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.

Thyroid Trifecta

A patient with no previous cancer history presented to the Head and Neck Clinic after imaging performed for radial nerve palsy detected multiple (4) thyroid nodules. The 1.9 centimeter isthmus nodule met biopsy criteria due to its size, hypoechogenicity, solid composition, and punctate echogenic foci, placing the nodule into a TI-RADS risk category of TR5. An ultrasound-guided fine needle aspiration (FNA) was performed, and Rapid Onsite Evaluation (ROSE) determined that the sample contained atypical follicular cells. Two additional passes were collected in Veracyte Afirma fixative to be reflexed for a Gene Sequencing Classifier in the event of an indeterminate final diagnosis, such as follicular lesion of undetermined significance (FLUS), atypia of undetermined significance (AUS), or suspicious for follicular neoplasm. The Diff-Quik smears, featuring nuclear inclusions and papillary formations, are presented below.

Images 1-2. Thyroid, Isthmus, Ultrasound-guided FNA. Diff-Quik-stained smears.

The following day, the Pap-stained smears demonstrated more pronounced cytologic features of papillary thyroid carcinoma, including nuclear invaginations, nuclear grooves, papillary clusters, and limited colloid. Rare tumor cells were also identified on cell block sections (not shown), and immunohistochemical (IHC) stains showed that the cells of interest were positive for thyroglobulin and TTF-1. Although there were a few pleomorphic and histiocytic areas on the smears which appeared different than classic papillary thyroid carcinoma, the thyroid isthmus FNA was signed out as papillary thyroid carcinoma (Bethesda Category VI), and correlation with clinical and radiological findings was recommended.

Images 3-5. Thyroid, Isthmus, Ultrasound-guided FNA. Pap-stained smears.

Due to the final cytology diagnosis, the patient was scheduled for a total thyroidectomy and possible neck lymph node dissection within three weeks of the initial biopsy. During the total thyroidectomy, the 2 centimeter isthmus nodule was noted, and there was no gross evidence of extrathyroidal extension or suspicious lymphadenopathy, as this was diagnosed as non-invasive follicular thyroid neoplasm with papillary like nuclear features (NIFTP). Adjacent to the NIFTP, but still within the isthmus, was a 1.6 cm hyalinizing trabecular tumor, which alludes to the other cells of interest identified on the FNA smears. No tumor was identified in three lymph nodes, and a less than 0.1 cm incidental micropapillary thyroid carcinoma (follicular variant) was found. The margins were negative for all three elements.

Images 6-8: Thyroid, Isthmus, Resection. H&E sections (6: 40x, 7: 100x, 8: 400x).

IHC was performed on the dominant isthmus nodule showing that the tumor cells are positive for TTF-1, thyroglobulin, and PAX-8, while negative for calcitonin. Ki-67/MIB1 at 37 degrees Celsius demonstrated no membranous staining, supporting the diagnosis of NIFTP. The same stains were performed on the neighboring 1.6 isthmus tumor with all of the immunostains yielding the same results, except positivity in Ki-67/MIB1. This single stain differentiates a diagnosis of hyalinizing trabecular tumor, a very rare (less than 1%) follicular-derived thyroid tumor that has strikingly similar features to papillary thyroid carcinoma and NIFTP.1 While hyalinizing trabecular tumors are typically a histologic diagnosis due to the solid trabecular growth pattern, hyalinization of extracellular spaces and lack of vascular or capsular invasion, careful cytologic analysis could postulate the presence of this tumor especially in FNAs that do not demonstrate classic papillary thyroid carcinoma features.1 It is important to note, especially in hindsight thanks to this case, that the histiocytic cells identified on the FNA smears had elongated nuclei with abundant cytoplasm and cells radially arranged around hyaline globules. While nuclear grooves and intranuclear inclusions are prominent in both tumors, the nuclear shape and presence of hyaline can help cytologists morphologically distinguish between papillary thyroid carcinoma and hyalinizing trabecular tumors and trigger the inclusion of MIB1 IHC staining (LiVolsi, 2022).

References

  1. LiVolsi VA. (17 May 2022). Hyalinizing trabecular tumor. PathologyOutlines.com website. https://www.pathologyoutlines.com/topic/thyroidhtn.html. Accessed May 27th, 2022.

-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: A 47 Year Old with Eye Pain and Redness

Case history

A 47 year old male with an extensive ocular history including laser assisted in situ keratomileusis (LASIK), multiple ocular traumas with repair, and myopic degeneration with neovascularization for which he was prescribed hard and soft contact lenses presented for bilateral eye redness, watering, and stinging pain. Recently, he had forgotten his soft contacts and wore his hard lenses to his job in construction which he reported doing once every 1-2 months. Ocular exam revealed only his usual chronic changes. His symptoms improved with moxifloxacin eyedrops, but never fully resolved. A month later he returned with what was initially assessed as diffuse corneal edema and conjunctival injection in his left eye, but no ring infiltrate or epithelial defect. Two days later, a large epithelial defect with surrounding ring infiltrate and hypopyon (settling of white blood cells at the base of the anterior chamber) developed in his left eye. Confocal microscopy showed findings concerning for Acanthamoeba infection and the contact lenses and case were sent for culture. Environmental organisms including Klebsiella varicola, Chryseobacterium gleum, and Pseudomonas fluorescens were recovered. In addition, cultures for Acanthamoeba sp., where sample is overlaid on a lawn of E. coli grown on a non-nutrient agar plate, were sent to a reference laboratory.

The patient was treated with Brolene, polyhexamide biguanide (PHMB), and chlorhexidine for Acanthamoeba as well as with antibacterial agents. Three months later his LASIK flap failed and was removed and sent for cultures and pathology which both grew Acanthamoeba sp.(Image 1). He continued treatment for another two months, but the corneal defect expanded. He underwent a therapeutic penetrating keratoplasty, and the explant cornea was sent for pathology. Sections showed acute and chronic inflammation of the corneal epithelium and stroma with rare cysts of Acanthamoeba with atypical morphology possibly representing treatment effect or nonviable organisms (Image 2). The patient continued treatment for another month afterward with resolution of symptoms.

Image 1. Representative photomicrographs of cornea with multiple Acanthamoeba cyst forms at differing stages of development (H&E, 400x magnification) and trophozoite with associated acute inflammation (inset, 500x magnification, oil immersion).
Image 2. Photomicrograph of this patient’s explanted LASIK flap. A) Low power magnification demonstrating acute and chronic inflammation in a background of degrading corneal tissue. An empty cyst is highlighted by the arrowhead (H&E, 100x magnification). B and C) High power magnification of likely nonviable cysts indicated by the arrowheads (H&E, 400x magnification).

