Course:PATH417:2022W2/Case1

Case 1:

Tim is not really a cat person, but when he found a wounded stray cat in his back yard yearning for comfort, he could not resist. Tim did not want to bring the stray cat into his house, so he built a small shelter for the cat in his backyard and bought it some food. Over the course of a few weeks Tim noticed the cat was scratching itself a lot. He examined the cat closely and thought that it might have fleas. On one occasion Tim got a bit too close while the cat was scratching, and Tim accidently got scratched by the cat. He knew it was not a bite, so he was not worried about the injury too much. The wound was not that deep but did bleed a bit. Tim washed it with soap and water and continued about his day. About a week later he noticed a small papule form at the site of the scratch. The papule developed into a nodule and a week later he started noticing swelling in his armpit. He also noticed increasing fatigue, a mild headache, and felt sweatier at night, sometimes to the point where he needed to change the t-shirt he was sleeping in. He was worried about the swelling in his armpit and thought he might have cancer so he went to his family physician for examination. Upon hearing the story about the cat scratch and development of swollen lymph nodes, his doctor was concerned about Cat-Scratch disease. Blood work was sent and serology resulted positive for Bartonella henselae.

Guidelines for answers:

- 4 pages, double spaced, regular margins, 12 point font;

- up to 2 pages of extra space can be taken for tables/diagrams and references

1. The Body System

(i) Describe the signs (objective characteristics usually detected by a healthcare professional) and symptoms (subjective characteristics experienced by the patient). Are there any other signs or symptoms that could have been commented on but are not presented in the case? What are the key History of Presenting Illness elements presented? What laboratory samples are taken and why? What are the meanings of the laboratory results reported? (No need to describe physiology of the signs and symptoms and no need to describe the laboratory testing itself as these are the basis of other questions).

Signs reported in this case are a papule that developed into a nodule, and swelling in the armpit. These are signs because they can be assessed through a physical examination by health professionals. Tim also reported the following symptoms: fatigue, mild headaches, and increased sweating. They are symptoms because of the subjectiveness of their expression. Beyond those reported in this case, additional signs and symptoms include fever, poor appetite, brain fog, muscle pain, photophobia, tachycardia, bowel problems, OCD behaviour, anxiety, rapid relapse off antibiotics, psychiatric problems, pain behind the eyes, and no response to previous antibiotic treatments (1).

History of presenting illness elements:

Tim presents to his primary care physician with a chief complaint of swelling in his armpit. A couple of weeks prior, he reports being scratched by a stray cat who he suspected had fleas. A week later at the site of the scratch, he developed a papule which then became a nodule which is when he noticed the swelling in his armpit. Tim also reports increasing fatigue, a mild headache, and feeling sweatier at night. Time does not report any pain currently.

The key elements here are the cat scratch, as well as the incubation time for the bacteria to become active in his body and develop into an infection (1). It is also important to note the progression of the infection, from a nodule to a larger papule, and then to the lymph node swelling that brought him in to see his physician.

Tim’s blood was taken as the sample and it was analyzed via serology testing. Species of Bartonella are difficult to isolate from patient specimens and grow in blood cultures so serology has become the first-line of testing for CSD. Serology testing through enzyme-linked immunosorbent assay (ELISA) or indirect fluorescent assay (IFA) detects elevated concentrations of immunoglobulin M (IgM) and immunoglobulin G (IgG) as a response to B. henselae infection. IgG titers between 1:64 and 1:256 suggest possible CSD, values greater than 1:256 represent current or recent infection. Presence of IgM, though brief, also represents acute infection. Tim’s blood work results could have shown high IgG and IgM values suggesting active CSD. (2, 3, 4)

Cutaneous reactions will be swelling, nodule/papule, and possible tenderness at the site of injury. Due to the lymphadenopathy at the site of inoculation, the lymphatic system is infected. The immune system is also affected as the bacteria may trigger a vascular-proliferative response. Cat scratch disease can also affect the eye, spleen, and CNS.(5)

References

1. Bartonellosis [Internet]. Centers for Disease Control and Prevention. Centers for Disease Control and Prevention; 2022 [cited 2023Jan15]. Available from: https://www.cdc.gov/bartonella/index.html?CDC_AA_refVal=https%3A%2F%2Fwww.cdc.gov%2Fbartonella%2Fsymptoms%2Findex.html

2. Klotz SA, Ianas V, Elliott SP. Cat-scratch Disease. Am Fam Physician. 2011 Jan 15;83(2):152-5. PMID: 21243990.

3. Bennet JE, Dolin R, Blaser MJ. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. 9th Ed. Philadelphia, PA: Elsevier; 2020.

4. Alizond V, Costa C, Sidoti F, Scutera S, Bianco G, Sparti R, Banche G, Dalmasso P, Cuffini AM, Cavallo R, Musso T. Serological and molecular detection of Bartonella henselae in specimens from patients with suspected cat scratch disease in Italy: A comparative study. PLoS ONE. 2019 Feb 8;14(2): e0211945. Available at https://doi.org/10.1371/journal.pone.0211945

5. Baranowski K, Huang B. Cat Scratch Disease. [Updated 2022 Jun 21]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK482139/

(ii) Which body systems are affected? In what way has the normal physiological functioning of this body system been disturbed by the infection (specifically looking at the physiological changes without detailing the bacterial mechanism of this disturbance as that is the basis of another question). Representing this diagrammatically is helpful to demonstrate understanding.

The skin is affected as a vesicle/lesion forms at the site of infection. The lymphatic system is also affected as lymph nodes swell due to the activation of lymphocytes in order to combat the infection. This also leads to the fever observed. In most cases, the Bartonella henselae infection will not invade other body systems as it is self-limiting.

If the patient is immunocompromised, the infection may affect other body systems. For example, the liver and spleen may be affected as Bartonella henselae infection may lead to multiple granulomatous lesions in the liver and spleen in rare cases (2).

Figure 1. Parinaud oculoglandular syndrome

The eye may also be affected, specifically Parinaud oculoglandular syndrome, where granulomatous conjunctivitis is seen as well as lymphadenopathy near the eyes and inflammation of the neural retina (1). Some may also experience macular degeneration and blockage of the vessels supplying blood to the eye (3). Other possible developments include edema of the optic disc, vision loss, and retinal detachment (4).

Moreover, the brain may also be affected as encephalopathy was seen in 80% of patients with Bartonella henselae infection and cranial and peripheral nerve was affected in 20% of patients. This may result in symptoms such as convulsions (46%) and combative behavior (40%) (1).

References

1. Clinicalkey. ClinicalKey. (n.d.). Retrieved January 18, 2023, from https://www.clinicalkey.com/#!/content/book/3-s2.0-B9780323612692000150?scrollTo=%23f0285

2. Ventura A;Massei F;Not T;Massimetti M;Bussani R;Maggiore G; (n.d.). Systemic bartonella henselae infection with Hepatosplenic involvement. Journal of pediatric gastroenterology and nutrition. Retrieved January 18, 2023, from https://pubmed.ncbi.nlm.nih.gov/10400104/#:~:text=Background%3A%20Systemic%20manifestations%20of%20Bartonella,lesions%20in%20liver%20and%20spleen

3. Ghazi, N. G., & Sams, W. A. (2012). A case of cat-scratch disease with unusual ophthalmic manifestations. Middle East African journal of ophthalmology. Retrieved January 18, 2023, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3353677/

4. Annoura, K., Sano, I., Makino, S., & Kawashima, H. (2020, December 15). Multiple ocular manifestations in a case of cat scratch disease without systemic signs. GMS ophthalmology cases. Retrieved January 18, 2023, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7745642/

(iii) What antibiotics/treatments might have been given (i.e., what are antibacterial treatments and how do these treatments work to help the body clear the organism)? Representing this diagrammatically is helpful to demonstrate understanding.

Figure 2. Axillary Lymph Node Swelling (1)

Mild cases of CSD typically do not require antibiotic treatment. CSD will resolve spontaneously in 90-95% of children just by controlling its symptoms with analgesics, antipyretics, and warm compresses. Management of patients with mild to moderate cases includes analgesics for pain and reassurance that the disease is not life-threatening. In some cases, aspiration of suppurative lymph nodes to relieve pain may be needed but incision and drainage is not recommended. (1, 3, 5) Physicians sometimes prescribe azithromycin at 10mg/kg on day 1 and 5mg/kg on the next four days for patients with moderate cases or with severe or multiple lymphadenopathies. Rifampin, ciprofloxacin, and trimethoprim-sulfamethoxazole may also be used as treatments. Antibiotic use still remains controversial as guidance differs between various authorities. Despite this, patients with severe cases of CSD recovered more quickly with antibiotic therapy. (2, 3, 6)

When antibiotics are prescribed, azithromycin is the antibiotic of choice. Azithromycin belongs to a class of antibiotics known as macrolides. They work against a wide range of gram-positive and gram-negative bacteria (eg. B. henselae) by binding to the 50S ribosomal subunit and inhibiting RNA-dependent protein synthesis. Azithromycin is more effective against gram-negative bacteria than other macrolides such as erythromycin and clarithromycin due to its better penetration of the bacterial outer envelope. It is also effective against pyogenic and beta-lactam-resistant bacteria. However, macrolides are just considered bacteriostatic agents in vivo, bactericidal activity is only demonstrated in vitro among select bacteria (4, 7).

