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.

Laboratory Ergonomics: Safe Today, Healthy Tomorrow

Ergonomics is a safety topic that gets little respect in the laboratory, but it can become very important over time. The effects of poor ergonomics are cumulative, and they can appear suddenly. When they arise, the pain and treatment are often difficult, and as people age, healing is slower as well. Because the consequences of repetitive motion injuries are slow to appear, it can be a challenge to raise concerns and create solutions regarding ergonomics. Education and action today can prevent a great deal of future injuries and staff shortages.

There are several areas in the lab where a focus on ergonomics can create benefits, and creating healthy movement and comfort does not need to be expensive or difficult. Laboratory workstations have a primary and secondary work zone.  Keep the most frequently used objects in the primary zone (within 18 inches of reach) and less frequently used in the secondary zone (within three feet).  Every employee is a different size. Teach staff to take a minute before beginning work to adjust the chair and other work items to make the workstation more comfortable.  Eliminate clutter beneath the workstation to allows room to stand or sit allowing for foot and leg comfort.

Chairs should have 4-way and preferably 6-way adjustability and come in a variety of sizes to fit the employees who work in the lab.  Chairs should have five legs with casters that are appropriate for the surface being used (e.g.: hard casters on carpet and soft casters on tile).  The backrest should flex between 90 and 113 degrees with arm rests removed on chairs in the technical area to allow the chair to get closer to the benchtop. 

The tops of computer monitors should be at eye level.  Since many employees may use the same monitor, having it on a movable arm will help each user move the monitor to an acceptable level.  Any glare on the monitor screen can be reduced with a glare screen or by reducing the light in the department.  Keyboards should lay flat to allow the hands and wrist to work in a neutral position and the arms to work at a 90 degree level for comfort.

When using a centrifuge, stand directly in front and work over the top when loading and unloading, and use two hands to close the lid.  Centrifuges should be placed low enough so that employees can see into the body of the machine easily. Place antifatigue mats in front of laboratory equipment that requires standing for long periods of time. These mats relieve lower back and leg discomfort.  When bending and lifting, employees should lift using their thighs and not the back. Teach staff to hold objects close to the body when lifting.  Never lift more than 50 pounds without assistance from other employees or an assistive device such as a hand truck.

Capping and uncapping tubes for an extended period, phlebotomy, and transcription are laboratory tasks that require the use of the same muscle groups in the hands.  When working in these areas, it is important to vary the tasks every 2-3 hours per day and take mini-breaks to stretch fingers and arms in order to prevent carpal tunnel issues.

Breaks are an important part of overall ergonomic health.  It is better to take a five minute break every hour than to take a 15 minute break every four hours.  It is especially important if you are using a microscope or a computer for an extended period of time.  Remember the 20-20-20 rule: Every 20 minutes look up to focus on something 20 feet away and blink your eyes 20 times.  This will allow you to moisturize your eyes and give them a short rest. This can help to prevent ergonomics issues such as Computer Vision Syndrome which can result in neck pain, vision problems, and headaches.

Ergonomics safety is important on all areas of the laboratory, and the best way to ensure good work practices is to perform an ergonomics assessment. An ergonomic assessment should include identifying physical work activities or conditions of the job that are associated with work-related musculoskeletal disorders (MSDs) and how to eliminate these hazards.  For additional information, review the Occupational Safety and Health (OSHA) laboratory ergonomics fact sheet (https://www.osha.gov/sites/default/files/publications/OSHAfactsheet-laboratory-safety-ergonomics.pdf).

Over one third of all U.S. worker injuries are related to MSDs caused by poor ergonomics. Laboratory employees are valuable resources, now more than ever, and preventing time away from work, surgeries and medical bills for laboratorians should be a priority. The results of poor ergonomic practices in the lab do not show up today, but they will have effects tomorrow if we don’t pay attention to them. Those effects can be career-altering, career-ending, and they can interfere with the happy and healthy retirement that we all want to enjoy. Take steps today to prevent that future- provide training, raise awareness, and perform ergonomics assessments to make sure staff remains comfortable and healthy for all of their tomorrows.

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: How to “Pin” a Diagnosis

Case History

A 7 year old female presented to the emergency department with left sided abdominal pain and a temperature of 103 degrees Fahrenheit. Labs drawn showed mild leukocytosis with a CT scan suggestive of acute appendicitis. The patient underwent uncomplicated appendectomy with no complication. Gross examination of the appendix revealed an unremarkable, non-perforated serosa and a fecalith within the lumen. Representative tissue sections submitted for microscopic analysis per grossing policy. The findings below led to the submission of the entire appendix to be evaluated.

Figure 1. Low power image of an appendix demonstrating mild acute inflammation, lymphoid hyperplasia and congestion.

Figure 2. High power image, Cross-section of an adult female E. vermicularis from the same specimen shown in Figure 1. Adherent to the appendiceal surface. Note the presence of the alae (blue arrow), and the presence of almond shaped eggs (red arrow).

Discussion

The nematode Enterobius vermicularis, widely known as the human pinworm, is one of the most common parasitic worm infections today in the United States, infecting approximately 40 million people. The patient population is often children who are infected via fecal-oral transmission, with autoinfection being common. Humans are the only known host of this nematode. Once E. vermicularis embryonated oocytes are ingested, the larvae hatch and inhabit the gastrointestinal system. At night, the larvae migrate down to the anus, lay their eggs, and the cycle recurs.

The clinical presentation can be asymptomatic or can present with perianal pruritus at night, which can be explained via the life cycle of the parasite as stated above. The method of choice for diagnosing E. Vermicularis is microscopic examination of the eggs via cellulose tape slide test. A piece of scotch tape collects the eggs near the perianal area of the patient, which is then used for analysis and identification of the eggs. Microscopically, E. Vermicularis can be identified by the spines or ‘alas’ on the surface with oval shaped, thick capsuled oocytes within, seen in figure 2. To improve the sensitivity of the scotch tape test, it is best to do this test in the early morning, when there is an increased chance of sampling the eggs.