Discussion

Acanthamoeba sp. are free-living amoebae found ubiquitously in the environment including in water, soil, dust, and air conditioning ducts.1 Over 20 species of Acanthamoeba have been identified, with eight known to cause human disease. A. castellani and A. polyphaga are the most common species identified from clinical infections.2 Acanthamoeba sp. are a primary reservoir of Legionella pneumophilia and can serve as vectors for other bacterial infections.3 These organisms may colonize the nasal passages of normal hosts.4 Acanthamoebal infections have varied clinical presentations depending on the route of transmission, organ(s) infected, and immune status of the host. These include amebic keratitis, granulomatous amebic encephalitis, and disseminated disease.3 Of these, Acanthamoeba keratitis (AK) is the most frequently encountered clinically.

AK can occur when the organisms are inoculated into corneal micro-abrasions, most often from contaminated hard contact lenses rinsed with tap water. AK represents 5% of all cases of contact-lens-associated keratitis, and 70-85% of AK cases are associated with contact lens use.1 Diagnosis of AK is heavily dependent on a high index of suspicion as AK presents with nonspecific ocular symptomology including blurred vision, photophobia, inflammation, and eye pain. A corneal ring infiltrate is characteristic, but only present in 50% of cases.1 Although historically culture is the gold standard for diagnosis, advanced technologies like confocal microscopy and PCR have greatly improved sensitivity and time to diagnosis.5 Cultures are usually grown on agar plates coated with gram negative bacilli such as E. coli.2 If Acanthamoeba are present, trails of bacterial clearing can usually be seen within days but may take up to several weeks.2 They have dormant cyst and active trophozoite forms. Microscopically they appear as round heterogeneous bodies with a distinct nucleus and surrounded by ruffled membrane and are 15-35 μm in length.3 PCR, given its analytical sensitivity, specificity and turn around time, is the more common method of diagnosis of AK and has replaced many instances of culture today.

AK has a poor prognosis and is potentially sight threatening. Factors contributing to disease severity include delayed diagnosis, pathogenic factors, and lack of effective medical management.1 Nearly 40% of patients fail initial therapy.1 Factors that contribute to Acanthamoeba pathogenicity include production of enzymes including elastases and proteases, adhesion molecules, and physiologic tolerance to different temperatures, osmolarities, and pH.6 The cyst stage confers resilience to many therapies which is compounded by poor tissue penetration of the antimicrobial agents often used in therapy.6 Repeated exposure to therapeutic antimicrobials can also lead to the development of resistance.6 In our patient’s case, treatment was successful following the LASIK flap removal, facilitating increased drug penetration and supported by pathologic findings of treatment effect in the explanted cornea.

References

  1. Somani SN, Ronquillo Y, Moshirfar M. Acanthamoeba Keratitis. 2021 Aug 11. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan–. PMID: 31751053.
  2. Maycock NJ, Jayaswal R. Update on Acanthamoeba Keratitis: Diagnosis, Treatment, and Outcomes. Cornea. 2016 May;35(5):713-20. doi: 10.1097/ICO.0000000000000804. PMID: 26989955.
  3. Marciano-Cabral F, Cabral G. Acanthamoeba sp. as agents of disease in humans. Clin Microbiol Rev. 2003 Apr;16(2):273-307. doi: 10.1128/CMR.16.2.273-307.2003. PMID: 12692099; PMCID: PMC153146.
  4. Clarke B, Sinha A, Parmar DN, Sykakis E. Advances in the diagnosis and treatment of Acanthamoeba keratitis. J Ophthalmol. 2012;2012:484892. doi: 10.1155/2012/484892. PMID: 23304449; PMCID: PMC3529450.
  5. Hoffman, J.J., Dart, J.K.G., De, S.K. et al. Comparison of culture, confocal microscopy and PCR in routine hospital use for microbial keratitis diagnosis. Eye (2021). https://doi.org/10.1038/s41433-021-01812-7
  6. Lorenzo-Morales J, Khan NA, Walochnik J. An update on Acanthamoeba keratitis: diagnosis, pathogenesis and treatment. Parasite. 2015;22:10. doi: 10.1051/parasite/2015010. PMID: 25687209; PMCID: PMC4330640.

-Tim Kirtek is a fourth year AP/CP resident at UT Southwestern Medical Center in Dallas, Texas.

-Dominick Cavuoti is a professor at UT Southwestern Medical Center who practices Medical Microbiology, Cytology and Infectious Disease Pathology.

-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.

Lab Safety: It’s Not Monkey Business

The monkeypox virus is poorly named. The actual source of the virus is unknown, although it is possible that African rodents and non-human primates (like monkeys) might harbor the virus and infect people. Either way, the virus has entered the United States again recently and has caused new safety concerns for laboratories around the country.

As with the novel Coronavirus pandemic, the monkeypox outbreak has created new safety concerns among laboratorians. How easily can this be transmitted? How should samples be handled or packaged for transport? Will this create a critical lab staffing shortage? How should waste be treated? It is vital that lab leaders and safety professionals answer these questions for staff and relay as much information as possible to allay unnecessary fears.

First, one of the most important areas of focus for laboratorians potentially working with monkeypox patient samples is to continue to utilize Standard Precautions. As always, all specimens in the lab setting need to be treated as if infectious. When handling standard clinical specimens (blood, body fluids, etc.) from suspected monkeypox patients, no extra safety precautions or PPE should be necessary in the lab. The quantity of pox virus likely to be in clinical specimens is low, although procedures that generate aerosols should always be avoided.

Laboratory staff should also be trained to package and ship Category B specimens. The current West African strain (clade) of monkeypox in the U.S. is not considered Category A under the Hazardous Materials Regulations (HMR), so monkeypox swab specimens for virus testing should be shipped similarly to other clinical specimens. Use the packaging kit and follow the instructions from the receiving testing lab.

There may be concerns about the spread of monkeypox infection among employees in the laboratory. Any infected employee should be using PPE when working in the department, and the monkeypox virus is only spread by close physical contact, direct contact with the infectious rash, scabs, or body fluids, and touching items (such as clothing or linens) that previously touched the infectious rash or body fluids. If there was contact with infected PPE or if an employee had prolonged face-to-face contact with an infected co-worker, that should be reported. The CDC states that monkeypox can spread from the time symptoms start until the rash has fully healed and a fresh layer of skin has formed. The illness typically lasts 2-4 weeks. People who do not have monkeypox symptoms cannot spread the virus to others. Direct any concerns to the employee health practitioners.