Figure 3. Mechanism of action of Azithromycin (4)

References

Cat Scratch Disease [Internet]. [place unknown: Centers for Disease Control and Prevention]; 2020 Jan 17 [cited 2023 Jan 19]. Available from https://www.cdc.gov/healthypets/diseases/cat-scratch.html
Klotz SA, Ianas V, Elliott SP. Cat-scratch disease. American Family Physician. 2011 Jan 15;83(2):152-155.
Mazur-Melweska K, Mania A, Kemnitz P, Figlerowicz M, Sluzewski W. Cat-scratch disease: a wide spectrum of clinical pictures. Postep Derm Alergol. 2015 Jun;XXXII(3):216–220.
Bennet JE, Dolin R, Blaser MJ. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. 9th Ed. Philadelphia, PA: Elsevier; 2020.
Baranowski K, Huang B. Cat Scratch Disease [Internet]. Treasure Island, FL: StatPearls Publishing; 2022 Jan [updated 2022 Jun 21; cited 2023 Jan 19]. Available from https://www.ncbi.nlm.nih.gov/books/NBK482139/
English R. Cat Scratch Disease. Pediatrics in Review. 2006 Apr;27(4):123-127.
Heidary M, Samangani AE, Kargari A, Nejad AK, Yashmi I, Motahar M, Taki E, Khoshnood S. Mechanism of action, resistance, synergism, and clinical implications of azithromycin. J Clin Lab Anal. 2022 Mar 24;36:e24427. Available at https://doi.org/10.1002/jcla.24427

(iv) What is a reportable communicable disease? Is this a reportable communicable disease in BC? If treatment were not administered, how would the disease evolve? What signs and symptoms could develop? What additional testing or monitoring could help with diagnosis of later stage disease?

The Animal Health Act defines reportable communicable disease as an environmental toxin, infestation, syndrome, or transmissible disease that should be reported to implement preventative measures to aid in controlling the spread (1). Human-to-human transmission of Bartonellosis has not been documented (2) and is not present in the BCCDC list of reportable diseases (3); although this disease is endemic in parts of North America, as it is non-transmissible between humans, it is not a reportable disease in BC. For most, treatment is not required as the infection is self-limiting, but for those that are immunocompromised, the disease could cause several complications, especially for those suffering from HIV. Bacillary epithelioid angiomatosis (BA) manifestation has been observed in skin and regional lymph nodes as well as infection in various internal organs including the liver, spleen, bone, brain, lung, bowel, and uterine cervix (4) .

Other complications that can occur are encephalopathy, , and hematologic manifestations. Encephalopathy can occur in as many as 5% of patients with patients experiencing a sudden onset of neurological symptoms which include seizures, bizarre behaviour, and altered levels of consciousness. This generally is not fatal with recovery taking place over a span of a few months. Patients with stellate macular retinopathy show signs of bilateral loss of vision, optic disc swelling, and macular star formation. Recovery of vision is generally resolved completely within 3 months of infection. Hematologic manifestations include hemolytic anemia, thrombocytopenic purpura, nonthrombocytopenic purpura, and eosinophilia. In one case, leukocytoclastic vasculitis has been associated with Bartonella henselae, however; only one case of such nature has been documented (5). Prognosis for this disease is generally positive with clinical resolution occurring over weeks to months but in the case of chronic infections, serological tests for immunofluorescent fluorescent antibodies for IgM and IgG antibodies can be employed (6).

References

1. Dinulos, J. G., Baughman, R. D., & Dinulos, P. (2019). Infestations and Bites. Journal of the American Academy of Dermatology, 80(4). https://doi.org/10.1016/j.jaad.2018.11.054

2. Food, M. of A. and. (2022, August 12). Reportable and notifiable diseases. Province of British Columbia. Retrieved January 18, 2023, from https://www2.gov.bc.ca/gov/content/industry/agriculture-seafood/animals-and-crops/animal-health/reportable-notifiable-diseases#:~:text=Reportable%20diseases%20include%20transmissible%20diseases,can%20be%20transmitted%20to%20humans

3. Canada, P. H. A. of. (2011, August 19). Government of Canada. Canada.ca. Retrieved January 18, 2023, from https://www.canada.ca/en/public-health/services/laboratory-biosafety-biosecurity/pathogen-safety-data-sheets-risk-assessment/bartonella-henselae.html

4. Rolain, J. M., Brouqui, P., Koehler, J. E., Maguina, C., Dolan, M. J., & Raoult, D. (2004, June). Recommendations for treatment of human infections caused by bartonella species. Antimicrobial agents and chemotherapy. Retrieved January 18, 2023, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC415619/

5. Clinicalkey. ClinicalKey. (n.d.). Retrieved January 18, 2023, from https://www-clinicalkey-com.ezproxy.library.ubc.ca/#!/content/book/3-s2.0-B9780323529501002364?scrollTo=%23hl0000154

6. Bartonellosis. Lyme Disease. (2019, August 22). Retrieved January 18, 2023, from https://www.columbia-lyme.org/bartonellosis

2. The Microbiology Laboratory

(i) What are potential causes of fever and swollen lymph nodes? Provide examples of both bacterial and non-bacterial pathogens, as well as non-infectious causes.

Various infectious and non-infectious etiologies can cause fever and swollen lymph nodes. Lymph nodes swell from immune cells' migration to a localized area, while fever is stimulated by cytokine release [1]. Factors contributing to these symptoms can include autoimmune disorders, malignant neoplastic diseases, lymphoproliferative disorders, and infections [2]. Swollen lymph nodes, or lymphadenopathy, can be categorized into two groups: localized or generalized [3]. A localized presentation likely indicates a local cause originating from an area of lymph drainage while a generalized presentation may suggest a root systemic cause [3]. Autoimmune disorders that mimic these symptoms include sarcoidosis, systemic lupus erythematosus, and rheumatoid arthritis [2]. Malignant cancer neoplasms that may cause fever and swollen lymph nodes include lymphoma and leukemia [2]. Specifically, Hodgkin and non-Hodgkin lymphomas can result in lymphadenopathy as well as B symptoms that include weight loss, fever, and drenching night sweats [4]. Hemophagocytic lymphohistiocytosis is a lymphoproliferative disorder that mimics these symptoms [2]. Infectious causes may be bacterial, viral, fungal, or protozoan parasites [2]. Bacterial causes may include Bartonella henselae (B. henselae), brucellosis, bacterial pharyngitis, syphilis, tuberculosis, and typhoid fever [2]. Tularemia is another bacterial infection caused by Francisella tularensis that can be caused by a cat bite, though infection through this route is rare [5]. Viral causes may include hepatitis, cytomegalovirus, herpes simplex, mononucleosis (Epstein-Barr virus), rubella, and viral pharyngitis [2]. Examples of fungal and protozoan parasite causes of fever and swollen lymph nodes are blastomycosis and toxoplasmosis, respectively [2]. Common considerations in the differential diagnosis of B. henselae infection include atypical mycobacterial diseases, coccidioidomycosis and valley fever, leishmaniosis, Lyme disease, lymphogranuloma venereum, nocardiosis, sarcoidosis, sporotrichosis, syphilis, and toxoplasmosis [6].

References

Goldman AS, Prabhakar BS. 1996, posting date. Chapter 1, Immunology Overview. In Baron S (ed), Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston, Texas. Retrieved January 19, 2023 from
Maini R, Nagalli S. Lymphadenopathy. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan [cited 2023 Jan 19]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK558918/
Gaddey HL, Riegel AM. Unexplained Lymphadenopathy: Evaluation and Differential Diagnosis. Am Fam Physician. 2016;94(11):896-903.
Kaseb H, Babiker HM. Hodgkin Lymphoma. 2022 [Cited 29 Jan 2022] In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing. Available from: https://www.ncbi.nlm.nih.gov/books/NBK499969/
Fohle E, Smith BA, Guerrero DM. A Rare Case of Spontaneous Splenic Rupture Secondary to Tularemia Following a Cat Bite. Cureus. 2021;13(2):e13218. Published 2021 Feb 8. doi:10.7759/cureus.13218
Mada PK, Zulfiqar H, Joel Chandranesan AS. Bartonellosis. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan. Available from: https://www.ncbi.nlm.nih.gov/books/NBK430874/

(ii) What samples should be taken for diagnostic testing?

The diagnosis of a B. henselae infection relies on epidemiological, clinical, histological, and serological criteria [1]. Therefore, in selecting diagnostic samples, one must consider the signs and symptoms of the disease to determine which specimens likely contain the etiological agent [2]. Commonly, a B. henselae infection is detected and confirmed by sampling the lymph nodes or blood [3]. When presenting with generalized symptoms of lymphadenopathy and fever, infection is ruled out before malignancy due to the invasive nature of the tests required [4].

Blood collection enables serological testing [3] and bacterial culturing for systemic infections [5]. When collecting a blood sample for bacteriological testing, it is important to prevent cross-contamination with bacteria present on the skin and in the environment [6]. To maintain a sterile environment, practices such as washing hands, wearing gloves, using a single needle, and disinfecting the collection site should be practiced with each collection [6]. Blood culture is regarded as the best method for microorganism detection in cases of bacteremia [5]. Blood cultures identify which bacterium, if any, is present in a patient’s blood [5,7]. Blood culture also enables one to test the antibiotic susceptibility of the bacteria [5] through the introduction of antibiotics to the media in the form of disk diffusion or dilution [2]. The susceptibility of bacterial culture to the antibiotic may help to inform diagnosis [2]. When completing both blood culture and serology, the blood culture should be taken first to be added to the broth mixture, then a sample can be added to a tube designed for serum separation [6]. To prepare a blood sample for serological tests, blood must be added to a serum separation tube containing gel to assist with serum separation through centrifugation [6].