Rarely, is this worm associated with any severe symptoms but patients can present with abdominal pain, suggesting intestinal obstruction, extra intestinal manifestations like vulvovaginitis, or appendicitis. The relationship between E. Vermicularis and appendicitis is up for debate as to whether there is a causative relationship or if it is an incidental finding seen within appendicitis. Regardless of the relationship, once a diagnosis of Enterobius vermicularis is made, treatment with an anthelmintic needs to be given to the patient, such as Albendazole or Pyrantel Pamoate. In addition, treatment for everyone in the household needs to be considered in confirmed cases of infection.

Routine surgical specimens, such as appendices, can perhaps be overlooked once acute inflammation is noted. It is important to be able to identify organisms, such as pinworms, on such specimens to get the patient the appropriate treatment.

References

  1. https://www.cdc.gov/dpdx/enterobiasis/index.html.
  2. https://www.sciencedirect.com/science/article/pii/S204908012030412X
  3. https://www.uptodate.com/contents/enterobiasis-pinworm-and-trichuriasis-whipworm?search=enterobius%20vermicularis&source=search_result&selectedTitle=1~32&usage_type=default&display_rank=1#H12

-Alexandra Medeiros, MD, is a first year anatomic and clinical pathology resident at Medical College of Georgia at Augusta University. Her academic interests include Forensic pathology, and surgical pathology.

-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: A 44 Year Old Male Finds a Tick on His Leg

Case History A 44 year old male pulled this (image 1) off of his leg after dragging brush out of a tree line in Vermont.

Image 1. Ixodes scapularis under a microscope. Characteristic features such as eight black legs, dorsal shield, and dark red color can be appreciated.

Ixodes scapularis

Ixodes scapularis, also known as the blacklegged tick or deer tick, is commonly found in the eastern and northern Midwest regions of the United States as well as southeastern Canada. This species of tick is approximately 3 mm in length. Morphologically, females have a black head and a dorsal shield with a dark red abdomen, while males are entirely black or dark brown. Both sexes have eight black legs and a characteristic anal opening, appearing within a horseshoe-shaped ridge on the ventral lower abdomen. Unlike other tick species, Ixodes scapularis does not have ridges on the edge of the lower abdomen. Ixodes scapularis can live up to 2 years in the wild and die after reproduction.1

Life Cycle, Transmission, and Infection

Ixodes scapularis is a three-host tick with a different host at each stage of development. Their life cycle lasts approximately 2 years, where they undergo 4 distinct developmental/life stages: egg, six-legged larva, eight-legged nymph, and adult. After hatching from the egg, it should have a blood meal at every developmental stage to survive. Ixodes scapularis is known to parasitize and feed from mammals, birds, reptiles, and amphibians, and its best-known host is the white-tailed deer. This species is unable to fly or jump so it usually waits for a host while resting in the tips of grass or shrubs. Depending on the developmental stage, preparation for feeding can take between 10 minutes to 2 hours.2 Once the tick finds a feeding spot on the host, it grasps onto the skin and cuts into the surface inserting its feeding tube, which can have barbs and can secrete a cement-like surface for better attachment. Moreover, the tick can also secrete small amounts of saliva with anesthetic properties to remain undetected during the blood meal. If attached to a sheltered spot, the tick can remain unnoticed for long periods. Ixodes scapularis will attach to its host and suck on the blood for a few days. The lengthy feeding process makes them good at transmitting infection. If the host has a bloodborne infection (e.g., Lyme disease), the tick may ingest the pathogen and become infected. If the tick feeds on a human later, that human can become infected with the same pathogen if it is a prolonged blood meal. However, if the tick is removed quickly (~ 24 hours), the risk of acquiring disease is reduced.2 The longer the tick is attached, the greater the risk of becoming infected. The risk of human infection is greater during the spring and summer.

Ixodes scapularis as a Disease Vector

Babesiosis

The causative agent of babesiosis are Basebesia microti and other Babesia species. These parasites preferentially infect red blood cells. In the United States, most cases are caused by Babesia microti.3 Babesiosis is most frequently reported in the upper midwestern and northeastern regions of the United States, where Babesia microti is endemic. Although this parasite is generally transmitted by Ixodes scapularis, Babesia parasites can also be transmitted via blood transfusions and, in some cases, congenitally. Babesiosis can range from asymptomatic to life-threatening. Some of the common signs and symptoms include fever, chills, sweats, general malaise or fatigue, myalgia, arthralgia, headaches, anorexia, nausea, and dark urine. Less common symptoms include cough, sore throat, emotional lability, depression, photophobia, conjunctival infection.3 Not all infected persons are symptomatic or febrile. Clinical presentation usually manifests within several weeks after exposure, but may develop or recur months after infection. The incubation period for Babesia species parasites is approximately 1-9+ weeks. Laboratory findings associated with babesiosis include decreased hematocrit due to hemolytic anemia, thrombocytopenia, elevated serum creatinine and blood urea nitrogen values, and mildly elevated hepatic transaminase values.3 To diagnose babesiosis in the laboratory, identification of intraerythrocytic Babesia parasites by light-microscopic examination of a blood smear, positive Babesia (or Babesia microti) PCR analysis, or isolation of Babesia parasites from a whole blood specimen by animal inoculation in a reference lab are recommended procedures. Additionally, demonstration of a Babesia-specific antibody titer by indirect fluorescent antibody testing for IgG can be used as supportive laboratory criteria—although it is not enough evidence to support a diagnosis of an active infection.3 Treatment for babesia usually lasts 7-10 days with a combination of two drugs: atovaquone plus azithromycin or clindamycin plus quinine, with the latter being the standard of care for severely ill patients.

