Lablogatory

Quicker Than the Eye

Len began his shift in the hematology department. He liked to use the counter-mounted safety shield when opening specimens because he did not like to wear goggles over his eyeglasses. When it was time to read differential slides, he knew he could not look into the microscope with his glasses on, so he reached up with his gloved hands, grabbed his frames and set them on the dirty hematology workbench next to the scope.

OSHA’s Bloodborne Pathogens Standard was promulgated (put into effect as law) in 1991. Its purpose was to prevent employee exposures to infectious organisms that may be present in blood or body fluids. For those employers covered, that meant creating an Exposure Control Plan, providing certain vaccinations, educating staff about exposure follow-up, and providing personal protective equipment (PPE).

Much has changed in healthcare since 1991, but the standard remains unchanged. Changing an OSHA standard does not happen often, and it does not happen quickly. In many ways, for the Bloodborne Pathogens Standard, that’s a good thing. The same protective measures must be in place in workplaces like laboratories, and despite the appearance of novel pathogens over the last 30 or so years, the basic required risk assessments and mitigation steps still apply.

Some people, however, complain that the standard doesn’t speak clearly enough about issues that have changed over time and that now need to be addressed. Do the regulations speak to personal electronic devices in the lab like smart phones, smart watches, and ear buds? There is mention of not having food or drink in the department, but what about chewing gum or candy? Sometimes you need to dig a bit deeper to discover that those issues are also addressed, even though some of those issues did not exist when the standard was written.

If you read the line, “Eating, drinking, smoking, applying cosmetics or lip balm, and handling contact lenses are prohibited in work areas where there is a reasonable likelihood of occupational exposure,” it seems very clear that OSHA is trying to prevent hand to face contact. While they did not cover every possible action, this likely includes gum chewing and touching cell phones which are then brought to the face (or worse, used at home by a toddler wanting to play). It can be argued that lab employees use telephones often on the job, and that gloved hands are near the face because of that.

So what other actions occur in your lab that could potentially create bloodborne pathogen exposure – actions that may occur every day or so quickly you don’t notice? Have you thought about wireless headsets or speaker phones in the lab? Do you look in cabinets and drawers for food or drink (especially during off-shifts)? Is gum chewing allowed in your lab (hint: if you’re in a CAP-accredited lab this is strictly forbidden)?

And what about poor Len with his glasses? Has anyone trained him to remove his gloves, wash his hands, and place his spectacles on a clean surface before using the microscope? There might be other things you did not notice. If you have an employee with hearing aids, do they remove them to answer the phone? Do some staff wear gloves when opening the lab exit door and others use bare hands? Are computer keyboards used with and without gloves? Is PPE worn into lab rest rooms? These are all instances where a lab-acquired infection could begin, and they happen in a flash. Perform risk assessments to not only locate the risks, but to implement ways to mitigate them. Magicians claim that their hands can move faster than the eye can see in order to work their tricks. Employees will perform “tricks” as well, but the outcomes may not be as entertaining. Providing safety education and observing people at work to see where other risks exist are important steps toward complying with the Bloodborne Pathogens Standard. The regulations are not new, but with updated lab policies and safety measures, they can be powerful tools to protect you and your staff from the new pathogenic threats headed our way.

–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: Abdominal Abscess from an Elderly Patient

An 85 year old female with past medical history of hypertension, hyperlipidemia and past surgical history of cholecystectomy presented to the emergency department (ED) with an abdominal pain in the left upper quadrant, which had been persistent for several days. Her vitals were BP:145/86 mm/Hg; pulse: 86 beats/minute; respiratory: 20/min; Temp: 98.3 °F (36.8 °C); SpO2-98%. Her medical history revealed that she had a diagnostic laparoscopy, common bile duct exploration, and stone extraction nine months ago. Since then, the patient had a chronically draining abdominal sinus for which she underwent diagnostic laparoscopy and multiple benign peritoneal implant biopsies 5 months prior to the current event.

