,16) These protrusions extend out and invaginate into adjacent cells to allow them to be internalized (4, 16). It can also allow R. rickettsii to enter the extracellular space (16).

Disruption of these cell membranes enables R. rickettsii to spread from cell to cell rapidly without being exposed to the extracellular environment (9,14). For instance, its intracellular lifestyle and cell to cell transfer allows R. rickettsii to evade the humoural immune response in primary infection (9). This is because antibodies circulating in the blood and extracellular space are unable to access these pathogens (9). Overall, this allows pathogen to avoid contact with immune cells and antibodies in the extracellular space, aiding their evasion of the innate and adaptive immune response (9, 16).

Type IV Secretion System (T4SS)

Several studies have suggested that Type IV Secretion Systems (T4SS) may have a potential role in R. rickettsii as an evasion mechanism to avoid the host immune response (4). T4SSs are multi-protein structures spanning the bacterial envelope and help aid in the survival and proliferation of bacterial pathogens within their hosts (4). Studies have shown that R. rickettsii have genes encoding for the T4SS, and thus, this system can act as an evasion mechanism (17). T4SSs in rickettsiae likely act in an “effector translocator” manner, which means they can deliver pathogenic molecules into the cytosol of host cells that will benefit the bacteria’s survival and transmission (18). The primary suspected role of the T4SS system in rickettsiae is the secretion of virulence factors (4). It has been postulated that there are certain rickettsial substrates that can be exported by the T4SS and aid in bacterial evasion mechanisms (4). These substrates include sec7 proteins, which can help establish replicative organelles for the bacteria by inhibiting phagosome-lysosome fusion to avoid degradation; and VipD proteins, which work alongside phospholipase A2 (PLA2) to perturb membrane trafficking and modulate intracellular bacterial growth (4). These factors secreted by the T4SS likely promote rickettsial survival by avoiding degradation, triggering synthesis of nutrients from the host cell or allowing adaptation of rickettsiae to the intracellular environment (4).

Arthropod Vector Support

It has been suggested that there may be some bacterial evasion mechanisms that can occur because R.rickettsii is a tick-borne pathogen and uses an arthropod vector to infect its hosts (19). As mentioned within the case study, Robert was bitten by ticks during a camping trip. When the ticks bit Robert, they would have thrusted their mouthparts into the dermis of his skin, allowing the saliva of the tick to come into contact with blood and lymphatic vessels, dendritic cells, and macrophages, as well as many other cells or soluble mediators of the immune system (19). However, the tick saliva can reduce or modulate host defences to create an environment potentially more favorable for pathogen establishment and development (19). In rickettsial infections, the tick saliva may influence T cell effector functions through its initial interaction with dendritic cells (15). The saliva of the tick can come into contact with dendritic cells present at the area of inoculation, which can inhibit DC maturation by decreasing the expression of costimulatory molecules (CD40, CD80 and CD86), which are needed for major histocompatibility complex (MHC) I and II upregulation to activate T cells (15). Therefore, DCs in contact with tick saliva are inefficient in the induction and activation of R. rickettsii antigen-specific T cells (15). This would adversely influence the acquired immune response against the bacteria, thereby allowing the pathogen to evade the immune response and lead to increased host susceptibility to severe and fatal rickettsial disease (15).

Figure 3: Interaction of rickettsiae with endothelial cells - highlighting phagosome escape, actin polymerization and cell to cell spread as important steps in infection.