Anaplasmosis

Anaplasmosis, formerly known as Human Granulocytic Ehrlichiosis, is caused by Anaplasma phagocytophilum. Anaplasmosis is commonly reported in the upper Midwest and northeastern regions of the United States. The incubation period for Anaplasma phagocytophilum is 5-14 days.3 Some of the common signs and symptoms of anaplasmosis include fever, chills, rigors, severe headaches, malaise, myalgia, gastrointestinal symptoms such as nausea, vomiting, diarrhea, and anorexia, and, in some cases, rash. The general laboratory findings for anaplasmosis during the first week of clinical disease include mild anemia, thrombocytopenia, leukopenia, and mild to moderate elevations in hepatic transaminases.3 Under the microscope, the visualization of morulae in the cytoplasm of granulocytes during examination of blood smears is indicative of diagnosis. However, to definitely determine diagnosis in the laboratory, detection of DNA by PCR of whole blood is recommended during the first week of illness. Additionally, demonstration of a four-fold change in IgG specific antibody titer by indirect immunofluorescence antibody assay in paired serum samples is recommended. The first serum sample should be taken during the first week of illness and the second serum sample should be taken 2-4 weeks after. Moreover, immunohistochemical staining of the organism from the skin, tissue, or bone marrow biopsies is also recommended for diagnosis.3 Anaplasmosis is treated with doxycycline. Treatment should be started once there is a clinical suspicion of disease, as delaying treatment may result in severe illness or in death.

Lyme Disease

The causative agents for Lyme disease include Borrelia burgdorferi and Borrelia mayonii. Lyme disease is most frequently reported in the Upper Midwestern and northeastern regions of the United States with some cases being reported in northern California, Oregon, and Washington. Data from 2015 shows that 95% of Lyme disease cases were reported in the following 14 states: Connecticut, Delaware, Maine, Maryland, Massachusetts, Minnesota, New Hampshire, New Jersey, New York, Pennsylvania, Rhode Island, Vermont, Virginia, and Wisconsin.3 The incubation period for Borrelia parasites is usually 3-30 days.3 Some of the early (3-30 days after a tick bite) signs and symptoms of Lyme disease include fever, chills, headache, fatigue, muscle and joint aches, and swollen lymph nodes may occur with an absence of rash. Erythema migrans is a characteristic rash of Lyme disease and it occurs in 70%-80% of infected people.4 This rash starts at the site of a tick bite after an average of 3-30 days (average is 7 days) and it gradually expands over several days reaching up to 30 cm across.4 As it enlarges, it can result in the characteristic “bulls-eye” appearance; it may feel warm to the touch and it is rarely itchy or painful. Some of the later (days to months after a tick bite) signs and symptoms include severe headache and neck stiffness, additional erythema migrans rashes in other areas of the body, facial palsy, arthritis with severe joint pain and swelling—especially in the knees, intermittent pain in the tendons, muscles, joints, and bones. It may also lead to heart palpitations or Lyme carditis, episodes of dizziness or shortness of breath, inflammation of the brain and spinal cord, nerve pain, and shooting pains, numbness, or tingling of the hands and feet.4 Laboratory diagnosis for Lyme disease includes the demonstration of IgM or IgG antibodies in serum and a two-step testing protocol is highly recommended.5 Moreover, isolation of an organism from a clinical specimen is also recommended. Treatment for Lyme disease includes antibiotics such as doxycycline, cefuroxime axetil, or amoxicillin.

When assessing a patient for any tick-borne diseases, the clinical presentation should be considered alongside the likelihood that the patient has been exposed to an infected Ixodes scapularis tick, or any other tick. Moreover, if a tick is found, engorgement of the tick should be considered when assessing for the possibility of disease transmission.

References

  1. Thevanayagam S. Ixodes scapularis [Internet]. 2012. Available from: https://animaldiversity.org/accounts/Ixodes_scapularis/.
  2. Centers for Disease Control and Prevention. Lifecycle of Blacklegged Ticks [Internet]. 2011 [updated November 15, 2011]. Available from: https://www.cdc.gov/lyme/transmission/blacklegged.html.
  3. Centers for Disease Control and Prevention. Tickborne Diseases of the United States: A Reference Manual for Healthcare Providers [Internet]2018. Available from: https://www.cdc.gov/ticks/tickbornediseases/TickborneDiseases-P.pdf.
  4. Centers for Disease Control and Prevention. Lyme Disease – Signs and Symtoms [Internet]. 2021. Available from: https://www.cdc.gov/lyme/signs_symptoms/index.html.
  5. Mead P, Petersen J, Hinckley A. Updated CDC Recommendation for Serologic Diagnosis of Lyme Disease. MMWR Morb Mortal Wkly Rep. 2019;68(32):703. Epub 2019/08/16. doi: 10.15585/mmwr.mm6832a4. PubMed PMID: 31415492; PubMed Central PMCID: PMCPMC6818702 potential conflicts of interest. No potential conflicts of interest were disclosed.

Amelia Lamberty is a Master’s student in the Pathology Master’s Program.

-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.

Hematology Case Study: Temporal Arteritis or COVID-19?

What is your least favorite test in hematology? The first things that come to my mind are those tests that are time consuming, tedious, and manual. I’ve worked in a hematology lab that did Kleihaur betke (KB) tests, and whenever I worked, I seemed to get one, or sometimes more, in a given shift. And when I worked in blood bank, we did KBs in blood bank, and I certainly did my share there, too. KBs seem to follow me around! Those, I must admit, are probably my least favorite, but I know that many techs dread parasite smears or % parasitemia, reviewing 150 or more fields or counting thousands of cells on a smear. Manual body fluid counts, manual reticulocyte counts, and manual platelet counts are likely some others on our lists of “not favorites.” Basically, anything that requires a lot of time, manual counting, and math!