Laboratories should have an emergency management plan in place which includes how to handle staffing shortages. That plan may include sending routine testing to an alternate location, using point-of-care testing or reducing services to a limited test menu. In most laboratories, however, this monkeypox outbreak is unlikely to create a massive staffing outage. The virus does not spread quickly or in public, and a pandemic of monkeypox is not expected.

Handling monkeypox waste is another consideration for laboratories. Normally, the waste associated with monkeypox virus is considered a Category A waste (waste contaminated with a known highly infectious substance). However, waste from patients infected with the current West African strain of monkeypox is considered exempt from the category A Infectious Substance Regulations according to the Department of Transportation. It can be managed as regulated medical waste. Soiled laundry, including lab coats, should never be shaken or handled in manner that may disperse infectious particles. Laundry should be contained (bagged) at the point of use. Organizations should contact their local public health authority for more information if needed. As the past few years have shown, new threats will continue to emerge, and they will raise safety questions in the laboratory. As always, laboratorians should stay vigilant, pay attention to the work they do every day to avoid injuries and exposures when handling any specimens. Communicate with the hospital departments to ensure proper internal specimen transport of clinical and diagnostic (swab) specimens. Handling laboratory specimens has never been monkey business- the use of Standard Precautions and safe work practices will keep employees safe through this outbreak, and for whatever comes next.

Dan Scungio, MT(ASCP), SLS, CQA (ASQ) has over 25 years experience as a certified medical technologist. Today he is the Laboratory Safety Officer for Sentara Healthcare, a system of seven hospitals and over 20 laboratories and draw sites in the Tidewater area of Virginia. He is also known as Dan the Lab Safety Man, a lab safety consultant, educator, and trainer.

Microbiology Case Study: A 75 Year Old Found Unresponsive

A 75 year old female with a past medical history of coronary artery disease, hypertension, pre-diabetes mellitus, chronic obstructive pulmonary disease, prior left lobe cavitary lesion of unknown etiology, and tobacco use presented to the ED after being found nonresponsive on the couch. Family reports the patient said she had emesis the night before and felt as if she had a “stomach bug”. MRI shows T2 hyperintensities in the right MCA distribution. CSF results as follows.

White Blood Cells300
Red Blood Cells12
Protein990
Glucose79
Cryptococcal antigenNegative
Fungal cultureNo fungi isolated
HSVNegative

Laboratory findings

CSF was sent to the microbiology lab for bacterial and fungal smears and cultures. No fungi were identified. Cryptococcal antigen was negative. HSV was also negative. CSF Gram stain shows gram positive bacilli. CSF culture showed a small, white, smooth, translucent appearance on sheep blood agar. In semi-solid agar after overnight incubation at room temperature, an umbrella shaped pattern of motility was seen. The organism was identified as Listeria monocytogenes by MALDI-TOF mass spectrometry.

Image 1. Listeria monocytogenes on sheep blood agar.
Image 2. Listeria monocytogenes showing “umbrella zone” pattern of motility on semi-solid agar.

Discussion

Listeria spp. is a genus of gram positive, aerobic, facultative intracellular, catalase positive bacteria. Listeria monocytogenes is a common colonizer in the environment (animals, soil, vegetable matter) and occasionally colonizes the human gastrointestinal tract. Listeria prefers colder environments and can be found as a food contaminant, most notably in milk, raw vegetables, cheese, and meats. In addition, colonized mothers can pass Listeria monocytogenes to the fetus.1

Listeria monocytogenes has 3 notable virulence factors:2

  1. Listeriololysin O: a hemolytic toxin that allows for survival within phagocytes
  2. Act A: induces actin polymerization that facilitate cell-to-cell spread
  3. Siderophores: organisms capable of scavenging iron from human transferrin to enhance cell growth

Neonates, immunocompromised individuals, and the elderly are more likely to acquire infection. Infection can present as bacteremia and CNS infections including meningitis, encephalitis, brain abscesses, and spinal cord infections. Listeria monocytogenes is the 3rd most common cause of meningitis behind Streptococcus pneumoniae and Neisseria Meningitidis. In neonates, an in-utero infection can cause granulomatous infantisepticum leading to systemic infection and stillbirth.3 Listeria monocytogenes can also present as gastroenteritis.

References

  1. Allerberger F. Listeria: growth, phenotypic differentiation and molecular microbiology. FEMS Immunol Med Microbiol. 2003;35(3):183-189. doi:10.1016/S0928-8244(02)00447-9
  2. Bailey & Scott’s Diagnostic Microbiology – Elsevier eBook on VitalSource, 14th Edition – 9780323433792. https://evolve.elsevier.com/cs/product/9780323433792?role=student
  3. Engelen-Lee JY, Koopmans MM, Brouwer MC, Aronica E, van de Beek D. Histopathology of Listeria Meningitis. Journal of Neuropathology & Experimental Neurology. 2018;77(10):950-957. doi:10.1093/jnen/nly077

-Nicholas Taylor, DO is a 1st year anatomic and clinical pathology resident 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: Young Male Patient Presenting with Severe Hip Pain

Case History

A young man presented to the emergency department with the primary complaint of severe right hip pain persisting for 2 days. This pain began after standing uncomfortably for hours at an event. His right hip was tender to palpation with some erythema and swelling. He had no recent fall or known injury. He denied recent fever, chills, aches, constipation, diarrhea, changes in urinary habits, chest pain, and shortness of breath. An ultrasound (US) of the right hip joint showed moderate amount of effusion (Image 1). Laboratory results also showed an elevated white blood cell count (WBC).

Image 1. US of the right hip joint showing moderate effusion.

The patient underwent incision and drainage of his right hip to relieve the swelling, and samples were collected for Gram stain and aerobic and anaerobic bacterial culture. Blood was also collected from two separate sites for culture. The blood cultures showed no growth following 5 days of incubation. Gram stain of the effusion showed 4+ WBC with no organisms seen (Image 2). The joint fluid was set up for aerobic and anaerobic bacterial culture. Anaerobic bacterial culture showed no organism growth. However, a few small, grey-ish mucoid colonies grew in the first quadrant on Chocolate agar in the aerobic culture (Image 3). Gram stain was performed on one of the colonies (Image 4). MALDI-TOF confirmed the identification of Neisseria gonorrhoeae.  

Image 2. Gram stain of effusion obtained from the right hip joint showing increased WBCs.
Image 3: Left) Bacterial growth on the Chocolate agar plate. Right) Bacterial growth subcultured on another Chocolate agar plate.
Image 4: Gram stain of one of the colonies that grew on the Chocolate agar. The Gram stain showed organisms shaped like coffee-beans.