Examination of the LUQ revealed a fluctuant lump in the LUQ, which was close walled with no purulence or drainage. The CT abdomen demonstrated an increased infiltration of the left rectus abdominis, left anterior abdominal wall muscles, and subcutaneous tissues in the upper abdomen, with a suspicion for infectious etiology.

The patient was evaluated by general surgery for abscess at the LUQ. The abscess was drained, the fluid was sent for a bacterial culture, and the patient was started on IV vancomycin and Zosyn. Blood cultures were collected but had no growth. The pathology report of peritoneum implants and soft tissue biopsies showed focal necrotizing granulomatous inflammation but negative special stain for fungi (GMS-F) and acid-fast bacilli (AFB). The Gram stain of her abscess fluid culture was negative with a few neutrophils. However, her culture grew spready colonies on blood and chocolate agar after 4 days of incubation (Figure 1). Since the initial Gram stain was negative, Kinyon stain was performed and was positive (not shown). It was identified by Matrix-assisted laser desorption ionization Time of Flight (MALDI-ToF) as Mycobacterium fortuitum species.

Figure 1. Dry spready colonies on Chocolate agar plate.

Discussion

There has been recent evidence of an increased prevalence of Nontuberculous Mycobacterium (NTM), and it is becoming a major public health concern.^1,2 NTM is a diverse group of ubiquitous, environmental, acid-fast organisms that can produce a wide range of diseases, most of which are found in skin and soft tissue infections (SSTI).³ Historically, NTM has been classified into Runyon groups based on the colony morphology, growth rate, and pigmentation.⁴ Identification is made with rapid molecular diagnostic technology. However, grouping the species of NTM is based on the growth rates and divided into rapidly growing mycobacteria (RGM) and slowly growing mycobacteria (SGM).

RGM includes species that grow on the media plates within 7 days and subdivided into 5 groups based on pigmentation and genetic similarity: Mycobacterium fortuitum, Mycobacterium chelonae/abscessus, Mycobacterium mucogenicum, and Mycobacterium smegmatis. Most SSTIs commonly associated with surgery and cosmetic procedures are caused by 3 RGM species: M fortuitum, M abscessus, and M chelonae. These infections are nonspecific in their clinical presentations and may present with abscesses, cellulitis, nodules, ulcers, panniculitis, draining sinus tracts, folliculitis, papules, and plaques. There is a delay in diagnosis of these infections, as mycobacterial cultures are not routinely performed on surgical wound infections or skin biopsy specimens which are essential for an accurate diagnosis, especially because the treatment varies depending on the species and its sensitivities.⁵

M. Fortuitum is a Gram positive, acid-fast, aerobic rod-shaped, saprophytic, rapidly growing NTM that is typically considered an opportunistic pathogen. They are widely distributed in the nature and can be isolated from soil, dust, natural surface and municipal water, wild and domestic animals, fish, hospital environment, contaminated medical instruments, and implants. Common culture media include Middlebrook 7H10 or 7H11 agar, BACTEC 12B broth and 5% sheep blood agar or chocolate agar. These organisms may not stain well with the Ziehl-Neelsen or Kinyoun method and may not be recognized readily with the fluorochrome method due to lipid rich long-chain mycolic acids in their cell walls. Because of the high mycolic acid content in the cell wall, it does not stain well by the Gram stain, which is likely the reason for the negative Gram stain results in our patient abscess culture.

It is well known that older biochemical tests are replaced by newer diagnostic methods including matrix associated laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and molecular methods, including line probe hybridization assays, as well as 16S ribosomal RNA sequencing. DNA line probe assays provide a rapid means of identification and currently there are two commercially available assays: INNO-LiPA MYCOBACTERIA v2 assay (Fujirebio Europe, Ghent, Belgium) and GenoType assay (Hain Lifescience GmbH, Nehren, Germany). However, neither of them is currently FDA-approved, and therefore, the use is largely restricted to the public health or reference laboratories in United States. Studies utilizing these lines probe assays have reported satisfactory sensitivity and specificity.^6,7,8,9 Notably, a study by Fida et al., reported a case of Mycobacterium smegmatis that was misidentified as Mycobacterium fortuitum by a DNA line probe assay.