One other test that probably doesn’t make many “favorites” list is the Erythrocyte Sedimentation Rate (ESR), or sed rate. Remember old fashioned Westergren Sed Rates that took an hour to do, while the ER doctor kept calling looking for their “STAT” results? There are still labs that set up manual sed rates that take an hour, and modified manual methods that take “only” 15 or 30 minutes. Some semi-automated methods can give us results in a couple minutes, but still require techs to fill a capillary tube and load the instrument. Fortunately, real help may have arrived, in the form of fully automated ESR instruments! There are instruments now that actually make ESRs almost fun. I’ve never seen techs so excited about a new instrument as they were when we got iSeds. This thing is amazing! It’s like a little Ferris wheel for sed rates. You pop the whole tube in, they go for a little ride around the Ferris wheel, then drop out, in less than 30 seconds. And you can keep loading tubes even while it’s running. A truly Stat ESR. Now that’s amazing!

Image 1. Alcor iSED Automated ESR Instrument

While these new instruments make ESR’s easier to run, with more reproducible results, and less hands-on time, they still don’t get much love, because, well…there are newer tests available for inflammation, and we know that the ESR is not a specific test for diagnosis. Across the years, some lab tests have become antiquated and obsolete…bleeding times come to mind, along with CK-MB. In 2010 an article was published that supported discontinuing laboratory tests that no longer have clinical utility in the lab. The ESR was on this list. Yet, many labs still perform ESRs. Should the ESR be phased out, or are there still valid reasons for ordering them?

Even though the test is considered non-specific, the ESR test is considered helpful in diagnosing two specific inflammatory diseasestemporal arteritis (TA) and polymyalgia rheumatica. A high ESR is one of the main test results used to confirm these diagnoses. It is also used to monitor disease activity and response to therapy in both these conditions. Almost all cases present with an elevated ESR, though a normal ESR should not be used to rule out these conditions.

Case 1: A 70 year old White female was admitted to the ER complaining of throbbing headache and blurry vision. She stated that the headache started 2 days ago, had been at her temples at first but in the past few hours was getting worse. She stated that she was prompted to come to the ER because now her whole scalp hurt, and her vision was blurry. A CBC, Basic panel, CRP and ESR were ordered. The CBC results were unremarkable, other than and increased platelet count of 480,000/µL. ESR was 110 mm/hr. Basic panel results were normal. CRP was 2.51 md/dL.

The patient was started on prednisone immediately, and a temporal artery biopsy was scheduled, with a suspicion of temporal arteritis (TA), also known as giant cell arteritis (GCA). TA is an autoimmune disease that causes inflammation of the temporal arteries. Under the microscope, the inflamed cells of these arteries look giant, which is how the disease got its name. The inflammation causes constriction of the arteries, can affect chewing and eating, and may cause blindness if not treated promptly. Treatment of choice are corticosteroids, often prescribed for at least a year. Symptoms are monitored frequently and lab results, including the ESR, can be used to monitor the condition and response to treatment.

If you are still wondering if the ESR should be discontinued as a useful test, we are now seeing patients with COVID infection and elevated ESRs. Over the past 2 years, several articles have been written about elevated ESRs in COVID-19 patients. One study aimed to evaluate the usefulness of ESR in distinguishing severe from non-severe COVID-19 cases. The study suggests that severe COVID-19 cases are associated with higher elevations of ESR, as compared to non-severe cases. A case report of a patient recovering from COVID described an increased ESR. The high ESR persisted for a long time even after the patient recovered from COVID-19, while no other inflammatory processes or other conditions known to raise ESRs were found.

Case 2: My second case is a case of a 58 year old woman who presented with an earache and a pulsing temporal headache. Ear infection was ruled out and the patient was referred to ophthalmology for possible TA. The patient’s CRP was elevated but her ESR and platelet counts were within normal reference range. The patient was COVID tested as part of a pre-op workup before temporal artery biopsy and the COVID-19 test came back positive. There have been cases in literature in the last year of this new set of symptoms in COVID-19 patients. The conclusion from these cases is that if a patient appears with symptoms consistent with TA with an elevated CRP but with a normal ESR and platelet counts, that the patients should be tested for COVID.

The ESR is one of the oldest laboratory tests still in use. The study of the sedimentation of blood was one of the principles on which ancient Greek medicine was based. In the 1700’s, physicians noticed that the rate of red blood cell sedimentation changed during illness. This theory was first introduced as a laboratory test over 100 years ago. Depending on the historic accounts and articles you read, it was first described by a Polish physician, Edmund Biernacki, in 1897, or by a Swedish physician, Robert Fahraeus, in 1915. Biernacki proved the connection between the rate of sedimentation and the amount of fibrinogen in the blood and suggested using the ESR in diagnostics. Alf Vilhelm Albertsson Westergren (a familiar name!) also presented a similar description of the ESR. In the early 1920’s. Dr Westergren went further to develop the blood drawing technique and defined standards for the ESR. To this day, the Westergren Erythrocyte Sedimentation Rate method is recognized as the gold standard reference method for ESR measurement.

Image 2. Manual Westergren ESRs

The sed rate measures the rate at which erythrocytes sediment by gravity, in mm/hour. RBCs usually repel each other due to zeta potential and aggregation is inhibited. In conditions with increased fibrinogen or immunoglobulins, these proteins coat the RBCs, promoting aggregation. The RBCs form rouleaux which settle faster than individual RBCs. In conditions such as anemia, the ESR will be high because with a lower hematocrit, the velocity of the upward flow of plasma is altered and red blood cell aggregates fall faster. In polycythemia the increased blood viscosity can cause a lower ESR. In sickle cell anemia, and other conditions such as spherocytosis, the RBCs are abnormally shaped and will not form rouleaux easily, thus decreasing the ESR.