Discussion

Neisseria gonorrhoeae is a fastidious, gram negative diplococci bacteria that can grow inside neutrophils after surviving phagocytosis. It is oxidase positive and aerobic, and generally transmitted through sexual contact such as vaginal, anal, or oral sex.1,2 After a gonococcal infection has resolved, the patient does not develop immunity to future infections from the bacteria. Reinfection is possible due to its ability to evade the immune system by varying its surface proteins, therefore making it appear novel to the immune system.3 Signs of septic arthritis include chills and fever, pain at the joint, inability to move infected joint, erythema, and swelling.4

Multiple factors increase the risk of septic arthritis, including a systemic blood-borne infection, IV drug use, osteoarthritis, past history of septic arthritis, rheumatoid arthritis, alcoholism, diabetes, HIV, lung or liver disorders, old age, and a suppressed immune system.4 Other forms of gonococcal infection are genitourinary infections, which are the most common, disseminated gonococcemia, and gonococcal ophthalmia neonatorum. Genitourinary infections can be particularly dangerous in women if left untreated, as this can lead to pelvic inflammatory disease that could result in infertility due to scarring of the fallopian tubes.5,6 Gonococcal infections of the eyes are one of the leading cause of blindness in neonates in the United States, but can be successfully prevented through treating the mother with antibiotics before birth and administration of eye drops to the baby at birth.7

Identification of N. gonorrhoeae can be done using Gram stain, aerobic bacterial culture on Chocolate or Modified Thayer-Martin (MTM) agar, or nucleic acid amplification test (NAAT). Testing can be done from a urethral swab, urine sample, or sample of body fluid from the area of suspected infection.8,9 Culture is slow with low recovery rates. In urogenital cases, where there is ample colonization of normal flora, genital flora may outgrow N. gonorrhoeae, reducing its recovery. MTM media is useful because it is a GC agar base that makes it selective for N. gonorrhoeae growth. It contains vancomycin, colistin, nystatin, and trimethoprim lactate, which suppresses growth of most other gramgnegative diplococci, gram negative bacilli, gram positive organisms, and yeast.10 The most common testing methodology for urogenital gonococcal infection is NAAT. Some FDA approved platforms also accept rectal or throat samples, however most only accept those from urogenital sources.11 Also, while NAAT is a quick and sensitive diagnostic test, it has the downside of not being able to distinguish between DNA obtained from living or dead bacteria.12

Intravenous (IV) or intramuscular (IM) ceftriaxone is the preferred treatment choice for N. gonorrhoeae infections. Alternatively, other third generation cephalosporins can be used as well, including cefotaxmine and ceftizoxime. Typically, patients with a beta-lactam allergy have been shown to tolerate ceftriaxone, and those who cannot should undergo desensitization due to its effectiveness against this infection. A single dose of azithromycin or a prescription of doxycycline taken twice daily for a week is usually added to the regimen to cover for a potential Chlamydia trachomatis co-infection. Patients presenting with purulent arthritis should also undergo drainage, either arthroscopically or through multiple joint aspirations.13,14

References:

  1. Ryan, K. J., Ray, G., and Sherris, J. C. (2004). Sherris Medical Microbiology: An introduction to Infectious Diseases, 4th edition. McGraw-Hill Medical.
  2. 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.
  3. Hill, S. A., Masters, T. L., and Wachter, J. Gonorrhea – an evolving disease of the new millennium. Microb Cell. 2016; 3(9): 371-89.
  4. Johns Hopkins Medicine. Septic Arthritis [Internet]. 2022. Available from: https://www.hopkinsmedicine.org/health/conditions-and-diseases/arthritis/septic-arthritis. Cited 2022 March 21.
  5. Levinson, W. (2014). Review of Medical Microbiology and Immunology, 13th edition. McGraw-Hill Medical.
  6. Jennings, L. K. and Krywko. D. M. Pelvic Inflammatory Disease. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021. Available from: https://www.ncbi.nlm.nih.gov/books/NBK499959/.
  7. Castro Ochoa, K. J. and Mendez, M. D. Ophthalmia Neonatorum. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022. Available from: https://www.ncbi.nlm.nih.gov/books/NBK551572/.
  8. Ng, L.-K. and Martin, I. E. The Laboratory Diagnosis of Neisseria gonorrhoeae. Canadian Journal of Infectious Disease and Medical Microbiology. 2005; 16: 1-11. Article ID: 323082.
  1. Van Der Pol, B., Ferrero, D. V., Buck-Barrington, L., Hook 3rd, E., Lenderman, C., Quinn, T., et al. Multicenter evaluation of the BDProbeTec ET system for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine specimens, female endocervical swabs, and male uerthral swabs. J Clin Microbiol. 2001; 39(3): 1008–16.
  2. Tankeshwar, A. Modified Thayer-Martin Agar: Preparation, Uses. Microbe Online [Internet]. Available from: https://microbeonline.com/thayer-martin-agar-composition-preparation-uses-colony-characteristics/. Cited 2022 March 29.
  3. Workowski, K. A., Bachmann, L. H., Chang, P. A., Johnston, C. M., Muzny, C. A., Park, I., et al. Sexually Transmitted Infections Treatment Guidelines, 2021. MMWR Recomm Rep. 2021; 70(4): 1-187.
  4. Janssen, K. J., Hoebe, C. J., Dukers-Muijrers, N. H., Eppings, L., Lucchesi, M., and Wolffs. P. F. Viability-PCR Shows That NAAT Detects a High Proportion of DNA from Non-Viable Chlamydia trachomatis. PLoS One. 2016; 11(11): e0165920.
  5. Guillot, X., Delattre, E., Prati, C., and Wendling, D. Destructive septic arthritis of the sternoclavicular joint due to Neisseria gonorrhoeae. Joint Bone Spine. 2012; 79(5): 519-20. 
  6. Zaia, B. E. and Soskin, P. N. Images in emergency medicine. Man with severe shoulder pain. Gonococcal arthritis of the shoulder. Ann Emerg Med. 2014; 63(5): 528-71.

-Marika L. Forsythe, MD is a PGY1 Pathology Resident at University of Chicago (NorthShore). Her academic interests include molecular diagnostics and its growing importance in the field of Pathology.

-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.