In our case, histopathology reported necrotizing granulomas with a negative AFB stain. There has been literature evidence reporting that these SSTIs cases present with a mixed suppurative-granulomatous inflammation, with only a few cases showing well-formed granulomas.¹⁰ In most of these pathological cases, mycobacterial stains, such as AFB or FITE, are negative. However, negative stains do not entirely exclude the diagnosis and hence medical management by clinicians should be based on the culture, which remains the gold standard method for identification of AFB.¹¹

There is limited literature evidence of M fortuitum as an opportunistic pathogen causing disseminated infection especially in immunosuppressed patients or receiving steroids.¹² A case report of chyluria caused by Mycobacterium fortuitum infection in a 64-year-old male, who was successfully treated with two weeks of amikacin, trimethoprim-sulfamethoxazole and levofloxacin followed by 24 weeks of levofloxacin and doxycycline.¹³ Another case of Mycobacterium fortuitum osteomyelitis of the cuboid bone following a penetrating plantar trauma. The patient underwent a single-stage surgery and resolved the infection after 5 months of treatment with gentamicin-/vancomycin.¹⁴ M. Fortuitum is resistant to all antituberculosis drugs but susceptible to macrolides, amikacin, doxycycline, fluoroquinolones, and trimethoprim-sulfamethoxazole. Therefore, an aggressive and prolonged NTM treatment is required to completely clear the infection and reduce the recurrence.

References

-Preeti Malik, M.D, MPH, PGY2 Pathology resident at Montefiore Medical Center.

-Phyu M. Thwe, PhD, D(ABMM), MLS(ASCP)CM is Associate Director of Infectious disease testing laboratory at Montefiore Medical Center, Bronx, NY. She completed her CPEP microbiology fellowship at the University of Texas Medical Branch in Galveston, TX. Her interest includes appropriate test utilization and extra-pulmonary tuberculosis.

Determining Time of Death: Separating Science from Pseudoscience

One of the most common questions I’m asked by family members is “do you know when they died?” If death occurs in the hospital, or is witnessed, the time of death isn’t controversial. It’s common though in forensics that people may not be found for hours, days, weeks, or more. Forensics television shows usually depict an investigator measuring body temperature at the scene, and then confidently declaring they’ve been dead for 44 hours. Unfortunately, there aren’t any existing methods that actually give that level of precision – but there is a way we can systematically approach the question.

When determining time of death (TOD), it’s most important to keep in mind that it will be an estimate. The estimate starts with the “window of death” – the time between when the decedent was last known alive and when their body was found. The smaller this window, the greater accuracy is possible.

Once the window is known, one can assess postmortem changes of the body. Livor mortis is the gravity-dependent settling of blood within vessels, which can appear as soon as twenty minutes after death. Sparing of lividity will be present in areas of pressure, such as parts of the body pressed against the floor or with tight clothing. Livor is initially blanchable, but after 8 to 12 hours blood extravasates from vessels and it becomes “fixed”. Clearly though, this only allows one to differentiate between ‘less than’ or ‘greater than’ 8 to 12 hours.

Rigor mortis (stiffening of the body after death) occurs because of postmortem ATP depletion. Muscle fibers require a supply of ATP to both contract and relax – once ATP levels are sufficiently low, muscle will remain contracted until the fibers are broken down by decompositional changes. Generally speaking, rigor starts to develop within an hour of death, peaks from 12 to 24 hours, and dissipates by 36 hours. However, these are average intervals. The onset of rigor is hastened by vigorous physical activity, seizures, electrocution, or increased body temperature, which preemptively deplete ATP. Rigor is also harder to detect in people with low muscle mass (e.g. infants), and can’t be assessed in frozen bodies with those with extensive thermal damage.