The ESR is an easy, inexpensive, non-specific test that has been used for many years to help diagnose conditions associated with acute and chronic inflammation. An elevated ESR is not associated with a specific diagnosis; therefore, it must be used in conjunction with other tests. Conversely, a normal ESR cannot be used to exclude the presence of significant disease. The ESR should also not be used as a screening test in asymptomatic patients. Since fibrinogen is an acute-phase reactant, the ESR is increased in many inflammatory and neoplastic conditions that increase fibrinogen, including diabetes, infection, pelvic inflammatory disease, lupus. rheumatoid arthritis, acute coronary syndrome, and neoplasms. However, noninflammatory factors such as older age, female gender, and pregnancy can also cause elevation of the ESR. 

Historically, the ESR was used to indicate inflammatory conditions and monitor disease progression or response to treatment. More specific tests have been developed for many of these conditions, but the ESR still has its advantages. Interestingly enough, for a test that 12 years ago was on the ‘antiquated’ list, in the past 2 years there have been over 50 scientific journal articles written about the ESR. The ESR can eliminate unnecessary testing and help decrease medical costs. It has its advantages in small labs and in rural areas because it can provide quick results without expensive instrumentation. For labs that do not perform more sophisticated tests such as CRP and procalcitonin, the ESR can provide answers without waiting for results from reference laboratories. Even though an ESR may take 1 hour, it is much faster than send out testing. It can therefore expedite a diagnosis, or normal results can give the physician and patient timely reassurance.

What is your favorite or least favorite test in hematology? Let me know and I can highlight it in a future blog!

References

  1. Au, Benjamin Wai Yin MBBS, MMed (Ophth); Ku, Dominic J. BMed, MSurg; Sheth, Shivanand J. MBBS, MS (Ophthal) Thinking Beyond Giant Cell Arteritis in COVID-19 Times, Journal of Neuro-Ophthalmology: March 2022 – Volume 42 – Issue 1 – p e137-e139
  2. Brigden ML. Clinical utility of the erythrocyte sedimentation rate. Am Fam Physician. 1999 Oct 1;60(5):1443-50. PMID: 10524488.
  3. Hale AJ, Ricotta DN, Freed JA. Evaluating the Erythrocyte Sedimentation Rate. JAMA. 2019;321(14):1404–1405. doi:10.1001/jama.2019.1178
  4. Pu, Sheng-Lan et al. “Unexplained elevation of erythrocyte sedimentation rate in a patient recovering from COVID-19: A case report.” World journal of clinical cases vol. 9,6 (2021): 1394-1401. doi:10.12998/wjcc.v9.i6.1394
  5. Tishkowski K, Gupta V. Erythrocyte Sedimentation Rate. [Updated 2021 May 9]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK557485/
  6. Alan H. B. Wu, PhDKent Lewandrowski, MD, et al. Antiquated Tests Within the Clinical Pathology Laboratory. The American Journal of Managed Care. September 2010, Volume 16, Issue 9
  7. https://emedicine.medscape.com/article/332483-workup
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.

The Cure for Expertise Loneliness

“The world is moving so fast, that we have few true experts on tomorrow. All we have are experts on yesterday.” –GYAN NAGPAL, The Future Ready Organization

One of my ingrained memories from my time at Harvard was the set of criteria needed to be promoted up the academic ranks within the medical school. At the “last” level—associate to full professor—one of the criteria was that you have an established international reputation and expertise in a particular area which is clearly demonstrated by your body of written work as well as the external opinions of 20+ people (in formal letters) who have never directly worked with you. Although that may sound like a tall order in words, it is something that musicians, actors, and influencers can easily demonstrate, although their “particular area” is not necessarily something tangible but certainly entertaining. In academia overall, achieving such documented prowess may appear easier for some fields than others. For example, behavioral psychology has spawned an entire industry of self-help books across every possible genre which all are based on the same principles; yet, no one really cares about the reproductive processes of Chytridiomycota. There are many people who call themselves experts in behavioral psychology and so many conventions, book signings, and meetings about the topic that those experts are probably never lonely. But our buddy who studies Chytridiomycota probably doesn’t have lunch with someone every day that includes a spicy discussion about mycelial separation. In certain areas of academia—for example, astronomy and physics—the inclusivity of publications and sharing of credit for collective work is so common that the above criterion really makes no sense but, at the same time, they are an integrated, harmonious community who all know each other and likely have raging keggers where nothing less than all of the known universe is discussed. Unfortunately, in the medical field, there are a plethora of expert cliques (Oncology, Dermatology, Surgery, Pathology, etc.) and then there are many individuals who are experts in their narrow area that the “cool kids” don’t really care about. COVID demonstrated that the cool kids do care when their house is on fire and those dejected experts are the only ones with a fire extinguisher—but I refuse to write any more about COVID. I am declaring it officially dead to me (but still staying on top of it). To quote a friend, I’ll just call it, “our recent unpleasantness.”

The take home is that expertise is excellent for society but for the individual who is burdened with a particular expertise that is uncommon (or unsavory), daily life can be lonely. Of course, there are people around that one can talk to, but they want to talk about mundane things like work, taxes, politics, Instagram, television shows, or actors. No one wants to get down and dirty into Chytridiomycota!! And we all know those folks—and love them—for their enthusiasm and their quirks. But this loneliness is much more common that we’d like to assume. When I was at Harvard, there were a cadre of people—all of different backgrounds and in different specialties—that were “global health” people. I have waxed on in previous blogs about the complex and expansive definition of “global health”, but the point is that anyone who identifies that way speaks a common enough language and has read the same books to have an engaging conversation about the topic. When I moved from Harvard to my current role at ASCP, the pool of immediate colleagues dwindled but my day-to-day job kept me in touch with so many global health people that I was even more engaged. Then the recent unpleasantness struck, and I relocated for remote working. I love my neighbors dearly but none of them have heard of global health. Moreover, because my day involves a computer terminal, innumerable meetings and emails, cross-coverage of activities, and evolving roles to meet the rapidly evolving world, the virtual global health quotient of my week has dwindled further. And sure, I could join a Facebook group or a WhatsApp thread or follow a Twitter thread about global health (and I do all of those things) but it still a little lonely most of the time.