Lymph node aspirates would also be beneficial in diagnosing this infection, due to the involvement of the lymph node [3]. Lymph node aspirates are useful for PCR-based diagnosis [8], and other methods. However, lymph node aspiration samples are not often readily available for diagnostic tests as the procedure is not commonly recommended [3]. Lymph node aspiration is only recommended in specific cases, to decrease pain and swelling in the lymph node or in cases of uncertain diagnosis [3]. In the case of B. henselae infection, there is often a primary inoculation lesion present on the skin that can be sampled to perform PCR testing, rather than sampling the lymph node directly [9].

References

Hansmann Y, DeMartino S, Piémont Yves, Meyer N, Mariet P, Heller Rémy, Christmann D, Jaulhac Benoît. 2005. Diagnosis of CAT scratch disease with detection of Bartonella henselae by PCR: A study of patients with lymph node enlargement. Journal of Clinical Microbiology 43:3800–3806.
Washington JA. Principles of Diagnosis. In: Baron S, editor. Medical Microbiology [Internet]. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996 [cited 2023 Jan 19]. Chapter 10. Available from: https://www.ncbi.nlm.nih.gov/books/NBK8014/
CDC Centers for Disease Control and Prevention. Bartonella Infection: Bartonella henselae infection or cat scratch disease (CSD) [Internet]. U.S. Department of Health & Human Services; 2022 Jan 10. Available from: https://www.cdc.gov/B./B.- henselae/index.html
Baranowski K, Huang B. Cat Scratch Disease. 2022 [Cited 2023 Jan 19]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; Available from: https://www.ncbi.nlm.nih.gov/books/NBK482139/
Opota O, Croxatto A, Prod’hom G, Greub G. Blood culture-based diagnosis of bacteraemia: state of the art. Clin Microbiol Infect [Internet]. 2015 Apr;21(4):313-322. Available from: https://doi.org/10.1016/j.cmi.2015.01.003
WHO Guidelines on Drawing Blood: Best Practices in Phlebotomy. Geneva: World Health Organization; 2010. 2, Best practices in phlebotomy. Available from: https://www.ncbi.nlm.nih.gov/books/NBK138665/
Reed JB, Scales DK, Wong MT, Lattuada CP, Dolan MJ, Schwab IR. Bartonella henselae neuroretinitis in cat scratch disease: Diagnosis, management, and sequelae. Ophthalmology [Internet]. 1998;105(3):459-466. Available from: https://doi.org/10.1016/S0161-6420(98)93028-7
Mada PK, Zulfiqar H, Joel Chandranesan AS. Bartonellosis. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan. Available from: https://www.ncbi.nlm.nih.gov/books/NBK430874/
Goaz S, Rasis M, Binsky Ehrenreich I, et al. Molecular Diagnosis of Cat Scratch Disease: a 25-Year Retrospective Comparative Analysis of Various Clinical Specimens and Different PCR Assays. Microbiol Spectr. 2022;10(2):e0259621. doi:10.1128/spectrum.02596-21

(iii) Explain the tests that can be performed on samples to detect any of the potential bacterial pathogens associated with this disease. Discuss any specialized needs for this pathogen and limitations/advantages of mentioned tests.

Direct and indirect detection methods are available for bacterial identification. Direct detection methods detect bacterial components and include, but are not limited to, microscopy, bacterial culture, gram staining, detection of pathogen antigens, histological spectrum, and polymerase reaction chain testing. Indirect detection methods often use serology to identify features, such as antibodies, that indicate current or prior infection [1].

Microscopy detects bacteria [2], but further testing is needed to identify the strain.

Bacterial culturing identifies which bacterial strain, if any, is currently infecting an individual [3]. Blood bacterial cultures are divided into two separate experiments, one aerobic and the other anaerobic, to determine which condition permits bacterial growth [3]. Cultures can utilize selective media, which contains inhibitory substances to permit the growth of only specific strains, or nonselective media [2]. Bartonella alpha proteobacteria growth medium (BAPGM) is an effective medium for B. henselae culture [4]. Bartonella spp. require additional growth supplements for culture such as hemoglobin, erythrocytes, or hemin due to their requirement for heme to proliferate [5]. Bacterial growth in culture is indicated by increasing pH, as bacteria produce carbon dioxide [3]; this can be visualized by a color change, fluorescent signal, or other visual cues [3]. Alternatively, visual detection of turbidity, discrete colonies, or other features may indicate the presence of bacteria [2]. Gram staining of blood culture aliquots allows individuals to identify the cell wall composition of bacteria [3,6]; gram-positive bacteria stain purple, whereas gram-negative bacteria stain pink [6]. A limitation of blood culturing is its lengthy procedure; the standard incubation time is five days but may be increased for certain strains [3], including B. henselae [1] Also, bacterial contamination may occur during the collection of the blood sample or during culture procedures [3].

Figure 1 Prolonged blood culture of B. henselae bacteria [4].

Enzyme-linked immunosorbent assays (ELISA) can be used as an indirect or direct detection method. Its direct detection of bacteria involves bacterial antigen detection in patient samples [2]. One coats wells with antibodies that bind a specific bacterial pathogen, adds patient samples to these wells, and then adds a second enzyme-linked, antigen-specific antibody to wells [2]. After adding an enzyme substrate to wells, those containing the specific bacterial antigen in question will be visualized by a colored product [2]. ELISA tests can only test for the presence of one bacterial antigen at a time, leading to a low throughput.

Serology is the best initial diagnostic test for B. henselae [1]. Serological testing can involve indirect fluorescent assays (IFA) or ELISA [1]. These assays detect pathogen-specific antibodies in patient samples [1]. The presence of specific antibodies indicates current or previous infection by a given microorganism. However, serological testing for B. henselae cannot differentiate between infections caused by closely related Bartonella bacterial species, due to the cross-reactivity of antibodies [7,8]. Further, individuals may continue to test positive for Bartonella infection, or others, by serology for years after clearance of the infection [7] due to the persistence of circulating antibodies. Early in the course of infection as the immune response develops, serological testing may not be able to detect antibodies [1], leading to false negatives [1,8]. IgM antibodies represent the first line of defense in active bacterial infection and their levels generally peak at 7 to 10 days and may not persist past the point of acute infection [9]. IgG antibodies can persist for months to years after active infection, making them less suitable to diagnose acute illness.

Histological Spectrum can be performed to confirm the presence of B. henselae in clinical cases [10]. Samples are primarily collected from a skin test, and ESSA from clinical cases or a lymph biopsy [10,11]. Samples are stained with Tissue gram, methenamine silver, Ziehl-Neelsen, auramine O, and Warthin-Starry [10]. The observation of the stained samples allows for the conformation of pathology through granulomatous lesions and surrounding parenchymal changes [10]. However, histological examination during early infection yields only lymphoid hyperplasia and arteriolar proliferation [10]. Therefore, confirming diagnosis is difficult and has a high potential for differential diagnosis.

Figure 2 Depicts the histological examination of cat scratch disease. Note that central stellate necrosis is surrounded by 3 layers: histiocytes (mid-bottom), lymphocytes (mid), and dense fibrosis (top) [6].

Polymerase Chain Reaction can be performed to confirm the presence of B. henselae. Upon the sample collection, the blood or lymph tissue is inoculated into cultures [12]. Afterward, DNA is extracted from the isolates, with methodology dependent on the sample utilized. DNA is amplified by PCR with alpha-proteobacterium range-restricted 16S ribosomal RNA gene primers p24E and p12B [12]. Negative and positive PCR controls – containing no DNA and B. henselae DNA – are ideal to include to confirm experimental results. The limitations of PCR are testing the specificity regarding time and conditions for the cultures. Identification of B. henselae through PCR analysis in the clinical setting is generally done through gel electrophoresis. The amplification product of PCR is run through an agarose gel (3%) and stained with ethidium bromide to visualize the results [9]. To identify B. henselae, the sample collected from the patient would be run against a negative control with no DNA present and a positive control with B. henselae DNA [11]. DNA moves through the gel via an electric field through pores, which separate the visualized bands by molecular weight [13]. PCR restriction fragment length polymorphism (PCR-RFLP) can be used to positively identify B. henselae within a sample. This process utilizes specific genes found in B. henselae to limit amplification to this bacteria [14]. These genes include the 16S rRNA gene, the 16S-23S rRNA ITS region 18, the citrate synthase (gltA) gene19, the riboflavin synthase (ribC) gene20, the groEL gene, or the RNA polymerase beta subunit gene [14].

References

Mada PK, Zulfiqar H, Joel Chandranesan AS. Bartonellosis. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan. Available from: https://www.ncbi.nlm.nih.gov/books/NBK430874/
Washington JA. Principles of Diagnosis. In: Baron S, editor. Medical Microbiology [Internet]. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996 [cited 2023 Jan 19]. Chapter 10. Available from: https://www.ncbi.nlm.nih.gov/books/NBK8014/
Opota O, Croxatto A, Prod’hom G, Greub G. Blood culture-based diagnosis of bacteraemia: state of the art. Clin Microbiol Infect [Internet]. 2015 Apr;21(4):313-322. Available from: https://doi.org/10.1016/j.cmi.2015.01.003
Maggi RG, Duncan AW, Breitschwerdt EB. Novel chemically modified liquid medium that will support the growth of seven Bartonella species. J Clin Microbiol [Internet]. 2005;43(6):2651-2655. doi:10.1128/JCM.43.6.2651-2655.2005
Okaro U, Addisu A, Casanas B, Anderson B. Bartonella Species, an Emerging Cause of Blood-Culture-Negative Endocarditis. Clin Microbiol Rev. 2017;30(3):709-746. doi:10.1128/CMR.00013-17
American Society for Microbiology. Gram stain protocols [Internet]. Washington, DC: The American Society for Microbiology; 2019 Aug 12. Available from: https://asm.org/Protocols/Gram-Stain-Protocols
CDC Centers for Disease Control and Prevention. Bartonella Infection: Bartonella henselae infection or cat scratch disease (CSD) [Internet]. U.S. Department of Health & Human Services; 2022 Jan 10. Available from: https://www.cdc.gov/B./B.- henselae/index.html
Reed JB, Scales DK, Wong MT, Lattuada CP, Dolan MJ, Schwab IR. Bartonella henselae neuroretinitis in cat scratch disease: Diagnosis, management, and sequelae. Ophthalmology [Internet]. 1998;105(3):459-466. Available from: https://doi.org/10.1016/S0161-6420(98)93028-7
Vainionpää R, Leinikki P. Diagnostic Techniques: Serological and Molecular Approaches. Encyclopedia of Virology. 2008;29-37. doi:10.1016/B978-012374410-4.00585-9
Lamps LW, Gray GF, Scott MA. 1996. The histologic spectrum of hepatic cat scratch disease. The American Journal of Surgical Pathology 20:1253–1259.
Hansmann Y, DeMartino S, Piémont Yves, Meyer N, Mariet P, Heller Rémy, Christmann D, Jaulhac Benoît. 2005. Diagnosis of CAT scratch disease with detection of Bartonella henselae by PCR: A study of patients with lymph node enlargement. Journal of Clinical Microbiology 43:3800–3806.
Koehler JE, Sanchez MA, Garrido CS, Whitfeld MJ, Chen FM, Berger TG, RodriguezBarradas MC, LeBoit PE, Tappero JW. 1997. Molecular epidemiology of bartonella infections in patients with bacillary angiomatosis–peliosis. New England Journal of Medicine 337:1876–1883.
Nature news. https://www.nature.com/scitable/definition/gel-electrophoresis-286/#:~:text=Gel%20electrophoresis%20is%20a%20laboratory,gel%20that%20contains%20small%20pores. Accessed January 27, 2023.
Diddi K, Chaudhry R, Sharma N, Dhawan B. Strategy for identification & characterization of Bartonella henselae with conventional & molecular methods. Indian J Med Res. 2013;137(2):380-387.

(iv) What are the expected results from these tests allowing for the identification of the bacteria named in this case. Discuss what would be interpreted as a positive result for each type of test; for culture discuss appearance on stains and growth characteristics.

Blood Culture: Though possible, blood culture is not an optimal method for B. henselae diagnosis [1]. B. henselae are slow-growing and require an incubation period of up to 21 days [1]. Further, B. henselae bacteria are difficult to culture due to difficult isolation and their fastidious nature [1]. Furthermore, blood culture-negative endocarditis, where the causative microorganism is not able to be cultured from the blood sample, occurs in up to 31% of all endocarditis cases [2]. Therefore, confirming the presence of B. henselae can be rather difficult. Following gram staining, a positive blood culture for B. henselae would contain bacteria that are pink (gram-negative) [3], pleomorphic [3, 4] and bacillary shaped [3, 4]. Cultures fitting this description, as seen in Figure 1, may indicate active B. henselae infection. Differentiation between Bartonella spp. is difficult as there are no unique phenotypic characteristics to distinguish them [5]. To identify B. henselae as the causative infective agent, an analysis of the bacterial DNA should be performed [5].

Figure 3 indicates the electrophoresis of PCR products using primers targeting the conserved gene sequence in B. henselae. Lanes 1 and 2 indicate the presence of B. henselae while lane 3 is a negative control. [10]

Enzyme-linked immunosorbent assays (ELISA): After coating wells with antibodies specific for a B. henselae antigen, adding patient samples, then adding a second enzyme-linked, antigen-specific antibody to wells, those containing the B. henselae will be visualized by a colored product [2]. Wells containing samples from the patient will appear colored if the infection is a result of B. henselae.

Serology: The presence of anti-B. henselae antibodies in a sample would imply an active or prior infection with the bacterium [3]. Anti-B. henselae IgM titres of 1:16 or higher indicate acute infection [1]. Alternatively, anti-B. henselae IgG titres of 1:256 or higher indicate current or previous infection [1]. While IgM titres more readily determine active infections, it is possible to determine by IgG titres as well. A marked increase in IgG levels between two consecutive samples taken 7 to 10 days apart would constitute an active B. henselae diagnosis [6]. Alternatively, if anti-B. henselae antibodies did not significantly change over this period, we may consider it to be a previous infection likely. Detection of B. henselae antigens would also indicate active infection.

Histological Spectrum: A molecular confirmation of a B. henselae infection by PCR allows for the histological examination of the bacteria [8]. A positive result for a histological spectrum is the hallmark depiction of cat scratch disease: a granuloma with stellate central microabscess – a localized collection of pus – surrounded by histiocytes, lymphocytes, and dense fibrosis [8, figure 2]. However, inconsistencies occur as the quantity of necrosis, fibrosis, and lymphocytes differ.

Polymerase Chain Reaction: When viewing a gel electrophoresis PCR test with both negative and positive controls, one would see different bands visualized in each lane of the gel [figure 3]. In the negative control, there would be no visible DNA band, while in the positive control and test sample lanes, there would be bands at the same molecular weight marker on the gel [9, figure 3]. Figure 3 shows two samples of B. henselae run on an agarose gel [10]. If the bands in the positive control and test sample are at the same marker, the sample that has been amplified is confirmed to be B. henselae [9]. A positive PCR-RFLP test would mimic the specific band profile on gel electrophoresis of a confirmed B. henselae sample [10]. PCR product digestion with TaqI and AciI yields distinct band formation on gel electrophoresis [10]. Comparing the PCR-RFLP profile between test samples and confirmed bacteria can yield a positive test result for B. henselae (Figure 4) [10].

Figure 4 shows the digestion products of TaqI (2) and AciI (3) with a marker DNA ladder (M) and positive control B. henselae DNA (1 and 4) [10]

References

Mada PK, Zulfiqar H, Joel Chandranesan AS. Bartonellosis. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan. Available from: https://www.ncbi.nlm.nih.gov/books/NBK430874/
Fournier PE, Thuny F, Richet H, Lepidi H, Casalta JP, Arzouni JP, Maurin M, Célard M, Mainardi JL, Caus T, Collart F, Habib G, Raoult D. 2010. Comprehensive diagnostic strategy for blood culture–negative endocarditis: A prospective study of 819 new cases. Clinical Infectious Diseases 51:131–140.
Reed JB, Scales DK, Wong MT, Lattuada CP, Dolan MJ, Schwab IR. Bartonella henselae neuroretinitis in cat scratch disease: Diagnosis, management, and sequelae. Ophthalmology [Internet]. 1998;105(3):459-466. Available from: https://doi.org/10.1016/S0161-6420(98)93028-7
Dolan MJ, Wong MT, Regnery RL, Jorgensen JH, Garcia M, Peters J, et al. Syndrome of Rochalimaea henselae adenitis suggesting cat scratch disease. Ann Intern Med [Internet]. 1993 Mar 1;118(5):331-336. Available from: https://www.acpjournals.org/doi/full/10.7326/0003-4819-118-5-199303010-00002
Zeaiter Z, Liang Z, Raoult D. Genetic classification and differentiation of Bartonella species based on comparison of partial ftsZ gene sequences. J Clin Microbiol. 2002;40(10):3641-3647. doi:10.1128/JCM.40.10.3641-3647.2002
Vainionpää R, Leinikki P. Diagnostic Techniques: Serological and Molecular Approaches. Encyclopedia of Virology. 2008;29-37. doi:10.1016/B978-012374410-4.00585-9
Mito T, Hirota Y, Suzuki S, Noda K, Uehara T, Ohira Y, et al. Bartonella henselae infective endocarditis detected by a prolonged blood culture. Intern Med [Internet]. 2016;55(20):3065-3067. doi: 10.2169/internalmedicine.55.7006
Lamps LW, Gray GF, Scott MA. 1996. The histologic spectrum of hepatic cat scratch disease. The American Journal of Surgical Pathology 20:1253–1259.
Hansmann Y, DeMartino S, Piémont Y, et al. Diagnosis of cat scratch disease with detection of Bartonella henselae by PCR: a study of patients with lymph node enlargement. J Clin Microbiol. 2005;43(8):3800-3806. doi:10.1128/JCM.43.8.3800-3806.2005
Diddi K, Chaudhry R, Sharma N, Dhawan B. Strategy for identification & characterization of Bartonella henselae with conventional & molecular methods. Indian J Med Res. 2013;137(2):380-387.

3. Bacterial Pathogenesis

(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

Geographic Distribution of B. henselae:

Figure 1: Distribution of CSD cases in The United States of America correlates with warm and humid climates (7)

Figure 2: B. henselae vectors and reservoir hosts (8)

The bacteria that causes Cat-Scratch disease (CSD) is B. henselae, a species within the genus Bartonella (1). It is a gram-negative, intracellular microbe that targets and infects mature red blood cells in humans (2). CSD has a worldwide distribution and it mostly affects children and young adults (2). B. henselae also infect dogs and horses (3). Infectious symptoms such as fever, swollen lymph nodes, and sore muscles will be elicited in response to the pathogen residing in the host’s blood, in contrast to cats, which are usually asymptomatic (1). There are two main genotypes of the B. henselae bacterium: Houston-1 (Type I) and Marseille (Type II) (4). Type I is the most common genotype found in Asia, while Type II is the most common genotype found in the United Kingdom, Australia, Western Continental Europe, and Western United States (4). Furthermore, there is a higher prevalence of CSD during the autumn and winter, which is most likely due to the seasonal breeding of domestic cats (5). In North America specifically, this disease is found in Southern populations and places where people do not have access to proper hygiene, such as refugees and the homeless. Around 20,000 human cases of CSD occur in the US where one-third of cats carry the bacteria (6). It is also more common for this infection to occur in warm and humid climates where cat fleas can thrive (7). Although the most abundant vector for B. henselae is cat fleas, many blood-sucking arthropods such as lice, ticks, and spiders, can also act as vectors for transmission (8).

How our patient has come in contact with B.henselae

Although Bartonella henselae infects non-human mammals, cats are the primary reservoir for B. henselae in Cat Scratch disease (CSD) (9). However, cats are typically asymptomatic despite a high B. henselae seroprevalence (10). B. henselae primarily infects endothelial cells and erythrocytes in mammalian host (11). A higher proportion of stray cats have B. henselae infection compared to domestic cats. Exposure to cats and cat fleas with B. henselae is a risk factor for and CSD (12).

Cat fleas, particularly Ctenocephalides felis, are arthropod vectors that facilitate cat-cat transmission and cat-human transmission of B. henselae (9). Humans can become infected when cat flea stool contaminated with B. henselae is exposed to non- intact human skin during a cat scratch, lick or bite (9). This was likely how Tim became infected, considering he was scratched and wounded by a stray cat that “might have fleas”. There is currently insufficient evidence of human-to-human transmission of B. henselae (9).

References:

1. Chaudhry R, Kokkayil P, Ghosh A, Bahadur T, Kant K, Sagar T, Kabra SK, Lodha R, Dey AB, Menon V. 2018. Bartonella henselae infection in diverse clinical conditions in a tertiary care hospital in North India. Indian Journal of Medical Research 147:189.

2. Nelson C. 2019. Bartonella infections - Chapter 4 - 2020 yellow book. CDC.gov. https://wwwnc.cdc.gov/travel/yellowbook/2020/travel-related-infectious-diseases/bartonella-infections.

3. Cheslock MA, Embers ME. 2019. Human Bartonellosis: An underappreciated public health problem? Tropical Medicine and Infectious Disease 4:69.

4. McCall A, Roberge A, Moffit B. 2019. Bartonella henselae. Mechanisms of Pathogenicity.

5. Nelson CA, Saha S, Mead PS. 2016. Cat-Scratch disease in the United States, 2005–2013. Emerging Infectious Diseases 22.

6. Jacomo V. 2002. Natural history of bartonella infections (an exception to Koch’s postulate). Clinical and Vaccine Immunology.

7. Cat-Scratch Disease in the United States, 2005–2013 - Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/Geographic-distribution-of-cat-scratch-disease-cases-by-US-census-division-United_fig2_308767076

8. Pulliainen, A, & Dehio, C. 2012. Persistence of Bartonella spp. stealth pathogens: from

subclinical infections to vasoproliferative tumor formation. Federation of European Microbiological Societies Microbiology Reviews, 36(3).

9. Bennett JE, Dolin R, Blaser MJ, Douglas RG, Mandell GL. Bartonella, Including Cat-Scratch Disease. In: Mandell, Douglas, and Bennett's principles and practice of infectious diseases. Philadelphia: Elsevier; 2020. p. 2824–43.

10. Yamamoto K, Chomel BB, Lowenstine LJ, Kikuchi Y, Phillips LG, Barr BC, et al. Bartonella henselae antibody prevalence in free-ranging and captive wild felids from California. Journal of Wildlife Diseases. 1998;34(1):56–63.

11. Dehio, C. (2005). Bartonella-host-cell interactions and vascular tumour formation. Nature Reviews. Microbiology, 3 (8), 621-31. doi:https://doi.org/10.1038/nrmicro1209

12. Gutiérrez R, Morick D, Gross I, Winkler R, Abdeen Z, Harrus S. Bartonellae in domestic and Stray Cats from Israel: Comparison of bacterial cultures and high-resolution melt real- time PCR as diagnostic methods. Vector-Borne and Zoonotic Diseases. 2013;13(12):857–64.

(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?

Through a break in the skin, B. henselae is able to easily enter the human host. For example, flea-infested cats carry flea feces that are contaminated with B. henselae (1). Through a cat scratch that disrupts the human’s endothelial barrier, the feces can come in contact with the open wound, resulting in the pathogen entering and infecting the human host (1). The pathogen may also be found in the host’s saliva. Therefore, a cat bite disrupting the human’s endothelial layer and exposing the blood vessels to the environment can also result in the pathogen entering the host through the infected host saliva (2).

Figure 3: Virulence Factors of B. henselae including Trw-system, FHA, BadA, VirB/VirD4 type IV secretion system and Beps (7).

It is speculated that the primary niche of B. henselae is erythrocytes and/or endothelial cells, which is a heavily debated area of research. There are knowledge gaps regarding the specific mechanism of entry. Interestingly, the direct inoculation of B. henselae into the bloodstream does not directly lead to infection, which suggests the bacteria has to be primed (3). Infected erythrocytes allow the B. henselae spread to distant sites through the systemic and lymphatic circulation (3). Moreover, host B-cell-rich granulomas trigger the recruitment and stimulation of macrophages leading to swelling of lymph nodes (3).

Figure 4: Entry of B. henselae into endothelial cells and the erythrocyte (8)

Bartonella adhesin A (BadA) is a trimeric auto-transporter adhesin that binds to host extracellular matrix and fibronectin during adherence (4). Moreover, BadA is involved with bacterial auto-agglutination and upregulation of proangiogenic factors in the host cell (4). The BadA precursors are secreted across the inner membrane into the periplasm through the secretory pathway (Sec) of the gram-negative bacteria (4). The head and stalk components of BadA are transported through a beta-barrel pore in the outer membrane where its assembly occurs (4). Filamentous hemagglutinin adhesin (Fha) follows a similar assembly process as BadA, but its role in B. henselae pathogenesis is unknown (6).

Entry into the host cell follows two pathways. The first pathway involves a VirB/ VirD4 type IV secretion system (T4SS) that transports Bartonella effector proteins (Beps) into host cells (6). BepC modifies host cell cytoskeleton arrangement for invasome- mediated internalisation of bacterial aggregates into the vascular endothelial cell (5). Bacterial attachment by BadA is speculated to enable VirB/VirD4 to function at an optimal distance (5). As shown in Figure 4, in the second route of entry, a single or a small group of bacteria are encapsulated into a Bartonella containing vaucole (BCV) (5,8). The BCV resists lysosomal fusion and destruction, allowing the bacteria to persist. Trw T4SS, Ia1B, hemolysin and deformin are reportedly involved in B. henselae adherence and altering the erythrocyte membrane, but their specific function is unclear (5). B. henselae proteins in the adherence and entry step are shown in Figure 3.

References:

McCall A, Roberge A, Moffit B. 2019. Bartonella henselae. Mechanisms of Pathogenicity.
Cheslock MA, Embers ME. 2019. Human Bartonellosis: An underappreciated public health problem? Tropical Medicine and Infectious Disease 4:69.
Harms A, Dehio C. Intruders below the radar: molecular pathogenesis of Bartonella spp. Clinical Microbiology Reviews. 2012 Jan;25(1):42-78.
Riess T, Andersson SGE, Lupas A, Schaller M, Schäfer A, Kyme P, et al. Bartonella adhesin a mediates a proangiogenic host cell response. Journal of Experimental Medicine. 2004;200(10):1267–78.
Franz B, Kempf VAJ. Adhesion and host cell modulation: Critical pathogenicity determinants of bartonella henselae. Parasites & Vectors. 2011;4(1).
Schmid MC, Schulein R, Dehio M, Denecker G, Carena I, Dehio C. The VIRB type IV secretion system of bartonella henselae mediates invasion, proinflammatory activation and antiapoptotic protection of endothelial cells. Molecular Microbiology. 2004;52(1):81–92.
Deng H, Pang Q, Zhao B, Vayssier-Taussat M. Molecular mechanisms of Bartonella and mammalian erythrocyte interactions: A Review. Frontiers in Cellular and Infection Microbiology. 2018;8.
Eicher SC, Dehio C. 2012. Bartonella entry mechanisms into mammalian host cells. Cellular Microbiology. 14(8).

(iii) Multiplication and Spread: does the organism remain extracellular or do they enter into cells? 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? (e.g. are there secondary sites of infection) and, if they spread, why do the bacteria hone in on particular secondary sites.

B. henselae is inoculated into the bloodstream at the site of infection (1). Interestingly, B. henselae disappears from the systemic circulation in the first 4 days post- inoculation (2). On the 5th day post-inoculation, B. henselae auto-agglutinate, reemerge and become extracellular in the bloodstream (2). The molecular mechanism of this step is uncertain. Phagocytes fail to initiate toll-like receptor TLR4 recognition due to the unconventional LPS structures of B. henselae (3). Furthermore, BadA can hinder complement activation (4). Afterwards, B. henselae penetrate and replicate in erythrocytes for up to weeks. The physiology of erythrocytes provides an intracellular niche for B. henselae to evade host adaptive and immune responses (2).

In-vitro studies demonstrate B. henselae can also invade endothelial cells, monocytes, macrophages and dendritic cells (5). Access to the blood enables B. henselae to spread to the lymph nodes and skeletal muscles resulting in lymphadenopathy and myalgia (1). Approximately half of CSD patients experience a single lymph node involvement, 20% develop multiple node development at one site and the remaining one third of patients have node involvement at multiple sites (1). Lymphadenopathy is the most prominent manifestation of CSD, which is consistent with Tim’s armpit swelling (1). In rarer cases, the bacteria may manifest in the brain and eye, resulting in encephalopathy and neuroretinitis (1). However, there is lack of research surrounding host-pathogen interaction and mechanism at these secondary sites of infection (1).

References:

Bennett JE, Dolin R, Blaser MJ, Douglas RG, Mandell GL. Bartonella, Including Cat-Scratch Disease. In: Mandell, Douglas, and Bennett's principles and practice of infectious diseases. Philadelphia: Elsevier; 2020. p. 2824–43.
Deng H, Pang Q, Zhao B, Vayssier-Taussat M. Molecular mechanisms of Bartonella and mammalian erythrocyte interactions: A Review. Frontiers in Cellular and Infection Microbiology. 2018;8.
(Focà A, Liberto MC, Quirino A, Matera G. Lipopolysaccharides: From Erinyes to Charites. Mediators of Inflammation. 2012;2012:1–6.
Riess T, Andersson SGE, Lupas A, Schaller M, Schäfer A, Kyme P, et al. Bartonella adhesin a mediates a proangiogenic host cell response. Journal of Experimental Medicine. 2004;200(10):1267–78.
Franz B, Kempf VAJ. Adhesion and host cell modulation: Critical pathogenicity determinants of bartonella henselae. Parasites & Vectors. 2011;4(1).

(iv) Bacterial Damage: what are the bacterial virulence factors and what role do they play in pathogenesis?

Figure 5: General schematic of B. henselae’s interactions with erythrocytes (2).

Figure 6: Hypothetical model for the Trw system (2).

Once the pathogen enters the human host, it first undergoes a priming period, a period of rapid replication of the pathogen, which will allow it to infect a greater amount of erythrocytes (1,2). The pathogen may also infect erythrocytic precursors, liver cells, and cells of other organs prior to infecting erythrocytes (2). Refer to figure 3, for an overview of the different virulence factors.

TrW Type IV secretion System:

Around five days after the date of initial infection, large amounts of B. henselae will begin to infect erythrocytes (3). A general diagram of the process is shown on Figure 5 (2). The initial adhesion of B. henselae to erythrocytes is mediated by the virulence factor, Trw type IV secretion system, which is a protein complex containing multiple components which span across the inner and outer membranes of the bacterial pathogen (2). A simple hypothetical model of the Trw system is shown on Figure 6 (2). Specifically, it is mediated by multiple copies of surface-exposed components, the TrwL and TrwJ variants (2). Once the bacterium has adhered to the erythrocyte, the deformation factor, deformin, and virulence factors, invasion protein B and hemolysin, of the B. henselae will result in the deformation of the erythrocyte membrane, thereby inducing internalization of the pathogen (2).

After the pathogen has successfully invaded the erythrocyte, it will begin replication inside a membrane-bound vacuole, which will provide protection against the host’s immune defense responses (2). The replication will continue until the critical number, eight daughter cells, are reproduced, after which the pathogen will remain in a non-dividing state for the remainder of its lifespan (2,3).

VirB/ VirD Type IV Secretion system

The VirB/VirD type IV secretion system will directly deliver Bartonella effector proteins (BEPs) into the cytoplasm of the host cell (1). This results in the human hosts producing an increased amount of erythrocytes, allowing for a greater amount of cells for the bacterial pathogen to invade and damage (1). Furthermore, BEPs also mediate intense immune responses that directly result in host damage and tissue damage (1). Infecting the host’s blood provides the ability for the pathogen to reach and infect other organs as well (1). Lymph nodes are a common site of B. henselae infection as the naive T cells become overwhelmed with B. henselae antigens that are presented by the macrophages, rendering the lymphatic tissue unable to keep up with the rate of T-cell differentiation (1).

Beps:

Beps modify host cell responses through the VirB/VirD T4SS to assist B. henselae pathogenesis (refer to figure 3). The recognition of Beps by the immune system triggers (NF)-kB pro-inflammatory signalling in the host. Intercellular adhesion molecule (ICAM-1) and E-selectin are upregulated, potentially strengthening the bacteria-host interaction (10). BepA inhibits endothelial cell apoptosis by elevating intracellular cyclic adenosine monophosphate (cAMP) (10). Therefore, BepA can infect neighbouring endothelial cells while evading the host immune response. BepC modifies host cell cytoskeleton arrangement for invasome- mediated internalisation of bacterial aggregates into the vascular endothelial cell (9)

BadA

Bartonella adhesin A (BadA) is a multifunctional virulence factor at multiple stages of B. henselae pathogenesis. Firstly, BadA is associated with fibronectin binding, extracellular matrix binding and bacterial auto-agglutination for adherence and entry into the host cell (4). Secondly, BadA upregulates proangiogenic factors in the host cell resulting in vascular proliferation (5).Thirdly, BadA inhibits complement activation and phagocytosis by macrophages (6). The host has difficulty detecting the bacteria and stimulating an appropriate immune response. B. henselae stimulates IL-10 production to dampen the function of T helper, monophages, macrophages and dendritic cells (4). Therefore, the human immune system initiates a weaker helper T-1 response to clear B. henselae infection (4).

Hemin binding proteins (Hbps):

Hemin binding proteins (Hbps) are another crucial virulence factor for nutrient uptake. Iron and hemin are relatively scarce in the human bloodstream. The porin-like outer membrane hbps not only acts as heme storage but improves the efficiency of heme acquisition for bacterial growth (6). Furthermore, HpbC protects against heme toxicity in the gut of arthropod vectors (8). Experiments with B. henselae Hbp knockdown mutants exhibit decreased ability to neutralise reactive oxidative species, reduced endothelial cell penetration and are less likely to survive in flea feces (9).

References:

McCall A, Roberge A, Moffit B. 2019. Bartonella henselae. Mechanisms of Pathogenicity.
Deng H, Pang Q, Zhao B, Vayssier-Taussat M. 2018. Molecular mechanisms of Bartonella and mammalian erythrocyte interactions: A Review. Frontiers in Cellular and Infection Microbiology 8.
Vayssier-Taussat M, Le Rhun D, Deng HK, Biville F, Cescau S, Danchin A, Marignac G, Lenaour E, Boulouis HJ, Mavris M, Arnaud L, Yang H, Wang J, Quebatte M, Engel P, Saenz H, Dehio C. 2010. The TRW type IV secretion system of bartonella mediates host-specific adhesion to erythrocytes. PLoS Pathogens 6.
Schmid MC, Schulein R, Dehio M, Denecker G, Carena I, Dehio C. The VIRB type IV secretion system of bartonella henselae mediates invasion, proinflammatory activation and antiapoptotic protection of endothelial cells. Molecular Microbiology. 2004;52(1):81–92.
Riess T, Andersson SGE, Lupas A, Schaller M, Schäfer A, Kyme P, et al. Bartonella adhesin a mediates a proangiogenic host cell response. Journal of Experimental Medicine. 2004;200(10):1267–78.
Deng H, Pang Q, Zhao B, Vayssier-Taussat M. Molecular mechanisms of Bartonella and mammalian erythrocyte interactions: A Review. Frontiers in Cellular and Infection Microbiology. 2018;8.
Focà A, Liberto MC, Quirino A, Matera G. Lipopolysaccharides: From Erinyes to Charites. Mediators of Inflammation. 2012;2012:1–6.
Roden JA, Wells DH, Chomel BB, Kasten RW, Koehler JE. Hemin binding protein C is found in outer membrane vesicles and protects bartonella henselae against toxic concentrations of hemin. Infection and Immunity. 2012;80(3):929–42.
Liu MF, Ferrandez Y, Bouhsira E, Monteil M, Franc M, Boulouis H-J, et al. Heme binding proteins of bartonella henselae are required when undergoing oxidative stress during cell and flea invasion. PLoS ONE. 2012;7(10).
Schmid MC, Schulein R, Dehio M, Denecker G, Carena I, Dehio C. The VIRB type IV secretion system of bartonella henselae mediates invasion, proinflammatory activation and antiapoptotic protection of endothelial cells. Molecular Microbiology. 2004;52(1):81–92.

4. The Immune Response

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

Innate: The innate immune response is able to prevent infection or reduce bacterial load. Components include phagocytes (including monocytes and macrophages), natural killer (NK) cells, cytokines, and infiltrating leukocytes.

The first and main line of defense against Bartonella henselae is macrophages, which have pattern recognition receptors (PRRs) on their surfaces (1). Specifically, toll-like receptor 4 (TLR4) recognizes lipopolysaccharide (LPS) (1). TLR-4 on T-lymphocytes recognizes LPS in bacteria such as Salmonella. Typically, LPS binds to the extracellular domain of TLR4, the receptors are activated by forming a homodimer and their TIR domain interacts with signaling proteins (2). The downstream signaling cascades activate the transcription factor, NF-kB, which transcribes pro-inflammatory cytokine genes including IL-1, IL-6, and TNF-a (3). However, LPS is presented in a different configuration in B. henselae making it difficult for TLR-4 to recognize (1). Namely, the LPS contains an unusual lipid A component with a long fatty acid chain and a lack of an O-chain polysaccharide (1). This allows B. henselae to escape the innate humoral response (2).

Monocytes and macrophages (i.e. phagocytes) are also involved in microbicidal, inflammatory, and angiogenic processes (4). They are also effectors of innate immunity and key angiogenic factors through the production of angiogenic cytokines (4). Effective phagocytic functions by these cell types have been proposed to be partly responsible for the localized expression of CSD, and uptake of bacteria by phagocytes is important to control and limit infection and CSD-associated bacteremia (4). Recruitment and activation of macrophages is also important in the type IV delayed-type hypersensitivity (DTH) response (4). The DTH response is involved in pathological granuloma formation, which is commonly observed in CSD infection (4). Alongside phagocytes, dendritic cells also present bacterial peptides on their MHC class II molecules to help activate CD4-naive T helper cells (4).

Other cells involved in the innate immune response include NK cells, which produce IFNγ (4). IFNγ activates antimicrobial functions of phagocytes; macrophage activation is particularly important (4). Cytokines, specifically macrophage-derived cytokines, are responsible as a part of the first line of defense against CSD infection (4). They initiate, regulate, and control various inflammatory cell functions (4). Secondary effector cells are also recruited by cytokines from locally activated cells (4). As a part of the innate defense, IL-8 levels are also elevated (5). There is also upregulation of proinflammatory cytokines such as IL-2 and IL-6, and anti-inflammatory cytokines such as IL-10 (5). Finally, activation of the complement system can cause lysis of B. henselae, which helps to clear the infection (6).

Adaptive: When physical barriers and innate immune system are breached, the adaptive immune system attacks pathogens using antigen specific cell-mediated and humoural responses. After the primary response, secondary adaptive responses become stronger and more efficient at clearing the pathogen.

The main cell-based adaptive immune response is the Th1 cell response: The helper T cell response is primarily responsible for the adaptive immune response against B. henselae (4). The Th1 cell response in particular is responsible for helping to develop cell-mediated immunity and stimulate B cells to release antibody classes IgG and IgM (4). Th-naive cells are driven to differentiation into Th-1 type cells through the release of cytokines by innate immune cells, including IL-12, IL-18, and IFNγ (4). CD4+ Th1 lymphocytes, in particular, become effector cells and are directly involved in the regulation of DTH and the elimination of B. henselae (4).

Hosts can mount a humoral response against B. henselae, however, the exact efficacy of this response is unknown (6). In bacterial infections, antibodies such as IgM and IgG are able to cause bacteriolysis and inactivation in gram-negative bacteria (3), including B. henselae. Serum antibodies affix to LPS of the bacteria, and activate complement proteins to form C3 and C5 complexes that will activate phagocytes to opsonization and digest the bacteria (3). Antibodies against B. henselae may play a role in eliminating bacteremia (4). IgG1 in particular creates a strong reaction in response to it, and is likely the primary type of antibody generated in response to CSD infection (7). IgG also appears to have a role in opsonization and clearance by phagocytic cells, although it does not seem to be involved in generating immunity (7). IgA, as a part of mucosal immunity, and IgM, are also predicted to be involved in humoral immunity responses against B. henselae (7).

Due to the presence of these antibodies and the nature of the adaptive immune response, the secondary response will be significantly less severe and less persistent than the primary response (2).

Immunocompetent vs. Immunocompromised hosts:

Lymphadenopathy (swollen lymph nodes) is a common symptom in immunocompetent patients with Cat Scratch Disease (CSD) (4), which means the innate immune response was not sufficient in containing a B. henselae infection. When lymph nodes enlarge, the adaptive immune system recognizes the pathogen and develops adaptive immunity against it in the lymph nodes by producing antibodies and remembering antigens specific to B. henselae. Immunocompetent patients typically do not need any antibiotic intervention as the infection often resolves on its own after 1-2 months (8). This is a sign of a successful adaptive immune response and the ability to set up an antigen specific immunity.

In immunocompromised patients, infections become vasoproliferative (9), meaning that the infection is widespread throughout the bloodstream. The lack of regularly functioning phagocytes can leave immunocompromised patients prone to more severe, long-lasting, and systemic diseases not often seen in immunocompetent patients such as bacillary angiomatosis (the creation of new blood vessels) (4). In HIV patients, bacillary angiomatosis can occur because CD4+ T cell numbers are suppressed which can lead to ineffective innate immune responses (10).

Reference List:

Deng H, Pang Q, Zhao B, Vayssier-Taussat M. 2018. Molecular Mechanisms of Bartonella and Mammalian Erythrocyte Interactions: A Review. Front. Cell. Infect. Microbiol. 8(431). doi: 10.3389/fcimb.2018.00431.
Rose SR, Koehler JE. 2020. Bartonella, Including Cat-Scratch Disease, 2824-2843. In Bennett J (ed), Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases, 9th ed. Elsevier, Philadelphia, PA.
Walker DH. Rickettsiae. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 38. https://www.3.nlm.nih.gov/books/NBK7624/
Resto-Ruiz S, Burgess A, Anderson BE. 2003. The Role of the Host Immune Response in Pathogenesis of Bartonella henselae. DNA Cell Biol. 22:431–440. https://doi.org/10.1089/104454903767650694.
Mosepele M, Mazo D, Cohn J. 2011. Bartonella Infection in Immunocompromised Hosts: Immunology of Vascular Infection and Vasoproliferation. Clin. Dev. Immunol. 2012:e612809. doi: 10.1155/2012/612809.
Karem KL, Paddock CD, Regnery RL. 2000. Bartonella henselae, B. quintana, and B. bacilliformis: historical pathogens of emerging significance. Microbes Infect. 2:1193–1205. doi: 10.1016/s1286-4579(00)01273-9.
Characterization of Human Immunoglobulin (Ig) Isotype and IgG Subclass Response to Bartonella henselae Infection | Infection and Immunity. https://journals.asm.org/doi/full/10.1128/IAI.66.12.5915-5920.1998.
Maggi RG, Ericson M, Mascarelli PE, Bradley JM, & Breitschwerdt EB. 2013. Bartonella henselae bacteremia in a mother and son potentially associated with tick exposure. Parasites & vectors. 6(1):1-9.
Biancardi AL, Land Curi AL. 2013. Cat-Scratch Disease. Ocu. Immu. Imflam. 22(2):148-154. https://doi.org/10.3109/09273948.2013.833631
Akram SM, Anwar MY, Thandra KC, Rawla P.. Bacillary Angiomatosis. January 2022, publishing date. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. https://www.ncbi.nlm.nih.gov/books/NBK448092/

(ii) Host immune response-associated damage - is it observed in CSD infection?

Given the mechanisms at play in B. henselae infection in immunocompetent hosts, it is likely that clinical symptoms associated with infection are attributable to the immune response instead of bacterial replication within the host (1). One of the clearest host immune response-associated damage observed in CSD is lymphadenopathy (swollen lymph nodes) at the site of inoculation which present as patchy necrotic, granulomatous change with stellate microabscesses (2). This is a sign of activity in the adaptive immune system. Another example is that of skin lesions. A primary cutaneous papule or pustule may develop 3-10 days post contact with a cat and can persist for 1-3 weeks due to inflammation associated with the innate immune response (3). However, 1-7 weeks after the infection, swollen lymph node(s) appears ipsilateral to the site of inoculation, indicating adaptive immune activity (3). Nearly half of the immunocompetent patients only have one swollen lymph node, 20% have multiple nodes in one site and the remaining 30% have swollen lymph nodes in multiple areas of the body (3). These enlarged lymph nodes can persist for 2 to 4 months and often resolve on their own (3).

While primarily targeting endothelial cells, vasoproliferative disease such as Bacillary Angiomatosis (BA) is very common in immunocompromised hosts such as in patients living with HIV, and B. henselae DNA has been amplified from these lesions (4,5). These lesions contain leukocytes, neutrophils, and fragmented nuclei (6) evidencing an incomplete inflammatory response. This damage is not directly caused by the host’s immune response and instead results from the incompetence of the cell-mediated immune response which results in host damage.

In the context of BA, bacterial interaction with endothelial cells stimulates upregulation of the receptor for IL8, an anti-inflammatory cytokine, on the cell surface. Bacterial replication in the peri-endothelial extracellular matrix leads to release of effector proteins BepA and BepA2, which increases intracellular cAMP, precluding NF-kB-dependent caspase activation, and also increases the Bcl2:Bax ratio (5). This autocrine signalling actively promotes an antiapoptotic state and downregulation of proapoptotic factors involved in the mitochondrial intrinsic apoptotic pathway. This prevents the release of damage associated molecular patterns (DAMPS), preventing their antigen presentation on MHC class II on monocytes, macrophages, and dendritic cells. Moreover, BepA also inhibits cytotoxic T-cell mediated lysis, thereby interfering with the cell-mediated immune response (7). Bcl2 is part of a positive feedback loop which stimulates the release of IL-8 and MCP-1 by monocytes and neutrophils which induces chemotaxis in macrophages and monocytes (5). These cellular events come together to stimulate the release of VEGF, thereby promoting angiogenesis and cell proliferation. As such, in the immunocompromised host, host damage results from the incomplete inflammatory response to the pathogen.

Reference List:

Resto-Ruiz S, Burgess A, & Anderson BE. 2003. The role of the host immune response in pathogenesis of Bartonella henselae. DNA & Cell Biology. 22(6):431-40.
Zangwill KM. 2021. Cat Scratch Disease and Bartonellaceae The Known, the Unknown and the Curious. Pediatric Infectious Disease. 40(5S):S11-S15.
Rose SR, Koehler JE. 2020. Bartonella, Including Cat-Scratch Disease, 2824-2843. In Bennett J (ed), Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases, 9th ed. Elsevier, Philadelphia, PA.
Jacomo V, Kelly PJ, & Raoult D. 2002. Natural history of Bartonella infections (an exception to Koch’s postulate). Clinical and Vaccine Immunology. 9(1):8-18.
Harms A, & Dehio C. 2012. Intruders below the radar: molecular pathogenesis of Bartonella spp. Clinical Microbiology Reviews, 25(1):42-78.
Biancardi AL, Land Curi AL. 2013. Cat-Scratch Disease. Ocular Immunology and Inflammation. 22(2):148-154.
Arvand M, Ignatius R, Regnath T, Hahn H, & Mielke ME. 2001. Bartonella henselae-specific cell-mediated immune responses display a predominantly Th1 phenotype in experimentally infected C57BL/6 mice. Infection and immunity. 69(10):6427-6433.

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

B. henselae is a facultative intracellular bacterium that is transmitted by arthropod vectors (1). To invade the host anatomical barrier, B. henselae penetrates the mucous membrane or the skin epithelial barrier (1). Then, the bacteria uses many virulence factors to generate transient and partial suppression of immunological activity (2). This may result in chronic infection and B. henselae remains persistent inside the mature erythrocytes (2). The persistent presence of B. henselae may lead to the formation of vasoproliferative cells in immunocompetent hosts (2).

BCVs (Bartonella-containing vacuoles)

After B. henselae penetrates the skin epithelial cells and enters the body, there are immune cells such as macrophages and neutrophils that phagocytose the bacteria (3). When B. henselae is phagocytosed by the immune cells, it induces the formation of Bartonella-containing vacuoles (BCVs) to invade phagocytes (4). The presence of BCVs delays lysosomal targeting which allows for B. henselae to escape from lysosomal fusion and acidification of phagosomes (4). As a result, the phagocytic activity of immune cells is reduced and antigen presentation is disrupted (4).

LPS (penta-acylation of LipidA)

LPS is a glycolipid expressed on the surface of gram-negative bacteria such as B. henselae (5). The three major components of LPS include the O antigen, Lipid A and the core oligosaccharide (5). The structure of LPS, particularly its Lipid A region, is recognized by TLR4 which stimulates downstream signalling pathways to trigger the secretion of pro-inflammatory cytokines (4). B. henselae is known to have a unique LPS structure that is less detectable by TLR4 which leads to reduced stimulation of signalling pathways to induce pro-inflammatory responses (4). Lipid A of B. henselae consists of unusually long fatty acid chains and a short carbohydrate region called penta-acylation of Lipid A (6). This unique structure of Lipid A is observed to be less immunogenic and inhibits the expression of TNF-α (6). The reduced simulation of inflammatory response allows B. henselae to invade the host’s innate immune response (4).

BadA (Bartonella adhesion A)

In order to acquire protection against the host immune response and initiate its replication process, B. henselae must adhere to erythrocytes (6). Many gram-negative bacteria consist of trimeric autotransporter adhesins (TAAs) which are bacterial outer membrane proteins that promote attachment to the host cells (7). B. henselae has a type of TAA called Bartonella adhesion A (BadA) which is involved in binding to endothelial cells and inducing the activation of hypoxia-inducible factor-1 (HIF-1) which stimulates the secretion of pro-angiogenic cytokines (7). The elevated level of pro-angiogenic cytokines such as vascular endothelial growth factor (VEGF) may contribute to bacterial colonization (7).

Moreover, BadA is known to induce autoaggregation of B. henselae and the variation of antigenic sites gives protection against phagocytosis by immune cells (4). Furthermore, BadA may be involved in preventing complement activation to disrupt the inflammatory response during innate immune responses, based on in vitro studies (4).

Agglutination

After B. henselae adheres and enters into erythrocytes, it is protected against immune components such as antibodies and complements (4). The bacteria begin their replication cycle inside the host red blood cells and generate persistent infection for weeks or months (8). For up to four days, B. henselae is able to colonize in the bloodstream without any detection from the immune system (8). To further invade the host immune response, replicating in large numbers is important (4). The large colonization of the bacteria causes agglutination which leads to biofilm formation (4). The biofilm formation gives protection against the immune components and adheres to the vascular walls (4).

BepD

Studies suggest that there are elevated levels of IL-10 cytokines during B. henselae infection (9). Il-10 is an anti-inflammatory cytokine that represses the activity of a pro-inflammatory immune response and prevents the maturation of immune cells such as macrophages and dendritic cells (10). BepD is one of the effector proteins that activate the host transcription factor called STAT3 (11). The activation of STAT3 triggers signalling cascades to secrete Il-10 cytokines (11). STAT3 is a cellular inflammation regulator that is known to trigger anti-inflammatory or pro-inflammatory responses (11). To activate STAT3, phosphorylation by the host c-ABL kinase is essential (11). BepD recruits c-ABL kinase near STAT3 and phosphorylates it (11). As a result, the pro-inflammatory immune response is repressed and the maturation of immune cells is suppressed (10, 11).

Reference List:

Zangwill KM. Cat Scratch Disease and Bartonellaceae: The Known, the Unknown and the Curious. Pediatr Infect Dis J. 2021 May 1;40(5S):S11-S15. doi: 10.1097/INF.0000000000002776.
Pulliainen AT, Dehio C. Persistence of Bartonella spp. stealth pathogens: from subclinical infections to vasoproliferative tumor formation. FEMS Microbiol Rev. 2012 May;36(3):563-99. doi: 10.1111/j.1574-6976.2012.00324.x.
Harms A, Dehio C. Intruders below the radar: molecular pathogenesis of Bartonella spp. Clin Microbiol Rev. 2012 Jan;25(1):42-78. doi: 10.1128/CMR.05009-11.
Deng H, Pang Q, Zhao B, Vayssier-Taussat M. 2018. Molecular Mechanisms of Bartonella and Mammalian Erythrocyte Interactions: A Review. Front. Cell. Infect. Microbiol. 8(431). doi: 10.3389/fcimb.2018.00431.
Bertani B, Ruiz N. Function and Biogenesis of Lipopolysaccharides. EcoSal Plus. 2018 Aug;8(1):10.1128/ecosalplus.ESP-0001-2018. doi: 10.1128/ecosalplus.ESP-0001-2018.
Ben-Tekaya H, Gorvel JP, Dehio C. Bartonella and Brucella--weapons and strategies for stealth attack. Cold Spring Harb Perspect Med. 2013 Aug 1;3(8):a010231. doi: 10.1101/cshperspect.a010231.
Tsukamoto, K., Shinzawa, N., Kawai, A. et al. The Bartonella autotransporter BafA activates the host VEGF pathway to drive angiogenesis. Nat Commun 11, 3571 (2020). doi:10.1038/s41467-020-17391-2
Schülein R, Seubert A, Gille C, Lanz C, Hansmann Y, Piémont Y, Dehio C. Invasion and persistent intracellular colonization of erythrocytes. A unique parasitic strategy of the emerging pathogen Bartonella. J Exp Med. 2001 May 7;193(9):1077-86. doi: 10.1084/jem.193.9.1077. PMID: 11342592; PMCID: PMC2193435.
Mosepele M, Mazo D, Cohn J. Bartonella infection in immunocompromised hosts: immunology of vascular infection and vasoproliferation. Clin Dev Immunol. 2012;2012:612809. doi: 10.1155/2012/612809.
Iyer SS, Cheng G. Role of interleukin 10 transcriptional regulation in inflammation and autoimmune disease. Crit Rev Immunol. 2012;32(1):23-63. doi: 10.1615/critrevimmunol.v32.i1.30.
Sorg I, Schmutz C, Lu YY, Fromm K, Siewert LK, Bögli A, Strack K, Harms A, et al. A Bartonella Effector Acts as Signaling Hub for Intrinsic STAT3 Activation to Trigger Anti-inflammatory Responses. Cell Host Microbe. 2020 Mar 11;27(3):476-485.e7. doi: 10.1016/j.chom.2020.01.015.

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

The most common symptom of cat scratch disease (CSD) is the enlargement of the lymph nodes which may last several months even if the infection has fully resolved (1, 2). In a majority of immunocompetent hosts, CSD is self-limiting and patients will recover fully by supportive care, without antibiotic treatment, in 2-4 months (1, 2, 3). It was reported that there is a faster improvement of lymphoid node size in patients who received antibiotic treatment (azithromycin) within the first month only when compared to placebo (1, 2). In the case of immunocompromised hosts, antimicrobial medications are usually recommended to eradicate B. henselae and prevent further complications such as liver issues, as the hosts have weaker immune systems compared to immunocompetent hosts (2, 3). Adaptive immunity includes antibody-mediated immunity (AMI) and cellular-mediated immunity (CMI) (4). In AMI, B-cells recognize the specific antigen, B. henselae, in first exposure and develop into antibodies (mostly IgG), which help in eradication of bacteria, and memory B-cells (4). Memory B-cells will recognize the same antigen in the future and act faster in producing antibodies when the host is re-infected with B. henselae (4). In CMI, both cytotoxic T-cells, which directly kill bacteria, and T-helper cells are activated (4). Moreover, memory T-cells will be developed by first exposure of bacteria which accelerate the recognition of the same antigen by reinfection (4, 5). There are different subtypes of memory T-cells which have specific functions. For example, effector memory T-cells are circulating in the bloodstream and residency memory T-cells remain in the tissues (5, 6).

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Conrad, D A. “Treatment of cat-scratch disease.” Current opinion in pediatrics vol. 13,1 (2001): 56-9. doi:10.1097/00008480-200102000-00010
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