Cooling of the body after death, known as algor mortis, is similarly prone to interfering elements. One can find many formulas for estimating the time of death based on the temperature of the body – unfortunately, none of them are particularly useful because of the assumptions that must be made. Change in temperature after death is affected by numerous variables, including body habitus, clothing, wind, actual body temperature at the time of death (not many people are constantly at 98.6℉), sepsis, terminal seizures, and many others. If the environment is warmer than the body, the temperature can even increase after death.

I’ll briefly mention vitreous potassium measurement, which is probably the most recently discovered (and debunked) “holy grail” of time of death. Similar to algor and rigor mortis, vitreous potassium does a reasonably decent job predicting time of death in a controlled experiment – but in this line of work, people don’t tend to die in controlled environments.

At the end of the day, time of death is best estimated by thorough scene investigation, correlated with the evidence the body provides. Newspapers or mail not retrieved from the mailbox, expiration dates on perishable groceries, last refills of prescriptions, and unreturned text messages or phone calls can all narrow down the window of death.

As stated earlier, the longer the interval between death and discovery of the body, the more difficult time of death determination becomes. In advanced decomposition, there is no rigor, livor, or algor remaining to assess (there may even be scant residual soft tissue). In one such situation, despite months of a potential “window of death”, dates on unopened bills and crossed-off calendar dates helped us place the time of death within one or two days. It’s not as flashy as multivariate equations for temperature or potassium levels, but it’s far more accurate and scientifically defensible.

*Image 1. The quilting pattern of this decedent’s mattress is visible in the livor mortis on his back*.

Image 2. This decedent’s right arm is defying gravity due to rigor – he was initially face down, and his arm musculature became temporarily fixed in this position. Rigor can be forcibly broken if needed, but will also break down as decomposition proceeds.

-Alison Krywanczyk, MD, FASCP, is currently a Deputy Medical Examiner at the Cuyahoga County Medical Examiner’s Office.

MRSA Testing

Methicillin-resistant Staphylococcus aureus (MRSA) is a well-known cause of bacteremia, pneumonia, skin and soft tissue infections, and osteomyelitis, resulting in significant morbidity and mortality worldwide.¹ Many testing methods (e.g. MALDI-TOF with susceptibility testing, molecular, chromogenic agar) have been developed for identification of MRSA and clinical microbiology laboratories will often use more than one. On occasion this leads to discrepant results which can be challenging to resolve and report.

How does methicillin resistance work?

Staphylococcus aureus (SA)has a peptidoglycan cell wall containing alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) molecules with peptide chains reinforced by crosslinks. Crosslinking is mediated by penicillin-binding proteins (PBPs), which are the targets of beta-lactam antibiotics such as penicillins and cephalosporins.² In methicillin-sensitive S. aureus (MSSA), these antibiotics bind PBPs and prevent formation of crosslinks, thus disrupting cell wall synthesis. However, methicillin resistance can occur if the PBPs are altered. MRSA produces PBP homologues such as PBP2a (encoded by the mecA gene) or more rarely, PBP2c (encoded by mecC), which don’t allow beta-lactam antibiotics to bind strongly so crosslinking occurs.^3,4

What tests are used to identify MRSA?

MRSA testing can be genotypic or phenotypic, but most cannot be performed directly on patient samples. With molecular testing, we can detect mecA and/or mecC, the genes most commonly responsible for methicillin resistance. However, positive molecular results on a direct specimen source (e.g., positive blood culture) cannot be definitively attributed to SAif other mecA-harboring organisms such as methicillin-resistant Staphylococcus epidermidis are also present.⁵

When there is a pure isolate of SA growing in culture, lateral flow assays and latex agglutination tests can be used to interrogate the presence of mecA. Both lateral flow assays and latex agglutination tests detect PBP2a using antibodies specific to this alternative penicillin-binding protein. Chromogenic agars are a modern-day biochemical test, taking advantage of specific enzymes produced by MRSA (e.g. phosphatase) which cleave chromogens in the media.⁶