Eureka! I was invited to give a talk—in person, ya’ll! With live people! —in Seattle in late April/early May at the behest of the Binaytara Foundation on Cancer Health Disparities. Most of the people who were invited to this summit were people I already knew but hadn’t seen in 2 years. I was happy to join but under the presumption that, “I’ve heard all this” but thought it would be great to see everyone. I was so wrong!!

The initial session was about health insurance coverage disparities in general and for cancer, and I savored every word. Many of you will read this and likely be highly informed in this area and others may know nothing about it. For me, I’ve been busy with global health disparities (primarily in Africa) and hadn’t paid enough attention to the continued disparities in my own back yard. I was humbled, a little ashamed, but delighted to learn. There were multiple specific projects and programs, presented by the leads, demonstrating how access to insurance programs and other payment programs massively reduced and resolved disparities in particular communities—minorities, inner city, homeless, etc. There were multiple data sets demonstrating how the Affordable Care Act had drastically increased access to care and reduced self-pay (a major barrier to proper cancer care outcomes). But it was not all “rainbows and butterflies”. There was a very upsetting presentation on the Medicaid expansion for cancer coverage which was allowed by the ACA that included descriptions of early expanding, slowly expanding, and nonexpanding states. It is important to note that this Medicaid expansion was money from the federal government to states to allow them to complete this coverage for more people with cancer including screening and early diagnosis. When the ACA became law, the federal government paid 100 percent of the cost of expansion coverage (from 2014 to 2016). After that, the federal share decreased, and now it pays 90 percent (as of 2020). Although the percentage has dropped from 100 to 90, the non-expansion states did have the opportunity to opt-in when there was 100% coverage. From Barnes et al (2021), “Early Medicaid expansion was associated with reduced cancer mortality rates, especially for pancreatic cancer, a cancer with short median survival where changes in prognosis would be most visible with limited follow-up.”1 What was also demonstrated was that, where expansion occurred, many health disparities were reduced. From Han et al (2018), “Disparities in the percentage of uninsured patients by race/ethnicity, census tract-level poverty, and rurality were diminished or eliminated in Medicaid expansion states but remained high in no expansion states, highlighting the promising role of Medicaid expansion in reducing disparities among sociodemographic subpopulations.”2 Medicaid expansion was free money from the federal government so why wouldn’t states take it if it can decrease cancer mortality and eliminate obvious disparities? According to familiesusa.org, Medicaid expansion has benefitted state economies, boosted job growth, and helped working but uninsured individuals improve their health and economic situations.3 The infographic shows the expanded, expanding, and nonexpanding states. Moreover, the decrease in the uninsured rates provided by the Medicaid expansion has provided offsetting savings (less uncompensated care provided by hospitals, more tax revenue on healthcare plans, etc.) that has more than covered state costs for the expansion. I will let you draw your own conclusions about why some states wouldn’t take free money from the government to care for minority groups and the impoverished. But all of that was just a taste of the conversations. And, for some of you, perhaps it sounds like the mating habits of Chytridiomycota. But these were my people and engaging with them for 3 days was an excellent cure for the mental loneliness of the past two years.

So, what did I learn from this event—other than a lot about health insurance, training people in disparities research, LGBTQ+ health access program, etc.? Convening is a very important part of the academic and professional and human process. Convening in person with other people in the same room creates safe dialog, allows for preposterous questions and new ideas, field tests opinions, and introduces people for more collaboration. Prior to our recent unpleasantness, with my global health team at ASCP and in my global health volunteer work at Harvard, we had been using video conferencing tools for years. I was a beta tester for Zoom before it was Zoom. I had been on 5 different meetings in one day using 5 different platforms. Videoconferencing was just a tool that we had to use to talk quickly and constantly with people all over the world. But we still had coffee in the break room, weekly in person meetings, and curbsides with other staff, etc. When the switch to complete videoconferencing occurred for all day work and, along with it, the inevitable virtual conference to replace a live meeting, the loneliness of expertise grew to an almost insufferable level. The cure, however, exists and it is in the live, in person meeting.

Note: Thanks to Matthew Schultz, Jeff Jacobs, and Suzanne Ziemnik of ASCP for their insightful ideas about this topic and input on this blog post.


  1. Barnes JM, Johnson KJ, Boakye EA, Schapira L, Akinyemiju T, Park EM, Graboyes EM, Osazuwa-Peters N. Early Medicaid Expansion and Cancer Mortality. J Natl Cancer Inst. 2021 Jul 14;113(12):1714–22. doi: 10.1093/jnci/djab135. Epub ahead of print. PMID: 34259321; PMCID: PMC8634305.
  2. Han X, Yabroff KR, Ward E, Brawley OW, Jemal A. Comparison of Insurance Status and Diagnosis Stage Among Patients With Newly Diagnosed Cancer Before vs After Implementation of the Patient Protection and Affordable Care Act. JAMA Oncol. 2018 Dec 1;4(12):1713-1720. doi: 10.1001/jamaoncol.2018.3467. PMID: 30422152; PMCID: PMC6440711.
  3. https://familiesusa.org/resources/momentum-on-medicaid-expansion/
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-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.

Microbiology Case Study: A 26 Year Old Female with Diarrhea

Case Description

A 26 year old female with a past medical history of Hemoglobin SC disease (Hb SC) and iron deficiency anemia presented to the emergency department with lower abdominal pain and diarrhea for three days. She began having multiple episodes of watery diarrhea, followed by bloody diarrhea after eating at a restaurant. During this time, she also had fever, chills, body aches, and headache. The patient had been on a course of ceftriaxone and metronidazole started three weeks prior for sore throat, ear infection, and bacterial vaginosis. She completed her metronidazole course prior to the current illness. Abdominal computed tomography revealed splenomegaly and a mildly dilated, fluid-filled appendix without evidence of infectious or inflammatory abnormalities. Hemoglobin on admission was 11.1 mg/dL (Reference Range: 11.2- 15.7 mg/dL) and MCV 62.9 fL (Reference Range: 79.4- 94.8 fL), which is similar to her baseline.

Laboratory Identification

The patient underwent work up for community-acquired diarrhea. Stool cultures grew non-typhoidal Salmonella (Image 1). Blood cultures performed at the time of admission flagged positive with gram negative rods which were also identified as Salmonella species by MALDI-TOF. The organism was susceptible to ampicillin, ceftriaxone, ciprofloxacin, and trimethoprim/sulfamethoxazole. The patient continued on intravenous ceftriaxone and responded to therapy. She was discharged home on oral ciprofloxacin.

Image 1. Salmonella Microbiologic Diagnosis using Xylose Lysine Deoxycholate agar and Triple Sugar Iron slant. A) Non-typhoidal strains of Salmonella are lactose non-fermenting, hydrogen sulfide producing (black colonies) enteric Gram-negative rods on Xylose Lysine Deoxycholate agar (XLD agar). B) Non-typhoidal strains of Salmonella are Alkaline (pink) over Acid (yellow) with the production of copious amounts of hydrogen sulfide on Triple Sugar Iron agar (TSI).

Discussion

Hemoglobin SC disease (Hb SC) is the second most common hemoglobinopathy after Sickle Cell Disease (SCD, Hb SS) globally.1 Hb SC disease occurs when a patient inherits both hemoglobin S and hemoglobin C alleles. Hemoglobin S and C variants are caused by point mutations in the hemoglobin beta- chain, and both variants lead to reduced affinity to the alpha-chain. While hemoglobin C is an abnormal form of hemoglobin that does not cause sickling on its own, when co-inherited with hemoglobin S, the beta chains polymerize, causing red cell sickling when oxygen tension is lowered in the blood.2 Patients develop anemia due to reduced red cell lifespan (27-29 days for Hb SC vs. 15-17 days for Hb SS) and subsequent destruction of red blood cells.3

Complications arise from vascular occlusion and destruction of red blood cells, leading to gallstones, pulmonary infarction, priapism, and/or cerebral infarction. Other complications include avascular necrosis of the femoral head, bone marrow necrosis, renal papillary necrosis, retinopathies, splenomegaly, and recurrent pregnancy loss. Although Hb SC patients often exhibit similar symptomology to sickle cell disease, symptoms are typically milder and present later in childhood.2,3 In comparison to patients with Hb SS, Hb SC patients have milder anemia, less frequent sickle cells, and less severe hemolysis. While Hb SC patients have fewer sickling episodes compared to Hb SS patients, Hb SC patients have more severe retinopathy and splenomegaly. It is also important to note that the enlargement of the spleen is often caused by red blood cell sequestration and the optimal function of the spleen is significantly reduced (functional hyposplenia), which can lead to increased risk of infection from encapsulated bacteria.

Diagnosis of Hb SC disease is typically made by performing hemoglobin electrophoresis (Image 2). Hemoglobin electrophoresis separates the differing varieties of hemoglobin by size and electrical charge. Capillary electrophoresis separates hemoglobin variants based on the “zone” of detection where each variant hemoglobin appears based on a reference pattern. Normal hemoglobin (A, F, A2) is easily discriminated from variant hemoglobins (S, C, E, D), and quantification allows for detection of beta-thalassemia (increased A2 fraction). While useful as a screening tool, the hemoglobin variants identified in the “zones” are not specific. For example, Hb C and Hb Constant Spring share a zone, and Hb A2 shares a zone with Hb O- Arab. Variants detected by capillary electrophoresis are confirmed by a second method, and in this case Hb SC was confirmed by acid agarose gel (Sebia Hydrogel). When subjected to acid gel electrophoresis, Hb C and Hb S migrate in separate bands, while Hb A, A2, D, and E comigrate in the “A” band, and the “F” band may contain F in addition to the glycated fraction of normal adult Hb A. Patients with Hb SC disease will have variants detected in the S and C zones in capillary electrophoresis and lack signal in the A zone.4

Image 2. Laboratory Diagnosis of Hb SC disease includes hemoglobin electrophoresis and peripheral blood smear review. A) Hemoglobin capillary electrophoresis (pH 9.4) separates F, S, C, A2, A (Sebia, Capillarys 2 Flex Piercing). B) Acid agarose gel (pH 6.0-6.2) separates hemoglobins F, A, S, and C (Sebia, Hydragel Acid QC lane).  C) Peripheral blood smear morphology showing characteristic Hb SC forms including target cells, boat shaped cells (single arrow), red cell with crystals (double arrow), and hemighost cells (triple arrow).

Examination of the peripheral blood smear from a patient with Hb SC disease (Image 2C) reveals frequent target cells, boat-shaped cells (taco shaped), and only rarely contains classic sickle cells. Hemoglobin C crystals can be seen, both free floating and inside red cells, a feature of CC and SC disease but not seen in SS disease. Hemi-ghost cells and cells with irregular membrane contractions are also more frequent in Hb SC disease. In contrast, sickle cells are rarely observed in peripheral smears from Hb SC patients.

Salmonellaeare flagellated gram negative bacilli that are members of the Enterobacterales. Salmonellosis is typically foodborne in nature and presents as a self-limiting acute gastroenteritis.5,6 However, these organisms can invade beyond the gastrointestinal tract resulting in bacteremia.6 This case presents Salmonella as a cause of bacteremia in a patient with Hb SC disease following a bout of gastroenteritis. Although there is a well-known association between SCD and invasive infections with Salmonella, the incidence of Salmonella infection in patients with Hb SC disease has not been well studied. Patients with SCD, particularly those in Africa, are at risk for developing invasive disease caused by non-typhoidal Salmonella, including osteomyelitis, meningitis, and bacteremia. It has been hypothesized that disruptions in the gut microbiome and increased permeability of enterocytes makes SCD patients more prone to invasive Salmonella infections.6 Furthermore, the compromised function of the spleen in both patients with SCD and Hb SC disease increases the risk of disseminated infection by encapsulated bacteria and Gram negative rods. The spleen plays an important housekeeping role removing old or damaged erythrocytes, but also has an important immunological function housing memory B cells, producing antibodies and macrophages that phagocytize circulating bacteria, particulates or other debris and then present the antigens to other immunological cells in the spleen.7 Although sepsis caused by Salmonella is an occasional progression of gastroenteritis, this patient’s Hb SC disease likely increased the likelihood of bacteremia because of her functional asplenia.

References

  1. Weatherall DJ. The inherited diseases of hemoglobin are an emerging global health burden. Blood. 2010;115(22):4331–6.
  2. Tim R. Randolph,24 – Hemoglobinopathies (structural defects in hemoglobin),Editor(s): Elaine M. Keohane, Catherine N. Otto, Jeanine M. Walenga,Rodak’s Hematology (Sixth Edition), Elsevier, 2020, Pages 394-423, ISBN 9780323530453, https://doi.org/10.1016/B978-0-323-53045-3.00033-7.
  3. (https://www.sciencedirect.com/science/article/pii/B9780323530453000337)
  4. Nathan, D. G., Orkin, S. H., & Oski, F. A. (2015). Sickle Cell Disease. In Nathan and Oski’s hematology and oncology of infancy and childhood (8th ed., pp. 675-714). Philadelphia, PA: Elsevier. Retrieved from https://www.clinicalkey.com/#!/content/book/3-s2.0-B9781455754144000206y.com/#!/content/book/3-s2.0-B9781455754144000206. Accessed 2022
  5. Bain, BJ. (2020) Haemoglobinopathy Diagnosis, Third Edition. Hoboken: John Wiley and Sons, Ltd
  6. Kurtz, J. R., Goggins, J. A., & McLachlan, J. B. (2017). Salmonella infection: Interplay between the bacteria and host immune system. Immunology letters190, 42–50. https://doi.org/10.1016/j.imlet.2017.07.006
  7. Lim, S.H., Methé, B.A., Knoll, B.M. et al. Invasive non-typhoidal Salmonella in sickle cell disease in Africa: is increased gut permeability the missing link?. J Transl Med 16, 239 (2018). https://doi.org/10.1186/s12967-018-1622-4
  8. Leone G, Pizzigallo E. Bacterial Infections Following Splenectomy for Malignant and Nonmalignant Hematologic Diseases. Mediterr J Hematol

-John Stack is a first year AP/CP resident at UT Southwestern Medical Center.

-Marisa Juntilla is an Assistant Professor in the Department of Pathology at UT Southwestern Medical Center. Dr. Juntilla is a board certified Clinical Pathologist and is certified in the subspecialty of Hematopathology.

-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.

-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: An Adult Presents with Hand Wound Following a Dog Bite

Case Presentation

An adult presented to the emergency department with a finger infection persisting for the past 14 days after being bitten by her dog. The finger was swollen, tender and red but the patient denied fever, chills, or purulent drainage. The patient was previously given 10 days of doxycycline and amoxicillin-clavulanic acid without any improvement. The patient underwent incision and drainage and the specimen was sent for aerobic culture and Gram stain. No organisms or WBCs were seen on the Gram stain. On day 3 of incubation, a yellow colony was observed on the chocolate agar. The colony was streaked out onto another chocolate plate for subculture (Image 1). MALDI-TOF identified this organism as Neisseria animoralis.

Image 1. Subculture of Neisseria animoralis.

Discussion

Neisseria animoralis and Neisseria zoodegmatis are primarily zoonotic organisms found as normal oral flora of cats and dogs. Both can cause wound infections in humans following animal bites. However, these organisms are under recognized animal bite pathogens, often leading to their identifications being dismissed as contaminants. While there are limited published studies on this organism, it is important to recognize its role in wound infections, as in our case. Due to lack of awareness and reduced recovery in culture, case studies have shown correlations with this organism and poor healing and chronic wound infections.

On Gram stain, N. animoralis appears as a Gram negative coccoid rod. In culture, N. animoralis is a slow growing organism that produces yellow or white colonies that are shiny and smooth. N. animaloris produces acid from glucose, but not lactose, sucrose, or maltose. MALDI-TOF is most commonly used for identification.

Limited N. animoralis treatment data are available currently. Most animal bite-related infections are polymicrobial in nature and thus, antibiotic treatment is broad spectrum to cover the most common aerobic and anaerobic organisms.

Resources

  • Johannes Elias, Matthias Frosch, and Ulrich Vogel, 2019. Neisseria, In: Carroll KC, Pfaller MA Manual of Clinical Microbiology, 12th Edition. ASM Press, Washington, DC. doi: 10.1128/9781683670438.MCM.ch36
  • Heydecke A, Andersson B, Holmdahl T, Melhus A. Human wound infections caused by Neisseria animaloris and Neisseria zoodegmatis, former CDC Group EF-4a and EF-4b. Infect Ecol Epidemiol. 2013;3:10.3402/iee.v3i0.20312. Published 2013 Aug 2. doi:10.3402/iee.v3i0.20312
  • Kathryn C. Helmig, Mark S. Anderson, Thomas F. Byrd, Camille Aubin-Lemay, Moheb S. Moneim, A Rare Case of Neisseria animaloris Hand Infection and Associated Nonhealing Wound, Journal of Hand Surgery Global Online, Volume 2, Issue 2, 2020, Pages 113-115, ISSN 2589-5141 https://doi.org/10.1016/j.jhsg.2020.01.003.
  • Merlino J, Gray T, Beresford R, Baskar SR, Gottlieb T, Birdsall J. Wound infection caused by Neisseria zoodegmatis, a zoonotic pathogen: a case report. Access Microbiol. 2021;3(3):000196. Published 2021 Feb 10. doi:10.1099/acmi.0.000196

-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.