Microbiology Case: A 35 Year Old Male with Left Leg Cellulitis

Clinical History

A 35 year old male with chronic bilateral lower extremity lymphedema due to obesity presented with a one-week history of subjective fevers and malaise with associated left lower extremity pain, swelling and erythema. The left leg was markedly edematous with erythema present above the knee down. The leg was tender to palpation, and multiple ruptured bullae and areas of severe desquamation with excessive serous drainage were observed. Importantly, no areas of purulence were noted (Image 2). A clinical diagnosis of severe non-purulent cellulitis was made, and the patient was admitted for parenteral antibiotic therapy of vancomycin and piperacillin-tazobactam. Necrotizing fasciitis was ruled out based on imaging, and significant clinical improvement was seen after 5 days of intravenous antibiotics. The patient was transitioned to oral therapy with amoxicillin-clavulanic acid and doxycycline for a total of 14 days of antibiotics.

Laboratory Workup

During the admission, urinalysis revealed turbid urine with elevated protein (30 mg/dL), and 2+ blood with 5 RBC/HPF on microscopic examination. Given the presence of protein with microscopic hematuria, causes of glomerulonephritis were investigated. Workup revealed a markedly elevated anti-streptolysin O (ASO) titer of 5310 (0-330) and a total complement (CH50) level of 14, which was low given his age. Urine sediment examination revealed red blood cell casts (Image 3). These clinical and laboratory findings were consistent with post-streptococcal glomerulonephritis (PSGN) due to Streptococcus pyogenes skin and soft tissue infection.

 
Image 1. Colony appearance and biochemical testing of S. pyogenes. A) Typical gram positive cocci in chains characteristic of streptococci. B) Growth on Sheep’s Blood Agar of small, translucent colonies with a wide zone of beta-hemolysis indicative of S. pyogenes. C) Catalase-negative S. pyogenes (left) compared to catalase-positive S. aureus (right). D) PYR-positive S. pyogenes (left) compared to PYR-negative S. aureus (right).
Image 2. Left lower extremity at presentation.
Image 3: Red blood cell cast seen in urine sediment.

Discussion

Streptococcus pyogenes are gram positive bacteria that appear in pairs and/or chains by microscopy (Image 1A). In culture, these organisms produce relatively small colonies which elaborate a large zone of beta hemolysis on blood agar plates; colonies are translucent with smooth edges (Image 1B). The beta-hemolytic activity of S. pyogenes is due to the activity of two hemolysins: Streptolysin-S (oxygen-stabile) and Streptolysin-O (oxygen-labile). S. pyogenes is the primary organism which expresses the Lancefield Group A carbohydrate antigen. Less frequently encountered strains of S. anginosus and S. dysgalactiae subsp. equisimilis may also express this antigen, so biochemical identification of S. pyogenes may be helpful for a definitive diagnosis. MALDI-TOF MS may also fail to discriminate between S. pyogenes and closely related β-hemolytic streptococci (including S. dysgalactiae and S. canis), necessitating adjunctive biochemical testing. Like other streptococci, S. pyogenes is catalase negative (Image 1C). Unlike other beta-hemolytic streptococci, S. pyogenes expresses pyrrolidonyl arylamidase (PYR) making this test a rapid and useful adjunctive diagnostic tool (Figure 1D). Bacitracin susceptibility was used historically but has been largely replaced by PYR testing due to concerns over specificity and prolonged turnaround time.

Globally, S. pyogenes is responsible for a large percentage of infection-related morbidity and mortality. The organism colonizes the skin and the nasopharynx of humans, but most colonized individuals do not develop active disease. Colonization however can lead to infection or dissemination to susceptible individuals. S. pyogenes infections exhibit a diverse range of clinical manifestations which can include pharyngitis, impetigo, erysipelas, cellulitis, necrotizing fasciitis, pyomyositis, streptococcal toxic shock syndrome, and bacteremia. S. pyogenes remains susceptible to penicillin, making β-lactams first-line drugs of choice for management. Conversely, rising levels of macrolide, lincomycin, tetracycline, and fluoroquinolone resistance has been observed. Susceptibility testing may be warranted if these agents are to be used, most often in the cases of severe penicillin allergy.

S. pyogenes infection can be complicated by multiple post-infectious immune-mediated sequelae including PSGN and rheumatic fever. Post-Streptococcus glomerulonephritis (PSGN) has a global incidence of > 470,000 individuals per year and occurs due to the deposition of immune complexes in the glomeruli resulting from previous S. pyogenes pharyngitis or soft tissue infection (as seen in this case). Typical clinical presentation of PSGN includes hematuria, proteinuria, edema, hypertension, elevated serum creatinine levels, hypocomplementemia, and general malaise. The elevated ASO titer (5310) was diagnostic of an S. pyogenes acute infection as the cause of this patient’s cellulitis. The development of proteinuria and hematuria following infection further supports a clinical diagnosis of PSGN. Treatment of PSGN is largely supportive with the focus on management of the underlying infection. Most individuals with kidney failure from PSGN recover to baseline renal function; however, there may be a link between PSGN and the later development of chronic kidney disease/end-stage renal disease.

References

  1. De la Maza LM, Pezzlo MT, Bittencourt CE, Peterson EM. 2020. Color Atlas of Medical Bacteriology, 3rd edition. ASM Press. Pg. 11-23
  2. Madaio MP, Harrington JT. 2001. The diagnosis of glomerular diseases: acute glomerulonephritis and the nephrotic syndrome. Arch Intern Med. 161(1):Pg. 25-34. doi: 10.1001/archinte.161.1.25.
  3. Stevens DL, Bisno AL, Chambers HF, Dellinger EP, Goldstein EJC, Gorbach SL, Hirschmann JV, Kaplan SL, Montoya JG, Wade JC. 2014. Practice Guidelines for the Diagnosis and Management of Skin and Soft Tissue Infections: 2014 Update by the Infectious Diseases Society of America. Clin Infect Dis. 59(2): Pg. e10-e52, https://doi.org/10.1093/cid/ciu296.
  4. Walker MJ, Barnett TC, McArthur JD, Cole JN, Gillen CM, Henningham A, Sriprakash KS, Sanderson-Smith ML, Nizet V. 2014. Disease manifestations and pathogenic mechanisms of Group A Streptococcus. Clin Microbiol Rev. (2): Pg. 264-301. doi: 10.1128/CMR.00101-13.
  5. Wong CH, Khin LW, Heng KS, Tan KC, Low CO. 2014. The LRINEC (Laboratory Risk Indicator for Necrotizing Fasciitis) score: a tool for distinguishing necrotizing fasciitis from other soft tissue infections. Crit Care Med. 32(7): Pg. 1535-41. doi: 10.1097/01.ccm.0000129486.35458.7d.

-John Markantonis, DO is the former Medical Microbiology fellow at UT Southwestern and has recently completed his clinical pathology residency. He is also interested in Transfusion Medicine and parasitic diseases.

-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.

Microbiology Case Study: Genotypic-to-phenotypic Discordant Results

Case History

Scenario 1: A 51 year old male with a history of diabetes, hypertension, coronary artery disease, gastric ulcer, chronic kidney disease and bilateral below knee amputation presented with epigastric pain, nausea, and vomiting. He was febrile and tachycardic. Computerized scan showed ascending/ transverse colitis and cholelithiasis. Blood cultures grew gram negative rods; the Biofire BCIDv2 panel reported Enterobacter cloacae with no genotypic, resistance markers detected. Phenotypic antimicrobial susceptibility testing (AST) from the Microscan Walkaway revealed resistance to ertapenem (>1mg/ml) but susceptibility to meropenem (£ 1mg/ml). Additionally, the isolate was resistant to 3rd-generation cephalosporins, fluoroquinolones, and intermediate-resistant to tetracyclines. Identification was confirmed by the MALDI-TOF MS upon growth on agar plates. The isolate was subbed with a meropenem disk to select for carbapenem resistance for further confirmatory testing. A Cepheid Carba-R test was ran on a sweep of the isolate growing near the carbapenem disk, which resulted in no carbapenemases detected. Results from E-tests with meropenem and ertapenem were consistent with original phenotypic result. Here, we reported the discrepant phenotypic result and genotypic results as is.

Image 1. Phenotypic testing results (E-test) for meropenem (MP,left) and ertapenem (ETP, right) of Enterobacter cloacae isolate described in scenario 1. E-test results were consistent with original phenotypic results which also identified the isolate as meropenem susceptible and ertapenem resistant. (Photo credit: Gizachew Demessie, Lead Tech, George Washington Hospital.)

Scenario 2: An 80 year old female underwent a Whipple procedure for a pancreatic mass. A wound culture was submitted from the operating room which grew both Streptococcus anginosus and Enterobacter cloacae complex. Phenotypic AST for the E. cloacae revealed susceptibility to ertapenem (≤0.5 mg/ml) but resistance to meropenem (4 mg/ml). The isolate was pan-susceptible to other drug classes (aside from intrinsic resistance). Similar to Case 1 above, identification was confirmed by the MALDI-TOF MS and the isolate was subcultured with selective pressure. A Cepheid Carba-R test did not detect any carbapenemases. However, upon repeating a phenotypic test, both ertapenem and meropenem were susceptible. Our investigation here led to the avoidance of reporting an incorrect phenotypic AST result.

Discussion

Genotype-to-phenotype discrepancies may occur in antimicrobial susceptibility testing. For example, an antimicrobial resistance (AMR) gene may be detected in a phenotypically susceptible isolate or an AMR gene may not be detected in a phenotypically resistant isolate. Such discordant results should be investigated so appropriate antimicrobial therapy is used on these patients. This leads us to an important question “What can laboratories do to solve these discrepancies?”

The first step in detection of discrepancies requires educating and teaching the lab staff to be vigilant in looking for odd susceptibility patterns (from results within a drug class and also the overall AST profile). Next, check if there was pure isolation of the organism on the purity plate; if not, each individual isolate should be subbed, identified and re-tested on both genotypic and phenotypic platforms. Of note, subbing the bacteria under selective antibiotic pressure (e.g. growing the isolate on agar plate with an antibiotic disk) can increase the potential of detecting resistance. Alternative methods (e.g. CarbaNP, mCIM, etc) could be considered if one is looking into specific resistant mechanisms. Due diligence in checking for clerical, transcription errors and contamination on equipment, especially when there is a consistent pattern of detection for a specific molecular target, is highly recommended. As such, a lab should maintain constant communication with the test manufacturer in case there are issues with batches or lots of reagents.1,2

While these rapid, genotypic panels tend to include the more common AMR mechanisms, there are still other mechanisms of resistance not on the panels. For gram negatives, AMR mechanisms such as AmpC beta-lactamases, porin mutations, efflux pumps and rare carbapenemases such as GES, IMI, and SME types are typically not included.3 Additionally, although the gene blaCTX-M is used as a marker for Extended Spectrum Beta-Lactamases (ESBL), different variants of ESBLs confer different cephalosporin (e.g. 3rd and 4th generation) phenotypes.4 A heteroresistant subpopulation, decreased or lack of expression of an AMR gene may also be potential explanations.

If a discrepancy is not resolved, it is suggested to report the isolate as resistant. If both the discrepant genotypic and phenotypic results are reported, one should consider recommending an infectious diseases consult or to contact the antimicrobial stewardship team.1 Additional information and suggested laboratory workflow can be found in Appendix H of the M100 guidelines from the Clinical Laboratory and Standards Institute.2 While molecular AMR approaches have many advantages such as a shorter turnaround time, phenotypic susceptibility testing can still offer valuable clinical information.5

  1. CLSI. Performance Standards for Antimicrobial Susceptibility Test. CLSI supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute; 2022, Edition 32
  2. Yee R, Dien Bard J, Simner PJ. The Genotype-to-Phenotype Dilemma: How Should Laboratories Approach Discordant Susceptibility Results? J Clin Microbiol. 2021 May 19;59(6):e00138-20.
  3. Tamma PD, Sharara SL, Pana ZD, Amoah J, Fisher SL, Tekle T, Doi Y, Simner PJ. 2019. Molecular epidemiology of ceftriaxone non-susceptible Enterobacterales isolates in an academic medical center in the United States. Open Forum Infect Dis 6:ofz353.
  4. Paterson DL, Bonomo RA. 2005. Extended-spectrum beta-lactamases: a clinical update. Clin Microbiol Rev 18:657–686.
  5. Dien Bard J, Lee F. 2018. Why can’t we just use PCR? The role of genotypic versus phenotypic testing for antimicrobial resistance testing. Clin Microbiol Newsl 40:87–95. 10.1016/j.clinmicnews.2018.05.003. 

Rami Abdulbaki, MD is a Pathology Resident (PGY-3) at The George Washington University Hospital. His academic interest includes hematopathology and molecular pathology.

-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.

What’s NOT New in Cancer Care?

In June of 2017 just at the start of the annual American Society of Clinical Oncology (ASCO) meeting in Chicago, Illinois, there were at least 7 new FDA approvals for immuno-oncology agents targeting PD-L1 in cancer. At that time (2017), there were 2030 potential agents targeting 265 different targets across cancer including the modalities of t-cell targeted and other immunomodulators, cell therapy, cancer vaccines, oncolytic viruses, and CD3-targeted bispecific antibodies. Just three years later (2020), prior to the COVID-19 pandemic, this landscape had increased to 4720 potential agents targeting 504 targets across the same spectrum. That represents a 233% growth in these agents. Although only a fraction of these is “approved” (i.e., FDA approved and in use in patients clinically), many these agents are in clinical trials that require patient recruitment using pathology and other testing data. What does this mean for pathologists and laboratory professionals? Depending on the tumor being targeted and the target, there may or may not be a specific laboratory test that needs to be performed which may be routine, like histology parameters or immunohistochemistry, or may require advanced methods, like unique antibodies/clones, specific quantification methods, or molecular testing. The range of testing is not even unique to a specific therapy—for example, pembrolizumab uses staining for PD-L1, MSI, or no testing at all depending on tumor type. For the sub-specialized pathologist that focuses on one or two organs only, mastering the rapid pace and required diagnostic-therapeutic pairings is still a challenge. Imagine what it is like to be a general surgical pathologist in a community setting serving a community cancer center. Moreover, the diagnosis of a specific tumor is often completely disconnected for any biomarkers that may be indicated at the time of collection or several months later depending on therapeutic outcomes. This poses a range of problems in logistics and processing that are still being worked out at the individual system level. Still, the plethora of new treatments for cancer patients is very exciting.

In 2017, the largest group of targets (which was heterogenous) were tumor associated antigens (TAA) which are molecules that are not normally found in the human body produced by tumor cells as the result of changes to cellular processes. Whether it is hybrid proteins, glycosylation, or phosphorylation products, etc., these unique antigens held amazing promise as something we could target and destroy without fear of hurting normal human cells. However, the bulk of these approaches were for tumor vaccines (>90%) in 2017, dropping to 58% in 2020 (and from a total of 265 to only 198). To date, however, only a handful of cancer vaccines have been fully approved including sipuluecel-T for metastatic prostate and T-VEC for advance melanoma. This example category creates a complex set of challenges for pathologists and laboratory professionals. What data is needed about a patient or their tumor before a vaccine can be used? Does it require special studies that are not easily available or are costly? After vaccination, what follow-up tissue or blood studies are needed to follow the patient? Who dictates which tests are required before treatment: industry or medicine? But the more important challenge is: When do we, as the laboratory, pull the trigger to develop and disseminate such information and on-board new tests? Certainly, we are not going to look at Phase I trials and start taking about needs for future diagnostics. But by Phase III (where there is still a high dropout rate before full FDA approval) the number of potential agents and tests may still be daunting. If we wait until approval, now we are behind because our clinical colleagues will start immediately wanting to use the therapy. Tumor vaccines are an interesting category because we assume, for the most part, that there is likely only a diagnostic role needed. But then consider targets like CD-19, PD-L1, PD-1, CD3, Her2, CTLA-4, CD20, MUC1, CD22 and so on which are very familiar to our laboratory family because we often have already a test for these markers.

But is it the correct clone?

Do we have to score or interpret it differently?

When the agent is for cell therapy (the largest growth area of therapy development with 294% growth alone), what role does the transfusion medicine team play in administering or monitoring the patient?

As with the prior example, at what point do we, as a specialty of diagnosticians, dig into the forthcoming clinical trial results to plan? If our colleagues are in academic centers and are part of the clinical trials, they often are aware of and are administering the very tests that determine trial entrance. But if one reads just a few clinical trials of these agents, you may find that the inclusion criteria require a large battery of tests; however, on the other end when it is clinical ready for prime time, only one biomarker may be needed. Such a clustered landscape of information poses frustrating challenges for the clinical team and laboratory team in trying to find the way forward to get patients the life-saving therapies that are quickly arriving.

There is no question that the collision of targeted therapeutics and evolving diagnostics (i.e., precision cancer medicine) has demonstrated phenomenal growth with ever increasing benefits for patients. Affordability and access to these therapeutics aside*, studies continue to be completed and published including combinations therapies and hybrid therapies which show incredible promise. At ASCO 2022, the results of the DESTINY-Breast04 Phase III trial showed that trastuzumab deruxtecan (HER2-directed antibody and topoisomerase inhibitor conjugate) show a 49% reduction in the risk of disease progression or death versus physician’s choice of chemotherapy for patients with HER2-low metastatic breast cancer. That finding should be read a few times to make sure that the impact of this statement is very clear for pathologists and the laboratory. Previously, how we report HER2 (0, 1+, 2+, 3+) was complicated and often required FISH for questionable cases to look directly for HER2 amplification. This new category of patients requires reporting accurately 1+ or 2+ (FISH negative) disease, as it has incredible implications for patients. This news follows the recent new indications for CDK inhibitors in breast cancer related to Ki-67 mitotic score. Just when we thought breast cancer was straightforward, there is more to know and, more importantly, more time and tedium and standardization needed to report it for each patient. And, of course, early triple-negative breast cancer can also be treated with checkpoint inhibitors after PD-L1 testing is performed…but that’s literally old news as the data was release in 2020 at the start of the pandemic.

Outside of therapeutics, diagnostics are evolving quite rapidly with the COVID-19-induced ability to use digital pathology more readily creating a super-highway for artificial intelligence products to be validated for clinical use. PaigeAI has two such products (one for prostate and the second for breast lymph node evaluation released March of 2022) and many others are sure to follow. In parallel, screening, imaging, and surgery have also had advancements that continue to improve patient care and outcomes. So, it seems that everything feels new in cancer but is that the case?

The bulk of tumors diagnosed in the US (and elsewhere) are done with simply H&E staining (up to 75%) with another 20% being further confirmed by a few IHC tests (bringing the total up to 95%). This is not new and, most importantly, is the standard of care for the time being that we use to classify tumors. That classification has dictated, to some degree, the correct NCCN or other cancer protocol that oncologists used to treat patients. At some point, however, sufficient data on the bulk of all tumor types will likely point precision medicine treatments at all cancers. At that point, will a tissue biopsy be necessary with full histology or will a fine needle aspiration with molecular testing dictate the care? The credible assumption is that standard histology and IHC will remain in practice for the foreseeable future because so much billing, accreditation, and compliance is tied closely to them. But we CAN envision a “histology-free” oncopathology approach that matches patients to treatments with a panel of biomarkers. Sounds amazing but also stressful from the point of view of your typical anatomic pathologist.

*But the final thought on this, and perhaps the most important, is cost. Much like the domestic energy market is facing a dwindling pool of customers who agree to pay more and more for “traditional power” while their neighbors pump excessive kilowatts into the grid with their solar panels and windmills enjoying essentially “free power”, progress in cancer screening, detection, and treatment should be dwindling the pool of potential patients and increasing the costs to deliver care to the remainder. However, data and trends suggest that cancer is increasing globally. Why, if we are spending so much money and development on cancer care? Poverty and access. Cancer care is both expensive (in the US) and relatively expensive (in LMICs) with a focus on a small group of patients (0.55% of a population per year develop cancer). Projections of populations who need certain therapeutics are calculated using payer pools and markets that are existing and reliable. That does not include the bulk of LMICs. So, when we consider the cost of the PD-L1 checkpoint inhibitor class per year per patient is upwards of $125,000 USD, how can we even consider that an option for impoverished patients living off $1 USD per day? But if we don’t sort that out and treat these patients, we are assuming that persons who are impoverished are less valuable than persons who can afford expensive care. That evil logic, however, doesn’t hold true because even individuals in the US often become destitute or lose the bulk of their fiscal well-being when they must pay for cancer care—a situation that simply does not occur in countries with socialized medicine and/or universal healthcare.

Cancer care is rapidly evolving and the new tools and therapies available are incredible and miraculous for many patient types who would have faced a death sentence even 10 years ago. But with this amazing progress, we cannot ethically let people with limited resources succumb to these diseases over something so trivial as money. To do so poses harm and sets us up for failure as a species. It is for these reasons that ASCP engages in global health outreach. We are excited to have recently launched the Access To Oncology Medicines (ATOM) program with UICC and more than 2 dozen partners which will rapidly bring high-quality generic cancer therapeutics to low- and middle-income countries. In parallel with the St. Jude/WHO efforts on pediatric cancer globally, we will deliver quality cancer diagnosis and treatment to all patients everywhere.

If you want to learn more about PD-L1 testing and/or overcoming barriers to I-O in persons of color, new education from ASCP is available at no cost at https://www.ascp.org/content/learning/immuno-oncology/.

You can also check out our free educational resources on HER2-low breast cancer and Ki-67 testing in breast cancer at https://www.ascp.org/content/learning/breast-cancer.

Special thanks this month the Kellie Beumer (instructional design) and Melissa Kelly (monitoring and evaluation) from the ASCP medical education grants team for their thoughtful inputs into this piece.

References

  1. https://www.cancerresearch.org/en-us/scientists/immuno-oncology-landscape
  2. https://www.mskcc.org/cancer-care/diagnosis-treatment/cancer-treatments/immunotherapy/cancer-vaccines
  3. https://www.astrazeneca.com/media-centre/press-releases/2022/enhertu-efficacy-results-in-her2-low-breast-cancer.html
  4. https://www.urmc.rochester.edu/news/story/what-is-ki-67-in-breast-cancer
  5. https://www.nejm.org/doi/full/10.1056/NEJMoa1910549
milner-small


-Dan Milner, MD, MSc, spent 10 years at Harvard where he taught pathology, microbiology, and infectious disease. He began working in Africa in 1997 as a medical student and has built an international reputation as an expert in cerebral malaria. In his current role as Chief Medical officer of ASCP, he leads all PEPFAR activities as well as the Partners for Cancer Diagnosis and Treatment in Africa Initiative.

A Quick Primer on BK Virus

While SARS-CoV-2 testing may be dominating discussions, I wanted to highlight other important, but lesser known molecular microbiology tests, starting with BK virus.

About BKV

BK virus (BKV), a member of the Polyomaviridae family, has a tropism for uroepithelial cells and causes disease in immunosuppressed patients, particularly those who have undergone renal transplants.1,2,3 The vast majority of immunocompetent adults are infected with BKV, with estimates up to 90%, and the bulk of cases are entirely asymptomatic.1,3 The exact method of transmission is unknown,3,4 but respiratory transmission is hypothesized. BKV can remain latent after initial infection and can reactivate when immunosuppressed.4 Intermittent asymptomatic viral shedding in urine is particularly common in pregnant individuals or elderly individuals.2

In renal transplant patients, BKV can lead to significant damage to the transplanted kidney and graft failure.1 Polyomavirus-associated nephropathy (PVAN) can occur.2 In bone marrow transplant recipients, hemorrhagic cystitis can occur as a result of this virus.2 Other organ systems can be impacted although much more infrequently.4

The Lab’s Role in Diagnosis and Monitoring BKV

Given the profound impact on renal transplant patients in particular, these patients are routinely screened for BKV both in the blood and the urine. Importantly, BKV can be shed asymptomatically in the urine and thus correlation with BKV detection in the blood is essential. Molecular testing is the method of surveillance. There are currently no FDA approved assays for BKV so labs that perform testing use laboratory-developed tests with analyte specific reagents or research use-only kits.1

Quantification is necessary for monitoring. As with any quantitative assay, there must be at least one negative control, one high positive control, and one low positive control included per run. All controls should fall within the linear range of the assay. To monitor for amplification inhibition, an internal control should be included for each sample.1

Image 1. Example of low (left) and high (right) positive controls.

We perform a BKV LDT assay here using Diasorin reagents and instrumentation. The green line is BKV target while the purple line is the internal control (IC). We run a low positive, high positive, and negative control with every run. Director review for all control and patient results is required. We use commercial BKV positive controls, which have established acceptable range that the quantification of controls must fall within for the run to be considered valid.

The Results and How They Impact Patient Care

Currently, there are no targeted treatments for BKV. In renal transplant individuals, modulation of immunosuppression is the main approach for managing BKV.3,4 A delicate balance must be achieved as reducing immunosuppression can lead to organ rejection while high levels of BKV can cause organ failure.

References

  1. 2016. 12.3 Molecular Methods for Identification of Cultured Microorganisms, Leber AL Clinical Microbiology Procedures Handbook, 4th Edition. ASM Press, Washington, DC. doi: 10.1128/9781683670438.CMPH.ch12.3
  2. Gregory A. Storch and Richard S. Buller, 2019. Human Polyomaviruses, In: Carroll KC, Pfaller MA Manual of Clinical Microbiology, 12th Edition. ASM Press, Washington, DC. doi: 10.1128/9781683670438.MCM.ch108
  3. Furmaga J, Kowalczyk M, Zapolski T, et al. BK Polyomavirus-Biology, Genomic Variation and Diagnosis. Viruses. 2021;13(8):1502. Published 2021 Jul 30. doi:10.3390/v13081502
  4. Mark D. Reploeg, Gregory A. Storch, David B. Clifford, BK Virus: A Clinical Review, Clinical Infectious Diseases, Volume 33, Issue 2, 15 July 2001, Pages 191–202, https://doi.org/10.1086/321813

-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.