Disk diffusion and broth/agar dilution are the standard phenotypic methods for quantitating antimicrobial resistance in SA growing in bacterial culture. Despite the name, methicillin is no longer used for testing or treatment of MRSA. Per Clinical and Laboratory Standards Institute, oxacillin-resistant and cefoxitin-resistant SA should both be reported as MRSA and considered resistant to all beta-lactam antibiotics.⁷

Why don’t my test results match?

Although detection of the mecA gene or its protein product PBP2a are the standard⁷, mixed MSSA and MRSA cultures can lead to discrepant results. Another source of genotypic-phenotypic discrepancy are mecA mutations where the gene is still present and detected, but functional PBP2a is no longer produced. PBP2c only shares ~70% homology to PBP2aand is not detected by latex agglutination assays^4-5, and mecC-mediated MRSA might be resistant only to cefoxitin and not oxacillin⁷. Other mechanisms of MRSA resistance are still being studied and not all are included on molecular test panels.

References

Turner, N.A., Sharma-Kuinkel, B.K., Maskarinec, S.A. et al. Methicillin-resistant Staphylococcus aureus: an overview of basic and clinical research. Nat Rev Microbiol 17, 203–218 (2019). https://doi.org/10.1038/s41579-018-0147-4
Sawa, T., Kooguchi, K. & Moriyama, K. Molecular diversity of extended-spectrum β-lactamases and carbapenemases, and antimicrobial resistance. j intensive care 8, 13 (2020). https://doi.org/10.1186/s40560-020-0429-6
Srisuknimit V, Qiao Y, Schaefer K, Kahne D, Walker S. Peptidoglycan Cross-Linking Preferences of Staphylococcus aureus Penicillin-Binding Proteins Have Implications for Treating MRSA Infections. J Am Chem Soc. 2017 Jul 26;139(29):9791-9794. doi: 10.1021/jacs.7b04881.
Ballhausen B, Kriegeskorte A, Schleimer N, Peters G, Becker K. The mecA homolog mecC confers resistance against β-lactams in Staphylococcus aureus irrespective of the genetic strain background. Antimicrob Agents Chemother. 2014 Jul;58(7):3791-8. doi: 10.1128/AAC.02731-13.
Lakhundi S, Zhang K. Methicillin-Resistant Staphylococcus aureus: Molecular Characterization, Evolution, and Epidemiology. Clin Microbiol Rev. 2018 Sep 12;31(4):e00020-18. doi: 10.1128/CMR.00020-18.
Flayhart D, Hindler JF, Bruckner DA, et al. Multicenter evaluation of BBL CHROMagar MRSA medium for direct detection of methicillin-resistant Staphylococcus aureus from surveillance cultures of the anterior nares. J Clin Microbiol. 2005;43(11):5536-5540. doi:10.1128/JCM.43.11.5536-5540.2005
CLSI Performance Standards for Antimicrobial Susceptibility Testing M100, 32nd edition. (2022) Clinical and Laboratory Standards Institute

– Angelica Moran, MD, PhD is a clinical microbiology fellow at University of Chicago Medicine and NorthShore University Healthsystem and research fellow at the Duchossois Family Institute. She is interested in translational research developing clinical laboratory diagnostics for precision medicine and the microbiome.

-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.

Dying in a Winter Wonderland: Staying Safe as the Temperature Drops

A 40 year old man was found deceased in a parking garage in a Midwest city. It was late October and had rained the previous evening. He was identified by his sister who was a tenant in the adjacent apartment building. Unknown to her, he had recently been discharged from the hospital after a one-week psychiatric admission. His sister stated he was homeless and would occasionally sleep in the parking garage for shelter.

At the scene the decedent was prone on the ground, clad only in a pair of boxers. His water-soaked shoes, socks, sweatpants, and shirt were strewn about him. Autopsy revealed an atraumatic, thin adult man. Prominent pink discoloration was noted over the hips and knees. Internal examination showed only patchy black-brown discoloration of the gastric mucosa and pale kidneys. Histology was remarkable for subnuclear vacuolization of the renal tubular epithelium. The cause of death was certified as environmental hypothermia, and the manner of death accidental.

Hypothermia is defined as a core body temperature below 95℉ (35℃) and can result from endogenous illnesses like hypothyroidism or sepsis. The most common cause, though, is exposure to cold environments. On exposure, the hypothalamus initiates shivering and increases cellular metabolism to produce heat. Another crucial survival response is vasoconstriction, particularly of vessels in skin and skeletal muscle. If the overall loss of heat overtakes the body’s ability to produce or retain heat, hypothermia will result.

Developing hypothermia doesn’t require frigid weather – in dry air, temperatures of 50℉ can still result in hypothermia. Wind removes warmed air surrounding the body, and water conducts heat three times faster than air; therefore, with either of these factors present, people can develop hypothermia at even warmer temperatures,

The autopsy findings of hypothermia are not specific. External examination may show bright pink discoloration of the skin over joints (“frost erythema”). There may be black-brown spots on the gastric mucosa, (“Wischnewsky spots”), thought to result from terminal vasodilation of submucosal vessels. The kidneys may be pale with microscopic subnuclear vacuolization of the tubular epithelium (the “Armanni-Ebstein” lesion). Acute hemorrhagic pancreatitis has also been described. However, these findings require a period of survival to develop—many cases, particularly if the decedent succumbs quickly, show no findings at all. The diagnosis of hypothermia therefore relies heavily on scene investigation. “Paradoxical undressing” (demonstrated in this case), refers to the phenomenon of a terminally hypothermic person taking off their clothes. This is caused by a feeling of warmth resulting from failure of vasoconstriction in the skin, and contributed by altered mentation.

Those at greatest risk are people spending extended time outdoors, including the homeless and outdoor recreationalists. The elderly and very young have a lower ability to centrally regulate body temperature. Children’s increased body surface area also leads to more rapid heat loss. People who are intoxicated with alcohol or drugs may not sense the cold or lack judgment to seek shelter. Alcohol also acts as a vasodilator, impairing vasoconstrictive adaptation to cold.

As the weather cools down, be mindful of how easily hypothermia can develop. Temperatures can be above freezing, yet those who are vulnerable are still at risk of hypothermia. Prepare yourself well for any snowy excursions, and keep an eye on those in your community who may not be able to seek shelter.

*Stomach mucosa showing spots of black or dark brown discoloration*
*known as Wischnewsky spots. These are not specific to hypothermia and may just be an indicator of physiologic stress.*

*Bright pink discoloration over the knees, or “frost erythema”.*

*Pallor of the renal cortices corresponds to the microscopic “Armanni-Ebstein” lesion. This isn’t specific to hypothermia and can be seen in ketoacidosis from any cause.*

-Alison Krywanczyk, MD, FASCP, is currently a Deputy Medical Examiner at the Cuyahoga County Medical Examiner’s Office.

Microbiology Case Study: Bacteremia with Long, Gram Negative Rod in a 34 Year Old Patient

Case History

A 34 year old male presented to the emergency department (ED) with acute onset abdominal pain, nausea, vomiting, persistent fever, and chills. His physical examination at that time was consistent with appendicitis. Patient was treated with Zosyn for broad coverage. Imaging showed a normal appendix. Three days later after blood was drawn, his blood cultures flagged positive for gram negative, elongated, thin rods. Growth was determined to be Fusobacterium mortiferum by MALDI-TOF. Ampicillin/sulbactam was started and patient was given Amoxicillin/clavulanic acid for outpatient treatment. Further follow-up of the patient showed normal white blood count and normal urinalysis. Repeat blood cultures were negative.

Images of Gram stain demonstrating long, slender, gram negative rods (top) and bacterial growth on anaerobic plate (bottom) from positive blood culture bottle.

Discussion

Fusobacteria are anaerobic, gram negative, spindle-shaped rods with pointed ends. They are part of the upper respiratory and gastrointestinal flora in humans but can cause diseases ranging from tonsillitis to septic shock.¹ Fusobacterium nucleatum and necrophorum are commonly isolated in human diseases, although other species such as Fusobacterium mortiferum, as described in our case, have occasionally been documented as a secondary cause of septicemia ² or bacteremia ¹and in rare instances implicated in the development of thyroid abscess.³

F. nucleatum is a member of oropharyngeal flora and unsurprisingly involved in gingival and periodontal diseases.⁴ It has been also described as the most likely cause of extra-oral infections among oral anaerobes.⁵ F. nucleatum has been detected in various fetal and placental tissues associated with adverse pregnancy outcomes, such as preeclampsia, chorioamnionitis and preterm rupture of membranes.⁶ Recent studies have reported this species to be abundant in colon, esophageal carcinoma, pancreatic and breast cancers. It is associated with poor prognosis in colon, rectal, pancreatic and esophageal cancers by promoting pro-tumorigenic immune microenvironment and reduction in the number of tumor-infiltrating lymphocytes.^{7, 10} One of the proposed theories is the involvement of the Fap2 virulence factor that has been described to inhibit tumor cell clearance in colorectal cancer cells.⁸ The other commonly isolated species is F. necrophorum, which is associated with oropharyngeal infection followed by septic thrombophlebitis of the internal jugular vein with sepsis and metastatic diseases typically involving the lungs. This syndrome is known as Lemiere’s disease first described in 1936 by Andre Lemierre. F. necrophorum usually causes infection in young, otherwise healthy adults in contrast to F. nucleatum¹ which is associated more with the elderly population. According to Afra et al most of the mortality cases were due to F. nucleatum as opposed to F. necrophrum. This could be attributed to co-morbidities in elderly patients with positive F. nucleatum cultures.

Fusobacterium species can be identified using mass spectrometry MALDI-TOF. Typically, Fusobacterium species are resistant to vancomycin, but susceptible to colistin and kanamycin disk identification tests; however, F. nucleatum is susceptible to all three drugs. F. mortiferum and F. varium grow in the presence of bile. F. necrophorum shows positive indole and negative nitrate testing. Sequencing of the 16S RNA gene and 16S-23S rRNA gene spacer region can be used to determine the different species^3,9

Fusobacterium species are usually susceptible to penicillin, clindamycin, metronidazole, and chloramphenicol and resistant to macrolides. F. nucleatum and F. necrophorum may produce beta-lactamases.³ In rare cases, surgical intervention is warranted for abscess formation.

References

Afra K, Laupland K, Leal J, Lloyd T, Gregson D. Incidence, risk factors, and outcomes of fusobacterium species bacteremia. BMC Infect Dis. 2013;13(1). doi: 10.1186/1471-2334-13-264.
Prout J, Glymph R. Fusobacterium mortiferum septicemia. Clinical Microbiology Newsletter. 1985;7(4):29. doi: 10.1016/s0196-4399(85)80052-0.
Stavreas NP, Amanatidou CD, Hatzimanolis EG, et al. Thyroid abscess due to a mixed anaerobic infection with fusobacterium mortiferum. J Clin Microbiol. 2005;43(12):6202. doi: 10.1128/jcm.43.12.6202-6204.2005.
Moore WE, Moore LV. The bacteria of periodontal diseases. Periodontol 2000. 1994 Jun;5:66-77. doi: 10.1111/j.1600-0757.1994.tb00019.x. PMID: 9673163.
Bolstad AI, Jensen HB, Bakken V. Taxonomy, biology, and periodontal aspects of Fusobacterium nucleatum. