CIS 2 - Mycobacterium, HIV - Duus _ Santos 11-17-23.docx

Hannah Eng Blood Lymph 2023 - Immunodeficiency CIS Dr. Santos and Dr. Duus The Case of the Enlarged Lymph Nodes Maggie Townsend is a 2 and ½ year old female brought into clinic with diarrhea, weight loss, and poor appetite. Her parents live in a rural area where most families can trace their lineage back to the initial settlers of the area. The community is close and intermarriage has been common throughout the history of the area. Maggie’s parents think they may be related to each other, though they believe that to be a distant relation. Maggie has been otherwise healthy to this point. Maggie has enlarged lymph nodes based on palpitation. Ultrasound and CT further show enlarged lymph nodes in the mesentery and para-aortic region. Maggie was referred to the local children’s hospital. CBC results WBC 9400 μ L -1 55% Neutrophils 30% lymphocytes 15% monocytes Serum Immunoglobulin values IgG 1750 mg dL -1 IgA 450 mg dL -1 IgM 175 mg dL -1 1) Interpret these lab results. ↑ monocytes ↑ IgG and IgA 2) The decision is made to take a biopsy of the enlarged nodes. What would you be looking for in this biopsy (differential diagnosis)? Differentials: bacterial infections, gastroenteritis, tuberculosis, cancers (lymphoma, myeloma, B-ALL), granulomas or giant cells The results of the biopsy are shown below. There are increased numbers of histiocytes and macrophages, but no granulomas or giant cell formation. The slide was stained with an acid-fast stain. 3) How does an acid-fast stain work and what are you looking for with an acid-fast stain? An acid fast stain penetrates the cell wall with a lipid-soluble dye. Then the cells are decolorized which washes the stain out of all the cells except those that are acid-fast. This stain generally looks for Mycobacterium species. Page 1 of 6

Hannah Eng Blood Lymph 2023 - Immunodeficiency CIS Dr. Santos and Dr. Duus 4) What is the interpretation of the acid-fast staining? What does that mean this patient is infected with? Acid fast staining is positive, suggesting the presence of mycobacteria. 5) What type of host immunity is required to clear this type of pathogen? How does that relate to the blood work done previously on this patient? Monocytes in particular are important in the process of clearing Mycobacteria from the body, since monocytes process antigen and stimulate lymphocytes to produce antibodies. CD4 T cells release IFN- ɣ which activates macrophages for bacterial killing. The CBC results showed elevated monocytes at 15% when monocytes are typically 2-7%. This indicates that there is not an issue with the presence of the cells, but potentially an issue with the activation of the macrophages or the secretion of IFNy by the lymphocytes. 6) The male cousins of this patient have also presented with recurrent infections with this type of pathogen. What does that information tell you about this patient’s disease? Since family members have had recurrent infections with the same pathogen, we can deduce that the patient may have primary genetic immunodeficiency, placing her at higher risk for infection. The key word to suggest immunodeficiency is “recurrent.” 7) You decide to test your hypothesis about this patient’s condition. You take her immune cells and treat them with a cytokine and then test the ability to kill the pathogen you identified with the acid-fast stain. The cells fail to kill the pathogen. What cytokine did you use and which cells do you expect it to work on? The cytokine that was used was IFN- ɣ which typically activates macrophages for bacterial and intracellular killing. If IL-12 was used, NK cells activity would be expected - killing of MHC-I lacking cells. Deficiency of IL-12 would lead to decrease of NK cell activity, and over-activity of macrophages causing a granuloma formation. We would expect Mycobacterium to be killed by macrophages. Because they were not killed, there is a macrophages and IFN- ɣ receptor deficiency . If it was a NADPH oxidase issue, there would be more issues due to neutrophils. No granuloma formation. 8) What is this patient’s immunodeficiency? Possibly due to Mendelian susceptibility to mycobacterial diseases (MSMD) due to inborn errors causing (more commonly) IL-12 receptor dysfunction or IFN- ɣ receptor deficiency. The receptor of the cytokine is defective because the result was negative due to the defective receptor. Page 2 of 6

Hannah Eng Blood Lymph 2023 - Immunodeficiency CIS Dr. Santos and Dr. Duus 2) What information in the case description provides clues to the identity of a potential underlying major cause of Anna’s current condition? ● poor birth delivery conditions ● Lack of prenatal care ● mothers drug history ● failure to thrive ● figure 1 3) What diagnostic test could you perform on Anna to test for this major cause, and who else in this case would you also want to test for presence of this infection? Explain how your test of choice works. ● test for Rotavirus - infant stool sample ● r/o other viruses / bacterial causes ○ Neonatal Herpes Simplex - test via viral culture,, PCR ○ HIV - nucleic acid tests (NATS), PCR ■ cannot do immunofluorescence testing yet because infant does not have Ig ○ Candida - yeast culture ○ syphilis, other TORCH infections (Toxoplasmosis, Rubella, Cytomegalovirus, herpes simplex virus), hepatitis B, hepatitis C, or tuberculosis infection. ● Genetic disorders: SCID ● Also want to test mother and father for HSV, HIV, Candida You immediately call for transport for Anna to Sunrise Children’s Hospital for inpatient care, where Anna is given IV rehydration, empirical antimicrobial drug treatments, and a barrage of diagnostic testing. 4) From what infection(s) is Anna currently potentially suffering (differential diagnosis), based on the case information? ● Rotavirus ● HSV-2 (Neonatal Herpes Simplex) ● HIV ● respiratory infection of many potential etiologies ● Candida infection 5) What diagnostic test(s) would you choose to run on Anna to more knowledgeably treat her current infections? Fecal, sputum, blood, oral specimens 6) What can you do to diagnose and treat Christine, and protect her unborn child? HIV test, prenatal care; ART if she is HIV positive Page 4 of 6

Hannah Eng Blood Lymph 2023 - Immunodeficiency CIS Dr. Santos and Dr. Duus To confirm a positive diagnostic test for Christine, the clinical diagnostic lab performed a Western Blot test; the result for Christine is in Lane C, and that for her boyfriend, Lance Brown (also ELISA-positive) in Lane B: 7) How would you interpret Christine’s test? Why? HIV-positive; Env, and Gag protein bands present 8) How would you interpret Lance’s test result? Why? Indeterminant; p24 and some Gag, but no Env protein bands- he has not yet fully sero-converted 9) Why would this test not work to diagnose Anna’s infection? What test can you use in this type of situation? As a 6 month old infant, Anna does not have antibodies levels fully developed yet. Anna will still have maternal anti-HIV Abs circulating. PCR or nucleic acid tests (NATS) are better suited. 10) This test is no longer routinely performed as a confirmatory diagnostic test for HIV-1. Explain why. What type of test is now routinely used as a confirmatory test? Too many indeterminate tests early in primary infection. ELISA antigen/antibody test and NAT confirmatory test are much more specific and sensitive. NAT test is now routinely used as a confirmatory HIV-1 test. Because 6 months of age is early for this particular syndrome to manifest in a normal disease course with this pathogen, you want to further analyze Anna’s immune cells. 11) What type of test could quantitatively analyze Anna’s lymphocyte populations, and how does it work? Flow cytometry will distinguish and enumerate T cell populations by fluorescence of antibodies to cell markers (CDs) 12) Which populations would you be most interested in analyzing, given her current condition? CD4+ T cells 13) What antibodies would you request be used for this test? CD3, CD4, CD8 Page 5 of 6