References

Obino D, Duménil G. 2019. The many faces of bacterium-endothelium interactions during systemic infections. Microbiol. Spectrum. 7(2): 69-81. https://doi.org/10.1128/microbiolspec.BAI-0010-2019
Astrup E, Lekva T, Davı`G, Otterdal K, Santilli F,O, ie E, Halvorsen B, Damos JK, Raoult D, Vitale G, Olano JP, Oeland T, Aukrust P. 2012. A Complex Interaction between Rickettsia conorii and Dickkopf-1 – Potential Role in Immune Evasion Mechanisms in Endothelial Cells. PLoS ONE. 7(9): e43638. https://doi.org/10.1371/journal.pone.0043638
Sahni SK, Rydkina E. 2009. Host-cell interactions with pathogenic rickettsia species. Future Microbiol. 4(3): 323-339. https://doi.org/10.2217/fmb.09.6
Mansueto P, Vitale G, Cascio A, Seidita A, Pepe, Carroccio A, Rosa S di, Rini GB, Cillari E, Walker DH. 2012. New Insight into Immunity and Immunopathology of Rickettesial Diseases. Clin. Dev. Immun. 967852: 1-26 https://doi.org/10.1155/2012/967852
Sahni A, Fang R, Sahni SK, Walker DH. 2019. Pathogenesis of Rickettsial Diseases: Pathogenic and Immune Mechanisms of an Endotheliotropic Infection. Ann. Rev. of Path., 14(1):127-152. https://doi.org/10.1146/annurev-pathmechdis-012418-012800
Pauwels AM, Trost M, Beyaert R, Hoffmann E. 2017. Patterns, Receptors, and Signals: Regulation of Phagosome Maturation. Trends in Immun., 38(6):407-422. https://doi:10.1016/j.it.2017.03.006
Sahni SK, Narra HP, Sahni A, Walker DH. 2013. Recent molecular insights into rickettsial pathogenesis and immunity. Future Microbiol., 8(10):1265-1288. https://doi.org/10.2217/fmb.13.102
Valbuena G, Walker DH. 2009. Infection of the endothelium by members of the order Rickettsiales. Thrombosis and haemostasis. 102(6): 1071-1079. https://doi.org/10.1160/TH09-03-0186
Schroeder C, Chowdhury I, Narra H, Patel J, Sahni A, Sahni S. 2016. In S. Thomas (ed.), Rickettsiales. Springer International Publishing, Galveston, Texas. https://doi.org/10.1007/978-3-319-46859-4_19
Blanton LS, Walker DH. 2020. Rickettsia rickettsii and Other Spotted Fever Group Rickettsiae (Rocky Mountain Spotted Fever and Other Spotted Fevers. In J.E. Bennett, R. Dolin, M.J. Blaser (eds.), Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases. Elsevier Inc, Philadelphia, PA.
Clifton DR, Goss RA, Sahni SK, Antwerp DV, Baggs RB, Marder VJ, Silverman DJ, Sporn LA. 1998. NF-κB-dependent inhibition of apoptosis is essential for host cell survival during Rickettsia rickettsii infection. Proc. Natl. Acad. Sci. (PNAS), 95 (8) 4646-4651. https://doi.org/10.1073/pnas.95.8.4646
Joshi SG, Francis CW, Silverman DJ, Sahni SK. 2006. NF-κB activation suppresses host cell apoptosis during Rickettsia rickettsii infection via regulatory effects on intracellular localization or levels of apoptogenic and anti-apoptotic proteins. FEMS microbiol. lett., 234(2):333-41.
Osterloh A. 2017. Immune response against rickettsiae: Lessons from murine infection models. Med. Microbiol. and Immun. 206(6): 403-417. https://doi.org/10.1007/s00430-017-0514-1
Walker DH. 1996. Rickettsiae. In S. Baron (ed.), Medical Microbiology. University of Texas Medical Branch at Galveston, Galveston, Texas.
Walker DH, Ismail N. 2008. Emerging and re-emerging rickettsioses: endothelial cell infection and early disease events. Nature Rev. Microbiol. 6(5): 375-386. https://doi.org/10.1038/nrmicro1866
Paddock CD, Alvarez-Hernández G. 2018. Rickettsia rickettsii (rocky mountain spotted fever). In S.S. Long, C.G, Prober, M. Fischer (eds). Principles and Practice of Pediatric Infectious Diseases. Elsevier US, New York, New York.
Riley SP, Pruneau L, Martinez JJ. 2017. Evaluation of changes to the Rickettsia rickettsii transcriptome during mammalian infection. PLOS one, 12(8):e0182290. https://doi.org/10.1371/journal.pone.0182290
Gonzalez-Rivera C, Bhatty M, Christie PJ. 2015. Mechanism and Function of Type IV Secretion During Infection of the Human Host. Microbiol. Spec., 4(3):10.1128. https://doi.org/10.1128/microbiolspec.VMBF-0024-2015
Wikel S. 2013. Ticks and tick-borne pathogens at the cutaneous interface: host defenses, tick countermeasures, and a suitable environment for pathogen establishment. Front. in Microbiol., 4:337. https://doi.org/10.3389/fmicb.2013.00337

iv. Outcome: is the bacteria completely removed, does the patient recover fully and is there immunity to future infections from this particular bacteria?

