Microbiology Case Study: A 67 Year Old with Foot Pain

Case description

A 67 year old male presented at the clinic with a primary complaint of foot pain; she has a previous medical history of M. tuberculosis infection of her prosthetic joint, osteoarthritis, and leukopenia. The patient described joint pains during the check-up and mentioned that she also started to have periumbilical pain two weeks ago, along with worm-like objects in her stool. The patient was in Ethiopia for 8 months in the past year and was very active. He has had some weight loss but no change in appetite; he denies any diarrhea, skin rashes, fever, or chills. The patient consumed undercooked meat products during the time she visited Ethiopia. No abnormal neurological symptoms presented at the time of the visit.

Orders were placed for H. Pylori antigen, fecal bacteria pathogen PCR, Giardia and Cryptosporidium antigen, and Ova & Parasite exam for the patient’s GI symptoms. The Ova & Parasite exam detected the objects in Image 1.

Image 1. Patient stool sample wet mount preparation.

Discussion

The Ova & Parasite exam was reported as Taenia species. The eggs had a diameter of around 37um. An infectious disease consult was ordered and a single dose of 600mg praziquantel was prescribed for the treatment. Repeat Ova & Parasite exams are ordered for 3 days post-treatment looking for dying parasites and 1 month post-treatment to confirm the cure (no eggs).

Taenia in the Taeniidae family of tapeworms (BioLib, n.d.). Three species are commonly found and most clinically important in human infection: Taenia saginata, Taenia solium, and Taenia asiatica; most Taeniasis is asymptomatic or has mild symptoms (Centers for, 2020b).

Taenia solium, or pork tapeworm often found in pork, is the most dangerous species to humans for two reasons. First, this is the only species that can cause the neurologic symptoms by cysticercosis in brain tissue; second, this species can take humans as intermediate hosts, which means it can cause human to human transmission within the household (Schmidt et al., 2009).

Taenia asiatica also lives in pigs, primarily in the liver instead of muscle. This species has a very similar genetic, morphology, and immunology to T. saginata. It is frequently found in Asia (Schmidt et al., 2009).

Taenia saginata, or beef tapeworm, is what our patient was assumed to have in this case. The life cycle is shown below in Figure 2. The patient presented because his ankle pain started to impact his walking significantly; however, he was not seeking help for his worm-like objects in the clinic, probably due to the mildness of the symptoms. The parasite infection was brought into sight because of his travel history and stool observation. Per CDC, Eastern Europe, Russia, eastern Africa, and Latin America are the highest risk areas (Centers for, 2020a). The patient stayed for 8 months in Ethiopia in eastern Africa. Ethiopia has a relatively poor sanitation status and a high prevalence of taeniasis (Jorga, 2020). The major contributors for our infectious disease clinicians to assume this patient has T. saginata infection but not T. solium infection are: there are no neurological symptoms, and there is no pork exposure due to his religion. Visualization of the tapeworm eggs or segments is important for identification the species. In this case, many eggs were found on the wet mount slide from the patient’s stool sample.

Treatment of taeniasis is with Praziquantel. Praziquantel removes the tapeworms from the human body by detaching the worm suckers from vessel walls. The medication is safe to give to ≥1year old patients (UpToDate, 2022).

Image 2. Taeniasis life cycle. Alive Taenia eggs or gravid proglottids in the environment get ingested by farm or wild animals. Oncospheres develop in the GI tract, then hatch to the intestine wall and penetrate the wall to migrate to muscle tissue. In the muscle tissue, oncospheres develop into cysticerci (cysticercosis happens at this step). After the meat products (generally animal muscle) get ingested by humans, the cysticerci grow into adult worms in humans. Some segments/worms/eggs will be released into the environment through feces to complete the life cycle (which allows detection and diagnosis of human infections).
https://www.uptodate.com/contents/image/print?imageKey=ID%2F64879

References

BioLib: Biological library. Taenia | BioLib.cz. (n.d.). Retrieved from https://www.biolib.cz/en/taxon/id43806/

Centers for Disease Control and Prevention. (2020a, September 18). CDC – taeniasis – general information . Epidemiology & Risk Factors. Retrieved from https://www.cdc.gov/parasites/taeniasis/epi.html

Centers for Disease Control and Prevention. (2020b, September 18). CDC – taeniasis – general information . frequently asked questions. Retrieved from https://www.cdc.gov/parasites/taeniasis/gen_info/faqs.html

Jorga, E., Van Damme, I., Mideksa, B. et al. Identification of risk areas and practices for Taenia saginata taeniosis/cysticercosis in Ethiopia: a systematic review and meta-analysis. Parasites Vectors 13, 375 (2020). https://doi.org/10.1186/s13071-020-04222-y

Schmidt, G. D., & Roberts, L. S. (2009). Chapter 21 Tapeworms. In Foundations of Parasitology, eighth edition (pp. 346–351). essay, McGraw-Hill Higher Education.

UpToDate. (2022). Praziquantel: Drug information. UpToDate. Retrieved from https://www.uptodate.com/contents/table-of-contents/drug-information

-Sherry Xu is a Masters student in the department of Pathology and Laboratory Medicine at the University of Vermont Medical Center.

-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Associate Professor at the University of Vermont.

Microbiology Case Study: A 46 Year Old with Chest Pain

Case History

A 46 year old male with a history of cystic fibrosis and bilateral lung transplant two years prior presented to the hospital with chest pain and hemoptysis. The patient was recently diagnosed with COVID-19, and a CT chest revealed multiple rounded, mass-like opacities with central cavitation. As imaging was not consistent with COVID-19 pulmonary disease and no clear risk for tuberculosis could be identified, a bronchoscopy with transbronchial biopsy was performed. Tissue and bronchiolar lavage fluid were collected and submitted to the microbiology laboratory for analysis. Viral etiologies including influenza A/B, Parainfluenza 1-3, Adenovirus, RSV and metapneumovirus were ruled out through molecular studies. Galactomannan was negative from the BAL fluid, as were fungal and mycobacterial cultures and Mycobacterium tuberculosis PCR. GMS staining of the biopsy was negative but organizing pneumonia and mononuclear infiltrate was noted. The patient had a history of recurrent multidrug-resistant Pseudomonas aeruginosa infection and was being managed with empiric ceftazidime/avibactam.

Laboratory Identification

Gram stains of both the tissue and BAL fluid were generally unremarkable. Histopathological analysis of the transbronchial tissue revealed changes suggestive of organizing pneumonia with mononuclear infiltrate (Image 2, left). Bacterial growth of a predominant organism from both the BAL and biopsy tissue was observed on plates after 48 hours on blood and chocolate agars but was absent on MacConkey agar. At 96 hours, the colonies of the organism had become mucoid, slightly pink and had coalesced (Image 1, right). Gram staining of the growth revealed short, poorly staining gram positive coccobacilli with a beaded appearance. Due to the incomplete gram staining of this isolate, modified acid-fast staining was attempted which was positive (Image 1, left). The organism was both catalase- and urease-positive. The isolate was subsequently identified by MALDI-TOF MS as Rhodococcus equi, and the patient was discharged from the hospital on imipenem and linezolid.

Discussion

Rhodococcus equi is a zoonotic pathogen which primarily causes infections among immunocompromised hosts. Infrequently isolated clinically, the organism is a primary pathogen of horses causing pneumonia with abscess formation in foals, often with dissemination into peripheral sites due to high organism burden. The organism is excreted in feces of infected animals, leading to contamination of soils from farms, ranches, and other agricultural environments from which the organism is either aerosolized and inhaled or acquired via direct inoculation.1 While human infections are classically associated with exposure to horses or their environment, there is a growing body of literature to suggest that many patients with microbiologically proven cases of R. equi infection lack such environmental exposures. This patient falls into the latter category, with no known exposure to livestock.

                R. equi is a member of the aerobic actinomycetes. Like Nocardia sp., the cell wall of R. equi contains mycolic acids which lead to positivity when stained with a modified acid-fast stain. The organism is a facultative, intracellular pathogen surviving within macrophages and histiocytes, leading to granulomatous inflammation, eventually leading to necrosis.2 Immunosuppression (including HIV infection or immunosuppressive therapy) is a major risk factor for R. equi infection, as most clinical cases are reported in this setting. In immunocompromised hosts, the spectrum of disease manifestations of R. equi are diverse, but most commonly (approx. 80%) include pulmonary involvement3 with upper lobe cavitary pneumonia.4 Characteristic malakoplakia (an infiltration of foamy histocytes with intracellular bacteria and basophilic inclusions name Michaelis-Gutmann bodies)1 can be associated with R. equi infection. These structures were noticeably absent in this patient’s case despite the observed histocyte aggregation and mononuclear infiltrate (Image 2, center, left).

R. equi pneumonia among solid organ transplant recipients, such as the patient in this case is associated with low overall morbidity and mortality, but require protracted antibiotic therapy regimens.1 Susceptibility testing is warranted to guide therapy of R. equi due to unpredictable resistance patterns among isolates. This patient’s isolate was revealed to be susceptible to amoxicillin/clavulanate, ceftriaxone, imipenem, ciprofloxacin, moxifloxacin, clarithromycin, amikacin, tobramycin, minocycline, trimethoprim/sulfamethoxazole, vancomycin, linezolid, and rifampin. The patient was discharged on imipenem/linezolid. At follow-up, the patient had clinically improved with a resolution of symptoms, but his radiologic abnormalities persisted and thus remains on oral therapy with moxifloxacin and minocycline.

References

Yamshchikov, AV, Schuetz, A, and Lyon, GM. Rhodococcus equi infection. 2010. Lancet Infect. Dis. 10:350-359.

Prescott, JF. Rhodococcus equi: an Animal and Human Pathogen. 1991. Clin. Microbiol. Rev. 4(1):20-34.

Weinstock, DM, and Brown, AE. Rhodococcus equi: an emerging pathogen. 2002. Clin. Infect. Dis. 34:1379-1385.

Mutaner, L, et. al. Radiologic featuresof Rhodococcus equi pneumonia in AIDS. Eur. J. Radiology. 1997. 66-70.

-Andrew Clark, PhD, D(ABMM) is an Assistant Professor at UT Southwestern Medical Center in the Department of Pathology, and Associate Director of the Clements University Hospital microbiology laboratory. He completed a CPEP-accredited postdoctoral fellowship in Medical and Public Health Microbiology at National Institutes of Health, and is interested in antimicrobial susceptibility and anaerobe pathophysiology.

-Dominick Cavuoti is a Professor in the Department of Pathology at UT Southwestern Medical Center. Dr. Cavuoti is a board certified AP/CP who is a practicing Clinical Microbiologist, Infectious Disease pathologist and Cytopathologist.


-Clare McCormick-Baw, MD, PhD is an Assistant Professor of Clinical Microbiology at UT Southwestern in Dallas, Texas. She has a passion for teaching about laboratory medicine in general and the best uses of the microbiology lab in particular.

Microbiology Case Study: A 17 Year Old with Chest Pain

Case History

A 17 year old female who presented to the emergency department with complaints of fever, vomiting, diarrhea, and chest pain for the past two weeks. She also reported an unintentional weight loss of 20 lbs. Her medical history consisted of essential hypertension for which she was previously on medication, however had been discontinued two years ago due to normal blood pressure. The patient reported that she is sexually active with one male partner and denied use of protection. She denied any other sexual partners or any prior history of sexually transmitted infections. Her urine NAAT testing was positive for chlamydia, but negative for gonorrhea. Blood cultures collected at the time of admission resulted in growth of gram-negative diplococci on day 2 of admission (Image 1) and colony growth on chocolate agar (Image 2). The organism was positive for both catalase and oxidase and identified by matrix-assisted light desorption ionization- time of flight (MALDI-TOF) as Neisseria gonorrhoeae. Due to her chest pain complaints and QT prolongation on EKG, a trans-thoracic echo was performed that demonstrated a large aortic root abscess suggestive of infective endocarditis. Ceftriaxone was started as treatment for her gram-negative endocarditis, and she was emergently transferred to another facility where an aortic valve replacement and patch aortoplasty were performed.

Image 1. Gram stain of the blood culture showing gram negative diplococci.
Image 2. Neisseria gonorrhoeae on chocolate agar producing small gray-white colonies.

Discussion

Neisseria gonorrhoeae is a fastidious, oxidase positive, gram negative diplococcus, commonly transmitted through sexual contact.2,3 Neisseria uniquely grows on chocolate agar and VPN/Thayer Martin agar, and has virulence factors such as pilli that attach to mucosal surfaces, and many antigenic variations that make it a highly resistant organism prone to reinfection.

In the laboratory, N. gonorrhea grows well on chocolate agar after 24-48 hours of incubation (Image 2) with less robust or no growth on blood agar. It is positive for both catalase and oxidase. Traditionally, sugar fermentation was used to differentiate Neisseria species from one another, but more ore rapid identification methods (MALDI-TOF and PCR) are being increasingly used in most clinical laboratories

In men, Neisseria usually ascends the genitourinary tract to cause prostatitis. In women, the infection can disseminate to cause pelvic inflammatory disease, which can cause scarring in the fallopian tubes, resulting in infertility. Neisseria also can present as an asymmetric polyarthritis, most commonly to the knees. The main treatment of Neisseria gonorrhea is ceftriaxone. Gentamicin is an acceptable alternative in patients with severe cephalosporins allergy.

This case involves a rare presentation of infective endocarditis caused by disseminated gonorrhea infection. Previous reported cases of gonococcal endocarditis1,4 reported ad subacute presentation in around 2-4 weeks with generalized fatigue, fevers, arthritis, rash, renal dysfunction, and new cardiac murmurs. Because it can present without preceding genitourinary symptoms, disseminated gonorrheal can be difficult to recognize. The infection is usually aggressive, forming large vegetations and rapid valve destruction, despite antibiotic treatment. Most commonly it involves the aortic valve, as seen in the case presented above, but can also involve the mitral and tricuspid valves in some cases. The damage usually requires valve replacement surgery in addition to antimicrobial therapy.5,6 Lastly, this case demonstrates the limitations of the urine NAAT to diagnose gonorrhea specifically in females and/or asymptomatic patients due the possible presence of inhibitors and the need for further testing if clinical suspicion remains.7

References

  1. Said M, Tirthani E. Gonococcal Infective Endocarditis Returns. Cureus. 2021 Sep 14;13(9):e17955. doi: 10.7759/cureus.17955. PMID: 34660143; PMCID: PMC8515499.
  2. Ryan, K. J., Ray, G., and Sherris, J. C. (2004). Sherris Medical Microbiology: An introduction to Infectious Diseases, 4th edition. McGraw-Hill Medical.
  3. Centers for Disease Control and Prevention. Gonorrhea. Available from: https://www.cdc.gov/std/gonorrhea/stdfact-gonorrhea-detailed.htm. Last updated 2021 July 22; cited on 2022 March 21.
  4. Fenech, Marylou, et al. “Neisseria Gonorrhoeae Infective Endocarditis.” BMJ Case Reports, BMJ Specialist Journals, 1 May 2022
  5. Thompson EC, Brantley D. Gonoccocal endocarditis. J Natl Med Assoc. 1996 Jun;88(6):353-6. PMID: 8691495; PMCID: PMC2608094.
  6. Nie S, Wu Y, Huang L, Pincus D, Tang YW, Lu X. Gonococcal endocarditis: a case report and literature review. Eur J Clin Microbiol Infect Dis. 2014 Jan;33(1):23-7. doi: 10.1007/s10096-013-1921-x. Epub 2013 Jul 16. PMID: 23856883.
  7. Whiley DM, Tapsall JW, Sloots TP. Nucleic acid amplification testing for Neisseria gonorrhoeae: an ongoing challenge. J Mol Diagn. 2006 Feb;8(1):3-15. doi: 10.2353/jmoldx.2006.050045. PMID: 16436629; PMCID: PMC1871692.

-Olivia Piscano is a second-year medical student at the Medical College of Georgia. She is currently interested in Internal Medicine, Pediatrics, and Infectious Disease.

-Hasan Samra, MD, is the Director of Clinical Microbiology at Augusta University and an Assistant Professor at the Medical College of Georgia.

Microbiology Case Study: 57 year old Female with Altered Mental Status and Declining Health

Case Description

A 57 year old female presents to the emergency department with altered mental status, decreased appetite and chest, abdominal and pelvic pain. She has a complex medical history including end-stage renal disease, cardiovascular disease with pacemaker placement, and recurrent ascites. Her physical exam was notable for hypotension (65/52), hypothermia (31.7°C), and abdominal distention. A CT of her abdomen and pelvis revealed marked ascites, and bloodwork indicated leukocytosis (12.81, ref 4.22-10.33), elevated lactate (4.1, ref 0.5-2.2), and acidemia (7.27). Given the concern for septic shock, an infectious workup was initiated. A diagnostic paracentesis was undertaken which revealed rare yeast forms by Gram stain (Figure 1). Routine and fungal cultures of the ascites fluid grew yeast which was identified as Cryptococcus neoformans by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Bacterial blood cultures turned positive after 3 days and Cryptococcus neoformans was identified (Figure 2). Interestingly, serum cryptococcal antigen tests were negative.

The patient was treated with amphotericin B, vancomycin, and meropenem. Despite intervention, the patient’s clinical condition continued to deteriorate, leading to multiple organ failure. The patient was transitioned to comfort care and expired soon thereafter.

Figure 1. Gram stain of peritoneal fluid (ascites) demonstrating two yeasts with surrounding capsule and host cells (100x objective, oil immersion).
Figure 2.  Bacterial blood culture with encapsulated yeasts on Gram stain (100x objective, oil immersion).

Discussion

This case highlights two significant points: 1) atypical presentation of a cryptococcal infection and 2) the value of complimentary approaches in the diagnostic workup of an infectious etiology.

Cryptococciare environmental fungi with worldwide distribution, classily causing opportunistic central nervous system infection in patients with either uncontrolled HIV or other significantly immunocompromising conditions. In this patient population, a staggering 70-90% of cryptococcal infections manifest as meningitis, with a one-year mortality rate as high as 70% in some regions.1 Meningitis caused by Cryptococcus sp. accounts for nearly 1 in 7 HIV-related deaths worldwide.2 This case and others,3 serve as an important reminder that cryptococcal disease may present in different ways, including peritonitis in a patient without significant immunosuppression.

Several methods are available to aid in the identification of Cryptococcus sp.4 Direct microscopic evaluation of Cryptococcus in fluid and tissue samples reveals round, narrow-based budding yeast of variable size (2-20 um). The capsule of Cryptococcus is classically seen with India ink, though recent work has shown that Gram stain is as effective.5 In our patient, direct microscopy of the peritoneal fluid provided the first clue that Cryptococcus was the causative agent. Other useful stains used for histopathological analysis include mucicarmine and Fontana-Masson, which stain the capsule and melanin, respectively. In culture, strains of Cryptococcus sp. typically elaborate a robust capsule leading to the formation of mucoid colonies. However, acapsular strains have also been identified.

Biochemical hallmarks of Cryptococcus sp. include the production of urease and phenoloxidase leading to the formation of melanin which is absorbed into the cell wall. Phenoloxidase activity is exploited for diagnostic purposes as it leads to melanized pigmentation in the presence of caffeic acid, such as in either a caffeic acid disk test or on Bird Seed agar.

Non-culture-based methods for the diagnosis of cryptococcal infections include detection of cryptococcal capsular antigens by either ELISA, latex agglutination, or a lateral flow immunoassay (LFA). The LFA is a cost-effective test with rapid turnaround time which exhibits strong agreement with other antigen detection methods. While the LFA has proven useful in a variety of settings,6 our patient’s LFA was negative, underlining the importance of using orthogonal methods in parallel to identify microbes. In cases where patients are infected with an acapsular strain of Cryptococcus, the antigen testing will be negative since the antigenic target (i.e the capsule) is missing. An important consideration when assessing discrepancies between Gram stain/culture and ancillary immunoassay testing is “prozone effect”. Prozone, or Hook effect, is a phenomenon where overwhelming amounts of analyte impairs immunocomplex formation, causing a lack of analyte detection (i.e false-negative test). To account for prozone, a serial dilution series of the sample with repeat testing should be performed to ensure accurate correlation.

References

  1. World Health Organization. Guidelines for the diagnosis, prevention, and management of cryptococcal disease in HIV-infected adults, adolescents and children, March 2018: supplement to the 2016 consolidated guidelines of the use of antiretroviral drugs for treating and preventing HIV infection.
  2. Rajasingham R, Smith RM, Park BJ, Jarvis JN, Govender NP, Chiller TM, Denning DW, Loyse A, Boulware DR. Global burden of disease of HIV-associated cryptococcal meningitis: an updated analysis. The Lancet infectious diseases. 2017 Aug 1;17(8):873-81.
  3. El-Kersh K, Rawasia WF, Chaddha U, Guardiola J. Rarity revisited: cryptococcal peritonitis. Case Reports. 2013 Jul 10;2013:bcr2013009099.
  4. Mais DD. Quick compendium of clinical pathology. American Society for Clinical Pathology Press; 2018.
  5. Coovadia YM, Mahomed S, Dorasamy A, Chang C. A comparative evaluation of the Gram stain and India ink stain for the rapid diagnosis of cryptococcal meningitis in HIV infected patients in Durban: brief report. Southern African Journal of Infectious Diseases. 2015 Jan 1;30(2):61-3.
  6. Perfect JR, Bicanic T. Cryptococcosis diagnosis and treatment: What do we know now. Fungal Genetics and Biology. 2015 May 1;78:49-54.

AUTHORS

-Andrew T. Nelson, MD, PhD, is a Clinical Pathology resident at UT Southwestern Medical Center in his second year. He has an interest in Clinical Chemistry.

-Andrew Clark, PhD, D(ABMM) is an Assistant Professor at UT Southwestern Medical Center in the Department of Pathology, and Associate Director of the Clements University Hospital microbiology laboratory. He completed a CPEP-accredited postdoctoral fellowship in Medical and Public Health Microbiology at National Institutes of Health, and is interested in antimicrobial susceptibility and anaerobe pathophysiology.

-Clare McCormick-Baw, MD, PhD is an Assistant Professor of Clinical Microbiology at UT Southwestern in Dallas, Texas. She has a passion for teaching about laboratory medicine in general and the best uses of the microbiology lab in particular.

An Adult Patient Presents with Mild Penile Irritation and Discharge

Patient History

An adult male presented to the primary care office with mild penile irritation and discharge without fever, dysuria, or other lesions. He is sexually active and reported recent unprotected sex with multiple partners. He is on pre-exposure prophylaxis for HIV and tested non-reactive for HIV, HCV, and syphilis antibodies. Chlamydia and gonorrhea were detected in his urine, rectal, and throat specimens by PCR. The lab paged the director to review and verify the results. Is it possible to be positive for both chlamydia and gonorrhea?

Discussion

In the United States, chlamydia and gonorrhea are the most commonly reported sexually transmitted bacterial infections. While most cases of chlamydia and gonorrhea are sexually transmitted, neonates can become infected by perinatal transmission.1,2,3 To prevent long-term complications in women, all sexually active women aged <25 years and older women with increased risk of infection should get tested annually for chlamydia and gonorrhea. All pregnant women <25 years old or are considered high risk should be screened at the first prenatal visit and in the third trimester or at the time of delivery for both organisms. CDC recommends screening genital and extragenital sites at least annually for all sexually active MSM at risk for infection.4

Chlamydia trachomatis (C. trachomatis) is a gramvnegative, obligate, aerobic, coccoid or rod shape bacteria that does not grow in routine culture. C. trachomatis cannot synthesize ATP and humans are the only known natural host for C. trachomatis.4 Neisseria gonorrhoeae (N. gonorrhoeae) is a Gram-negative, facultatively intracellular, obligate aerobe diplococci. While this organism can be grown in culture, sensitivity is lower compared to routine molecular methods. Co-infection is common, with an estimated 10–40% of patients with gonorrhea also infected with chlamydia, and the data also suggested an interplay between these two pathogens. 5,6,7 Patients with chlamydia and gonorrhea co-infection can have increased gonococcal bacterial load, which might facilitate gonorrhea transmission compared with a single infection. Chlamydia can evade the host immune response by preventing neutrophil extracellular traps (NETs) production, which can help gonorrhea to establish intracellular infection.8 Studies in mice suggest that C. trachomatis induces changes in the genital tract immune environment, making it a more permissive environment for N. gonorrhoeae.9

Appropriate specimens include self- or clinician-collected vaginal swab, endocervical swab, urethral swab, and first catch urine. For chlamydial and gonococcal infection diagnosis, CDC recommends testing by nucleic acid amplification tests (NAATs). NAATs are more sensitive and specific compared to other methods. FDA has approved NAATs for urogenital specimens and only particular platforms are approved for rectal and oropharyngeal specimens. C. trachomatis does not grow in routine culture and diagnosis at this time relies solely on NAAT. For N. gonorrhoeae, culture and antibiotic susceptibility should be evaluated in case of suspected treatment failure. Our lab uses Abbott Real-time CT/NG assay, which is currently FDA approved for testing urogenital specimens only.

CDC recommends treating chlamydia with a seven-day course of doxycycline with sexual abstinence until treatment completion/resolution of symptoms. Azithromycin or levofloxacin can be used as alternatives. For gonorrhea, a single ceftriaxone intramuscular injection is recommended, and gentamicin with azithromycin can be used in case of cephalosporin allergy. Unfortunately, for pharyngeal gonorrhea, there is no reliable alternative available for ceftriaxone allergy. Sexual partner evaluation, testing, and presumptive treatment are recommended, along with patient treatment.10 In cases where the chlamydial infection has not been ruled out, patients should also receive anti-chlamydial therapy. A test-of-cure (follow-up testing) for gonorrhea is required in throat infections only after 14 days of the treatment.10

References:

  1. Kreisel KM, Spicknall IH, Gargano JW, Lewis FM, Lewis RM, Markowitz LE, Roberts H, Satcher Johnson A, Song R, St. Cyr SB, Weston EJ, Torrone EA, Weinstock HS. Sexually transmitted infections among US women and men: Prevalence and incidence estimates, 2018. Sex Transm Dis 2021; in press.
  2. CDC. Sexually Transmitted Disease Surveillance, 2020. Atlanta, GA: Department of Health and Human Services; April 2022.
  3. https://www.cdc.gov/std/chlamydia/stdfact-chlamydia-detailed.
  4. http://dx.doi.org/10.15585/mmwr.mm6950a6external icon

-Paige M.K. Larkin, PhD, D(ABMM), M(ASCP)CM is the Director of Molecular Microbiology and Associate Director of Clinical Microbiology at NorthShore University HealthSystem in Evanston, IL. Her interests include mycology, mycobacteriology, point-of-care testing, and molecular diagnostics, especially next generation sequencing.

Triaging Times

As a clinical instructor and lead cytologist at my institution, I like to remind our newer cytologists and cytology students of the importance of being prepared for FNA biopsies so they develop good habits or best practices as they become more experienced. This level of preparation helps to create a culture of ongoing learning and improvement, which is necessary for the laboratory. In my experience, I’ve met some cytologists who prefer to go into a case blind, with the mindset that knowing the patient’s clinical history in advance muddies their knowledge, skills, and abilities, limiting their mindset by excluding the possibility of other diagnoses. While diving into the unknown might seem exciting, it is also a hindrance and could result in errors, especially when the clinical history helps us triage the patient’s sample. For example, knowing that the patient has a history of lymphoma or that the presentation state includes bulky lymphadenopathy prompts us to collect additional needle passes to send for flow cytometry analysis. Another concern is not knowing whether the patient has a history of breast, gastric, or esophageal cancer, and consequently processing the specimen routinely, which may result in an extended cold ischemic time. This delay in fixation along with insufficient formalin fixation can yield false negatives on ER/PR IHC in breast cancers and HER2 FISH in breast, gastric, and esophageal cancers, which could restrict the use of hormone therapies, such as tamoxifen and aromatase inhibitors for hormone receptor-positive (HR+) cancers, or trastuzumab for HER2+ cancers. I cannot overemphasize the importance of familiarizing yourself with clinical history and communicating case specifics while you act as a mediator between clinician and pathologist.

Whether the clinical history impacts the pre-analytical phase, such as specimen collection (limiting cold ischemic time or collecting additional needle passes for ancillary studies) or the analytical phase, as such processing (formalin fixation) and diagnosis (selecting an appropriate immunoprofile), we must remain vigilant and proactive in laboratory medicine. In this case, knowing the patient’s clinical history was of the utmost significance as it helped to reduce the number of immunostains and ancillary studies necessary to make the diagnosis. Using morphologic criteria in tandem with the patient’s clinical history narrowed the differential diagnoses to just two possible types of cancer, presented below.

A 59 year old male patient presented to the emergency room after an automobile accident. On imaging, the X-ray and CT scan identified a left humerus mass and fracture, and bloodwork was performed. His medical record was sparse and uneventful with no recent visits or encounters. To build a more comprehensive wellness profile and prepare for surgery, he was also offered a one-time screening for Hepatitis C, as an adult who was born between 1945 and 1965.

The left humerus mass was biopsied via CT-scan guidance and two passes were obtained. The Diff-Quik stained smears demonstrate large polygonal cells, some with abundant, granular cytoplasm and some isolated cells with naked nuclei. Vessels also appear to traverse some of the cell groups.

Images 1-2: Bone, Humerus, Left, CT-guided FNA. Diff-Quik-stained smears.

The Pap-stained smears also demonstrate polygonal cells with granular cytoplasm, nuclei with coarse chromatin, and prominent nucleoli. An interesting feature frequently identified in this case is the intranuclear inclusions, and in hindsight, a focus on these may have further reduced the number of immunostains performed.

Images 3-5: Bone, Humerus, Left, CT-guided FNA. Pap-stained smears.

The H&E-stained cell block sections show trabeculae with endothelial wrapping around the cell cords. While renal cell carcinoma was listed as a differential diagnosis due to its telltale oncocytic cytoplasm and vascularity, hepatocellular carcinoma was favored.

Images 6-7: Bone, Humerus, Left, CT-guided FNA. H&E sections (6: 100x, 7: 400x).

Immunostains were performed using proper positive and negative controls on the cell block sections, and the tumor cells show positive staining for Arginase, cam5.2, and Hepar1, while negative staining for CK7 and PAX8 (not shown).

Images 8-10: Bone, Humerus, Left, CT-guided FNA. Cell block section immunohistochemistry. 8: Arginase-positive; 9: cam5.2-positive; 10: Hepar1-positive.

Fortunately, before ordering immunostains, both our cytologist and pathologist working on the case peered into the patient’s medical record and noticed that he had recent bloodwork which demonstrated a positive Hepatitis C screening. This diagnosis was as recent as the identification of his humerus mass. Had it not been for his car accident, I can’t imagine how long he would have gone undiagnosed for both hepatitis and metastatic hepatocellular carcinoma. Incidental findings save lives, folks.

Granted, in settings of unknown primaries with widespread metastatic disease, such as carcinomatosis, an extensive workup is almost always inevitable. Narrowing down possible etiology based on information such as gender, age, and environmental or occupational exposure can help, but that doesn’t always yield a definitive answer as time- or cost-effectively as possible. In this case, that one clue of untreated Hepatitis C was all the cytopathology team needed. A rarity, sure, but as we are asked to do more personalized tests with less material, think of the patient’s specimen as a puzzle and keep your eye out for a clue both under the microscope and behind the computer. You never know what you might find that reduces errors and unnecessary testing while efficiently leading to a definitive diagnosis.

-Taryn Waraksa-Deutsch, MS, SCT(ASCP)CM, CT(IAC), has worked as a cytotechnologist at Fox Chase Cancer Center, in Philadelphia, Pennsylvania, since earning her master’s degree from Thomas Jefferson University in 2014. She is an ASCP board-certified Specialist in Cytotechnology with an additional certification by the International Academy of Cytology (IAC). She is also a 2020 ASCP 40 Under Forty Honoree.

Microbiology Case Study: Severely Immunocompromised Female with Respiratory Failure

Case History

A 50 year old female with a complex medical history consisting of lymphoma, diabetes mellitus (type II), sarcoidosis, congestive heart failure, chronic renal failure (stage 3), and pancytopenia  presented to the emergency department with shortness of breath, cough, fever. She was found to be positive for SARS-CoV-2 and was transferred to the ICU due to hypoxic respiratory failure. She was treated for sepsis and respiratory failure, but her status continued to decline. The patient had multiple admissions due to COVID-19 in the past, received remdesivir and was on corticosteroid therapy due to the interstitial lung disease from last year. Initial evaluation included complete blood count which revealed anemia (hemoglobin=8.7 mg/dl), leukocytosis (WBC = 21,900/mcl), lymphopenia (910/mcl) and thrombocytopenia (Plt = 27000/mcl). The patient was treated with broad antibiotics and additional steroids. Additional tests revealed hyperproteinemia and hypoalbuminemia. Chest x-ray showed worsening infiltrates in lungs and chest CT scan revealed left apical hydropneumothorax, loculated left pleural effusion, pneumomediastinum, and chest wall subcutaneous emphysema. Lung biopsy revealed necrosis. Histopathology examination revealed broad, branching hyphae with sporulation in lung tissue biopsy and bronchoalveolar lavage. Respiratory cultures of lung biopsy and BAL grew rapidly and lactophenol cotton blue tape preps showed broad hyphae with round sporangium and rhizoids between the stolons. The patient was diagnosed with mucormycosis, infection with Rhizomucor, and was treated with Amphotericin B. Surgical debridement of the tissue was not possible due to her declining condition. She passed away after 5 days.

Figure 1. H&E stain of the lung biopsy (top, left) and Papanicolaou stain of bronchoalveolar lavage (top, right) revealed broad, ribbon-like, right-angle branching hyphae (visible in lung biopsy) with sporulation (credits to Dr. Elham Arbzadeh, George Washington University School of Medicine and Health Sciences). Rapid growth was observed from the respiratory cultures of the tissue biopsy by day 2 (bottom, left) where lactophenol cotton blue tape preps showed broad hyphae with sporangium (bottom, right) and intermodal rhizoids (not shown in this image).

Discussion

The term mucormycoses refers to infections caused by the Zygomycetes which is further separated into Mucorales and Entomophthorales. Some of the members of Mucorales are Rhizopus spp., Mucor spp., Lichtheimia (Absidia) spp., Syncephalastrum spp., and Rhizomucor spp.1,2 These organisms live in soil, dung, and vegetative matter. Infection is usually acquired by inhalation/ingestion of their spores or direct inoculation and contamination of wounds. The mold can invade the walls of the blood vessels causing angioinvasion and often results in dissemination of mycotic thrombi and development of systemic infection. Zygomycetes are most commonly known for causing rhinocerebral, pulmonary, cutaneous, and disseminated disease. Infections with Zygomycetes most commonly occur as opportunistic infections in immunocompromised hosts. Risk factors include diabetes, those with acidosis, neutropenia, and sustained immunosuppression such as after transplantation.

Zygomycetes grow very fast (within 48 to 72 hrs.) and is often called a “lid lifter”. The colonies have a wooly mycelium and can be described as cotton candy-like. Lactophenol tape preps of the mold would reveal broad hyphae, aseptate or pauciseptate, ribbon-like hyphae with irregular width. At the tip of the sporangiophore, there is a sack-like structure called a sporangia with contains all the spores. Fungal elements and hyphae seen on tissue biopsies from patients with mucormycosis typically have near right angle branching (usually >40o) broad, non-septate hyphae. In contrary, those with aspergillosis show acute angle branching (usually <45o) with narrow, septate hyphae.3  

Genus-level identification can be achieved by microscopic morphology. Rhizomucor is an intermediate between Rhizopus and Mucor. Rhizoids found in Rhizomucor are few in number and are located on stolons, between the sporangiophores, as opposed to Rhizopus where the rhizoids are often seen directly at the nodes and Mucor which does not produce rhizoids. Sporangia (40-80 µm in diameter) are brown in color and round in shape. Apophysis is absent, which allows for differentiation from Lichtheimia (Absidia) where apophysis can be seen.4 The genus Rhizomucor includes three species: Rhizomucor pusillusRhizomucor miehei, and Rhizomucor tauricus.5

Treatment of mucormycosis consists of antifungal and surgical therapy. Amphotericin B is the most commonly used antifungal agent. Liposomal amphotericin B has also been successfully used in some cases with zygomycosis due to Rhizomucor.6  Early diagnosis and treatment are crucial and mortality rate is high.7  Of note, Zygomycetes are intrinsically resistant to voriconazole.

References

  1. Rippon J W. Medical mycology. The pathogenic fungi and the pathogenic actinomycetes. Philadelphia, Pa: Saunders; 1974. Mucormycosis; pp. 430–447. 
  2. Scholer H J, Müller E. Beziehungen zwischen biochemischer Leistung und Morphologie bei Pilzen aus der Familie der Mucoraceen. Pathol Microbiol. 1966;29:730–741.
  3. Mohindra S., Mohindra S., Gupta, R., Bakshi, J., Gupta, S. K. Rhinocerebral mucormycosis: the disease spectrum in 27 patients. Mycoses. doi: 10.1111/j.1439-0507.2007.01364.x.
  4. de Hoog, G. S., J. Guarro, J. Gene, and M. J. Figueras. 2000. Atlas of Clinical Fungi, 2nd ed, vol. 1. Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands)
  5. Schipper M A A. On the genera Rhizomucor and Parasitella. Stud Mycol. 1978;17:53–71. 
  6. Bjorkholm, M., G. Runarsson, F. Celsing, M. Kalin, B. Petrini, and P. Engervall. 2001. Liposomal amphotericin B and surgery in the successful treatment of invasive pulmonary mucormycosis in a patient with acute T- lymphoblastic leukemia. Scand J Infec Dis. 33:316-319.
  7. Ribes, J. A., C. L. Vanover-Sams, and D. J. Baker. 2000. Zygomycetes in human disease. Clin Microbiol Rev. 13:236-301.

-Maryam Mehdipour Dalivand, MD is a Pathology Resident (PGY-1) at The George Washington University Hospital. She is pursuing AP/CP training.

-Rebecca Yee, PhD, D(ABMM), M(ASCP)CM is the Chief of Microbiology, Director of Clinical Microbiology and Molecular Microbiology Laboratory at the George Washington University Hospital. Her interests include bacteriology, antimicrobial resistance, and development of infectious disease diagnostics.

Answering Your Questions about Monkeypox

I have been hearing many concerned questions about Monkeypox lately, and I wanted to add onto the great job already done by Dan Scungio in his previous post on how laboratorians should be safe around Monkey pox suspected samples. As a part of the queer community, I’ve heard from several people who are very concerned as this is predominately spread among men who have sex with men. I’ll be focusing on what is new about Monkeypox, how it is different, where it is spreading, and what can be done about it so far. I’ll address questions like should we be sequencing Monkeypox like COVID-19 and does your smallpox vaccination will protect you.

What is Monkey Pox?

This is an orthopox virus that is from the same family as smallpox, which was so effectively cleared from human circulation that vaccines were discontinued in the U.S. in the early 1980’s. It causes a systemic disease characterized by lesions that start as a red, flat rash (macula) then form vesicles that break open, crust and resolve in 2-4 weeks. If you ever had chicken pox, you may recall how painful it was, and this is the major symptom that requires medical management.

Figure 1. Transmission electron microscope image of Monkey pox (purple). https://www.cdc.gov/poxvirus/monkeypox/index.html

Is it really that big of a concern?

Initially case increases were attributed to undiagnosed disease just as happened with COVID-19 initially. However, now that commercial labs are testing for it and access to testing is not an issue, we still see case counts increasing. This indicates that the rapid spread is real and concerning. That rapid spread is one reason that it has now been declared a national public health emergency.

How is it tested for?

Initially testing was sent out to one of the CDC regional testing centers. However, there were only 60-70 of these sites and they had limited capacity for high throughput testing. Then Labcorp and Quest they can each perform PCR testing, which has expanded access greatly. However they have different specimen types they accept:

  • Labcorp: Lesion swab in VTM sent frozen or refrigerated (room temp not acceptable)
  • Quest: Lesion swab in VTM

What is new?

  • It has been in Sub-Saharan Africa for a long time.
  • Early summer it began to spread into other continents like Europe.
  • U.S. now has the highest levels of Monkeypox cases.

This all reinforces the impact of communicable diseases in a global society.

Figure 2. Global distribution of cases Jan 1, 2022-Aug 9, 2022. https://www.cdc.gov/poxvirus/monkeypox/response/2022/world-map.html

How are symptoms different?

  • The rash may begin without the typical prodrome symptoms of fever, malaise, etc.
  • Spread occurs by skin to skin contact
Figure 3. Examples of Monkey pox lesions. https://www.cdc.gov/poxvirus/monkeypox/symptoms.html

Does my smallpox vaccine protect me?

  • Smallpox vaccines are 85% effective for 3-5 years
  • Unknown how well they work after many years
  • They likely decrease severity of disease even if it was given many years ago.

What can be done to prevent it?

  • Monkeypox vaccination: requires 2 shots. Space out 4 weeks apart.
  • FDA recently approved 1) decreasing the dose and 2) performing subcutaneous injection.
  • This would increase the effective doses by 5x and still produce a robust immune response.

Should we be sequencing it?

  • There are 3 major clades of the virus with the Congo clade being more severe (10% death rate) than the one we are seeing (West African: 3%)
  • These differences can be found by PCR based tests
  • There is no treatment difference based on the clade.
  • If this continues to spread and mutate, then there could be a reason to sequence the virus.
  • Some evidence suggests the mutation rate is 2x higher than would be expected.
    • However, the last known samples were sequenced >5 years ago and not many were sampled to get a very accurate measure of the mutation rate.
    • So this news about mutation rate should be taken with a grain of salt.

References

Jeff SoRelle, MD is Assistant Instructor of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX working in the Next Generation Sequencing lab. His clinical research interests include understanding how lab medicine impacts transgender healthcare and improving genetic variant interpretation. Follow him on Twitter @Jeff_SoRelle.

Tick Identification: Why Do We Do It and What Does It Tell Us?

During the warmer months here in the Midwest, ticks are abundant and our microbiology lab receives several tick submissions per day for identification. When possible, we provide species level identification as well as sex for any tick submitted. While this is common practice in most microbiology laboratories, our molecular laboratory accidently received a tick specimen and, in the process of routing it to the microbiology lab, was curious as to why the tick identification matters—what does that tell us clinically? This led to an impromptu plate rounds with both labs and prompted me to write this post.


How do we determine tick identity?

A tick is submitted in a cup and sent to the laboratory. Ideally the tick would be submitted whole without missing appendages or damaged in any way. The tick is placed in ethanol to kill the organism and to allow for examination under a microscope. The mouth parts, scutum, and festoons are examined for defining features. Thorough examination is challenging when the tick arrives damaged or only partially intact.

Why do we provide tick identification?

Certain ticks carry specific pathogens. For instance, Amblyomma americanum (lone star tick) can transmit ehrlichiosis, Francisella tularensis, Heartland virus, Bourbon virus, and Southern tick-associated rash illness, while Ixodes scapularis can transmit Borrelia burgdorferi & Borrelia mayonii (both are causative agents of Lyme disease), Anaplasma phagocytophilum, and Erhlicia muris as well as Powassan virus. Knowing which tick that the patient was bitten by can allow providers to understand what potential pathogens they may or may not have been exposed to. If Amblyomma americanum is submitted, for example, that tick does not carry Borrelia burgdorferi. However, it is important to note that the majority of patients who develop tick-borne illness have no recollection of a tick bite! So while one tick may be discovered and sent to the lab, the patient could still have been unknowingly bitten by a different tick, which could carry other pathogens. When a patient exhibits clinical symptoms that are consistent with a tick-borne disease, such as Lyme Disease, the patient should be tested for that disease regardless of their tick history.

The patient has an Ixodes tick! They are worried about Lyme Disease. Should we send the tick out for molecular testing?

We discourage the use of molecular testing on the ticks themselves because ticks carry a variety of pathogens and there is a high likelihood of carrying a particular pathogen in a high prevalence area. For Ixodes ticks in Lyme Disease endemic areas, 15-70% of ticks may carry the causative agent, Borrelia burgdorferi. However, just because a tick carries a particular pathogen, it does not mean that the patient is now infected. This can lead to unnecessary treatment and misdiagnosis. Moreover, ticks must feed for a certain amount of time before pathogens can be transmitted. For example, Ixodes ticks must typically feed for more than 24 hours before it can transmit Lyme Disease or other pathogens.

Image 1. A male Dermacentor variabilis (also known as the American dog tick) submitted by one of our patients.

In summary, tick identification can provide a glimpse into what the patient was potentially exposed to and if symptoms do arise days to weeks later, the tick identification may offer additional clues. However, just because a person was bitten by a tick does not mean that they are infected. Identification is just a piece of the puzzle!

References

-Paige M.K. Larkin, PhD, D(ABMM), M(ASCP)CM is the Director of Molecular Microbiology and Associate Director of Clinical Microbiology at NorthShore University HealthSystem in Evanston, IL. Her interests include mycology, mycobacteriology, point-of-care testing, and molecular diagnostics, especially next generation sequencing.

Lymphocyte Subset Panels (AKA T4T8 Assays)

While writing my last blog, I asked “What is your least favorite test to do in Hematology?” (I’m not ignoring our favorite tests! I will get to those in another blog.) And then, I started thinking about why we may not like certain testing. Is it because they are time consuming, or repetitive? Is it because they hurt our eyes, or necks, or fingers? Or is it because it’s a test that we perform but we may not be sure what the test is for, or we don’t understand the theory behind it? I started thinking about my coworkers and other tests that could be on those lists and I immediately decided that a good candidate in our lab is the T4T8 panel. Probably the primary reason is that the instrument we do these on has given us many problems over the last year. The instrument has spent most of the year with an “instrument out of service” sign on it. Service has been here many times, but the instrument just appears to have exceeded its life expectancy. In normal times, when the instrument was in its prime, setting up and running a T4T8 panel does require a number of steps, and some time. In the last year we have had to add lots of coaxing, even more time, and some luck to get the test to run. This can be frustrating in any lab situation, but is particularly frustrating when we are short staffed, trying to train new staff, and very busy. So, I don’t think it’s the test itself that techs dislike, it’s the time it takes, not being comfortable with setting up the test, juggling our other work while struggling with another instrument, and the fact that even after we get results, a percentage of the samples have results that don’t meet our criteria and still need to be sent out to the reference lab. 

Another reason why this test may be a little intimidating is its unfamiliarity. It’s not a test that is done in every lab. I have worked as a Medical Laboratory Scientist for many years. I’ve worked in 6 labs since the mid 1980’s and the introduction of CD4 testing for human immunodeficiency virus (HIV) patients, yet my current place of work is the first place that we have done these in house. Before this job, if you asked me or any of my coworkers what a T4T8 panel was, we probably would have answered “a send out test”. A few weeks ago, we had a call from a doctor asking questions about his patient’s T4T8 assay results. The tech answering the phone got a blank look on their face and quickly handed the phone to me. This told me that techs, and even doctors, may not really understand what this test is testing and what the results mean. This further confirmed to me that the lack of knowledge about these tests may be another reason why these don’t win any popularity contests in our lab.

So, what exactly is a T4T8 panel?

Some other names for the test are a Lymphocyte subset panel, an Immuno T-cell (CD3/4/8) assay, T-Cell subsets Percent and Absolute panel or T-Lymphocyte Helper/Suppressor Panel. As a quick review, we know that lymphocytes are either B-lymphocytes or T-lymphocytes. Immunotyping lymphocytes can provide information for disease diagnosis and monitoring. All T-lymphocytes express CD3 antigens on their surfaces, which can be used to differentiate B-cell disorders from T-cell disorders. T-lymphocyte subsets include T-helper/inducer cells which express both CD3 and CD4, and T-cytotoxic/suppressor cells, which express CD3 and CD8. In a T4T8 panel we are concerned with identifying T-lymphocytes, and the percentage of each subset both individually, and compared to one another.

The test we perform uses monoclonal antibodies, anti CD3, anti CD4 and anti CD8, which recognize specific human lymphocyte subsets. Our reagents come as antibody containing tubes and are run on the Cell-Dyn Sapphire. After performing a CBC on the sample, the instrument is programmed to add an aliquot of the sample to the CD3 +CD4 reagent tube and a second aliquot to the CD3 + CD8 reagent tube. Immunophenotyping is performed by flow cytometry on these 2 aliquot tubes. The CD3 antibody in both tubes separates out all T-lymphocytes, and the addition of the CD4 in the first tube identifies the cells which are also CD4 positive, the T4 or helper cells. The CD3 + CD8 tubes identifies the percentage of T cells that are T8 or suppressor cells. The assay uses the CBC results and the immunophenotyping runs to calculate the helper/suppressor ratio, also known as T4/T8 ratio or CD4/CD8 ratio.

Why is this test performed?

After the discovery of lymphocyte subset abnormalities in human immunodeficiency virus (HIV) patients in the 1980s, lymphocyte immunophenotyping has become widely used in this patient population for the evaluation of their prognosis, immune deficiency status, response to therapy, and diagnosis of AIDS. The test is most often done to assess HIV infection status but may also be useful in the diagnosis and monitoring of other diseases or after organ transplantation. Some examples of conditions in which this assay may be useful include other viral and bacterial infections, severe combined immunodeficiency, Hodgkin disease, certain leukemias, multiple sclerosis, and myasthenia gravis. A newer application of CD4/CD8 ratios are as potential biomarkers of cancer progression. The most interesting new use of T-cell subset testing that I have read about has been with the recent COVID-19 pandemic. Several studies have shown that CD4 and CD8 T- cell counts reflected disease severity and can predict clinical outcomes of COVID-19 infection. These studies have concluded that COVID-19 patients presenting with relatively low CD4 and CD8 T-cell counts are more severely infected and may have a worse prognosis. The Abbott test we use was designed to be used to monitor immune status in (HIV)-infected individuals. It is not intended for screening for leukemic cells or for phenotyping samples in leukemia patients.

What do the results mean?

The absolute CD4 count and CD4/CD8 Ratio can be used as a snapshot of immune system health. Normal absolute CD4 counts are 600 to 1200 /mm3. In immune suppression, values drop below 500/mm3 and in advanced infection, values of less than 200/mm3 are consistent with a definition of acquired immunodeficiency syndrome (AIDS). In advanced disease, some patients may have a normal CD4 count but experience a weakening immune system. Or the immune system can become exhausted and unable to produce sufficient T-cells. The CD4/CD8 ratio is useful for judging the strength of the immune system. A normal CD4/CD8 ratio is between 1.0 and about 3.0-4.0.

 T-helper cells start the defensive immune response by signaling other cells that infectious pathogens are present. At initial infection with HIV, T-suppressor cells increase in an effort to destroy infected cells. We see an increase in CD8 cells as the CD4 cells are destroyed. These events result in a low CD4/CD8 ratio. When HIV antiretroviral therapy (ART) is initiated, the ratio will usually, gradually return to normal. However, if ART is not started or if the immune system is severely affected, the body may not be able to make adequate new CD4 cells and the ratio may never return to normal.

With the availability of very effective therapies available for the treatment of HIV, the CD4/CD8 ratio has become more important in patients with long term HIV infection. Recent studies have suggested that people with a low CD4/CD8 ratio who have been on treatment for years are at an increased risk from non-HIV illnesses such as cardiovascular and renal disease.

CD4 counts are important in HIV management and used to guide treatment including the decision to initiate prophylactic treatment against opportunistic infections. It is recommended that CD4 counts be performed every 3-6 months after initiation of ART. After the first 2 years on ART, CD4 monitoring can be decreased in frequency to every 12 months for people whose CD4 count is between 300 and 500 and may be considered optional for those with CD4 counts over 500. Table 1 and 2 shown below are examples of patient reports for the T4T8 assay.

Table 1. Patient with AIDS, CD4 count 200, T4/T8 ratio 0.16*
Table 2. Patient with absolute CD4 within normal range, but CD4/CD8 below 1.0*

*There are times when the absolute or % CD3T may be less than the sum of the CD4T and CD8T. This is due to averaging of CD3T counts from the 2 monoclonal tubes

In our lab, these tests are performed daily, as they are received, from 7am to 7PM, 7 days a week. There are no commercial quality control materials available for the test, so we must choose negative and positive QC from our patient population. For the QC we choose patients with CBC and WBC differential values within normal ranges, with no flags. There are additional age and diagnosis/treatment related restrictions on samples that can be used as controls. Our in-house patients often have abnormal results, and our patient population also includes our large outpatient hematology/oncology center. Thus, at times, finding appropriate controls can be challenging. I can add this to the list of ‘problems’ with this test and why techs don’t like them. Call me weird, but I actually like doing these! I like the challenge of finding QC, I like that they are ‘different’ from the hundreds of CBCs we perform each day, and I look at them as a little change in routine and a chance to do something unique. Though I wish the instrument would run perfectly every day, I even (sort of) don’t mind troubleshooting when it’s not working. I like solving problems! I enjoy teaching others how to run these, and I enjoy answering questions about the test.

Many thanks to my great coworker Jacky Olive for her assistance always and inspiration for this blog. I know these are not your favorite test!

*There are times when the absolute or % CD3T may be less than the sum of the CD4T and CD8T. This is due to averaging of CD3T counts from the 2 monoclonal tubes

Becky Socha MS, MLS(ASCP)CMBB

References

  • Abbott Laboratories, Cell Dyn Immuno T-Cell (Cd3/4/8 )ReagentsPackage Insert. Abbott Park, Il.
  • Li Raymund; Duffee Doug; Gbadamosi-Akindele Maryam F.CD4 Count. NIH National Library of Medicine. May 8, 2022
  • Domínguez-Domínguez L, Rava M, Bisbal O, et al. Cohort of the Spanish HIV/AIDS Research Network (CoRIS). Low CD4/CD8 ratio is associated with increased morbidity and mortality in late and non-late presenters: results from a multicentre cohort study, 2004-2018. BMC Infect Dis. 2022 Apr 15;22(1):379.
  • Liu Z, Long W, Tu M et al. Lymphocyte subset (CD4+, CD8+) counts reflect the severity of infection and predict the clinical outcomes in patients with COVID-19. Journal of Infection. Vol 81, Issue 2. P318-356, AUGUST 01, 2020
  • Kagan JM, Sanchez AM, Landay A, Denny TN. A Brief Chronicle of CD4 as a Biomarker for HIV/AIDS: A Tribute to the Memory of John L. Fahey. For Immunopathol Dis Therap. 2015;6(1-2):55-64
  • McBride JA, Striker R (2017) Imbalance in the game of T cells: What can the CD4/CD8 T-cell ratio tell us about HIV and health? PLoS Pathog 13(11)
  • Sinha A, Mystakelis H, Rivera AS, Manion M, et al. Association of Low CD4/CD8 Ratio With Adverse Cardiac Mechanics in Lymphopenic HIV-Infected Adults. J Acquir Immune Defic Syndr. 2020 Dec 1;85(4)
  • Wang YY, Zhou N, Liu HS, Gong XL, Zhu R, Li XY, Sun Z, Zong XH, Li NN, Meng CT, Bai CM, Li TS. Circulating activated lymphocyte subsets as potential blood biomarkers of cancer progression. Cancer Med. 2020 Jul;9(14)
Socha-small

-Becky Socha, MS, MLS(ASCP)CMBBCM graduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 40 years and has taught as an adjunct faculty member at Merrimack College, UMass Lowell and Stevenson University for over 20 years.  She has worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. She currently works at Mercy Medical Center in Baltimore, Md. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.