The incubation period of R. rickettsii typically ranges from 2 to 14 days, with the earliest symptoms of fever, headaches, and myalgia (muscle aches), manifesting due to the rapid initial release of proinflammatory cytokines (1). Fevers occur in 99-100% of cases, headaches in 79-91% of cases, and myalgia in 72-83% of cases (1). These symptoms have all been noted in Robert, along with abdominal pain. In addition to abdominal pain, other symptoms involving the gastrointestinal system, such as vomiting, nausea, and diarrhea, collectively suggest gastroenteritis, or inflammation of the gastrointestinal tract (1, 2). Another common symptom of infection is a rash, which occurs in 88-90% of cases (1). Less common symptoms, occurring in less than half of infected individuals, include coughs, thrombocytopenia (low amounts of platelets), conjunctivitis (also known as pink eye), stupor (a lack of senses), and edema. Death can also occur in approximately 4-8% of cases (1).

Figure: Rash from RMSF (3)

Prognosis

The prognosis of individuals infected with R. rickettsii is largely dependent on the amount of time before appropriate therapy is administered. If left untreated, a typical R. rickettsii infection can cause death of the human host in 7 to 15 days after the onset of symptoms. In more severe cases of infection, death can occur within 5 days without treatment (1). During these periods without treatment, infection will progress rapidly, antibodies may not have time to develop, and a rash may or may not have time to develop. Mortality rates range from 20 to 30% in untreated cases, highlighting the need for prompt testing and treatment (2, 4). In most cases, if antibiotic treatment is not started within 5 days of symptom onset, hospitalization is more likely to be required (2).

Long-term sequelae of R. rickettsii infection can be reduced with earlier antibiotic treatment (1). The most common long-term sequelae include neurologic abnormalities and limb amputations following necrosis. Severe cases of RMSF typically involve vascular damage to the point where it has become ischemic (shortage of oxygen) and irreversible; this results in the requirement of amputation for damaged tissues (5, 6). Most cases of acute R. rickettsii infection with neurologic involvement have poor prognoses (1). Following recovery from infection, there is a potential for residual neurologic abnormalities, such as ataxia, blindness, and global encephalopathy. Ataxia includes disorders involving impaired muscle coordination and encephalopathy involves damage, disease, and malfunction of the brain (1). In severe cases, other potential lifelong sequelae can include paralysis, mental disability, and hearing loss (11).

Figure: Rocky Mountain Spotted Fever rash on a foot (2)

Treatment and Recovery

The antibiotic treatment of choice for R. rickettsii infections is doxycycline, which is a type of tetracycline antibiotic (1, 4). The recommended dosage is 100 mg every 12 hours for 7 days, or for 2-3 days following fever recovery, and can be administered orally or intravenously; however, the dose given can depend on the patient and severity of disease (1, 4, 7, 8). Chloramphenicol is another antibiotic that can be used to combat infection. The typical dosage of chloramphenicol is 50 mg/kg per day for 7 days (1, 8). Rifamycins, such as rifampin, may also be effective against rickettsiae and work by inhibiting bacterial RNA polymerase, and consequently RNA transcription (9). Although the introduction of doxycycline and chloramphenicol into the treatment of R. rickettsii has decreased the lethality of RMSF from 30% to 3%, both treatments have drawbacks (10). Tetracyclines should not be administered to pregnant women, as there is a risk of teeth and bone malformation in their fetuses. Additionally, tetracyclines are associated with the risk of hepatitis in pregnant women (1, 11). Chloramphenicol use has the potential side effect of aplastic anemia, which is characterized by the decreased production of new blood cells, causing fatigue, excessive bleeding, and immunodeficiency. Gray syndrome in neonates, involving circulatory collapse, and inflammation of optic and peripheral nerves are other potential complications of chloramphenicol use (11).

However, R. rickettsii is resistant to many other antibiotics, such as beta-lactams, erythromycin, sulfamethoxazole-trimethoprim, and aminoglycosides (1). As a result, studies have shown that doxycycline and chloramphenicol are the most effective treatments for pathogen removal and patient recovery, and are associated with the lowest fatality rates of RMSF compared to other antibiotics (2). Doxycycline reversibly binds the 30S ribosomal subunit in bacterial cells, preventing association with tRNA and consequently inhibiting bacterial protein synthesis (12). Additionally, doxycycline is able to bind to the 70S ribosomal subunit in mitochondria, further inhibiting protein synthesis. Doxycycline is generally well-absorbed and distributes well throughout tissue (12). In most cases, individuals treated with doxycycline within the first 5 days of symptom onset will recover (13). Fluid management to maintain organ perfusion and Swan-Ganz catheter to monitor hemodynamics due to increased vascular permeability can also be used for optimal recovery and treatment outcomes (7). However, complete removal of the bacteria is not guaranteed; in some cases, R. rickettsii can persist in the human host despite antibiotic treatment and even cause a relapse of R. rickettsii infection. One study observed that viable R. rickettsii were isolated from the lymphatic tissues of a patient who was infected with RMSF, one year after recovery from their initial infection (14).

Figure: Necrosis of fingers due to fatal, untreated Rocky Mountain Spotted Fever (2)

In addition to G6PD-deficiency that was previously mentioned as a risk factor for severe infection, old age, alcoholism, treatment involving sulfonade, diabetes mellitus, and male sex are also risk factors for more severe illness and lethal outcomes of R. rickettsii infection (1, 15). However, death is possible with inadequate treatment even in healthy individuals, so prompt treatment measures should always be prioritized (4, 13).

Future Immunity and Prevention

Survivors of R. rickettsii infection typically acquire strong immunity to reinfection through the production of IgG and IgM antibiotics against bacterial OmpA and OmpB proteins by the humoral immune response in the later stages of infection (1, 15). Antibodies against LPS are not sufficient to confer protection against R. rickettsii infection. Due to the intracellular nature of the pathogen and late emergence of antibodies during primary infection, it is likely that antibodies against OmpA and OmpB protect against reinfection but are not highly important during primary infection (15, 16). In the later stages of primary infection, anti-inflammatory cytokine IL-10 will inhibit the production of TNF-α and IFN-γ by CD4+ Th1 cells to suppress the inflammatory response. At this time, CD4+ Th2 cells will release cytokines that assist in the production of antibodies by B cells (17). IgM antibodies appear approximately 2 weeks following symptom onset and will persist until 4 weeks before declining. IgG antibodies will similarly appear at approximately 2 weeks following symptom onset and will persist for months (17). IgG antibodies against OmpA and OmpB proteins will likely contribute to long-term protection against future infections by R. rickettsii; if the pathogen re-enters the host, the recognition of these proteins will result in the release of neutralizing antibodies; therefore, the pathogen can be eliminated before infection begins (17).

The prevention of R. rickettsii infection mainly involves measures to avoid tick bites. As RMSF is not transmissible between human hosts, there is no need for isolation of infected individuals (4). There are currently no vaccines available for the treatment of R. rickettsii infection, however they are desirable due to the increasing incidence of infection, increasing antibiotic resistance in R. rickettsii strains, and the potential for recurrent disease in cases where the antibiotic treatments do not successful remove all of the bacteria (4, 18). In a recent study, monoclonal antibodies directed against R. rickettsii, that most likely recognize OmpA and OmpB, protected mice from a lethal short-term challenge with large and toxic doses of homologous bacteria and prevented fever and rickettsia in guinea pigs (9). The mechanism for protection is unclear but it is thought that these antibodies opsonized the bacteria for uptake by phagocytic cells (9). This is because in mice, polyclonal antiserum resulted in enhanced killing of rickettsial species by macrophages (9). Additionally, recent in vitro studies indicate that monoclonal antibodies that recognize OmpB may also induce complement mediated killing of the bacteria (9). Thus, vaccination with recombinant proteins and peptides of OmpA and OmpB appear to be a promising future vaccine candidate, however there is still not enough evidence that vaccines against these bacterial proteins can confer full protection to the host (9).

References

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14. Parker RT, Menon PG, Merideth AM, Snyder MJ, Woodward TE. 1954. Persistence of Rickettsia Rickettsii in a Patient Recovered from Rocky Mountain Spotted Fever. The journal of Immunology, 73 (6) 383-386. https://www.jimmunol.org/content/73/6/383

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17. Osterloh A. The neglected challenge: Vaccination against rickettsiae. PLoS neglected tropical diseases. 2020 Oct 22;14(10):e0008704.

18. Osterloh A. Immune response against rickettsiae: lessons from murine infection models. Medical microbiology and immunology. 2017 Dec;206(6):403-17.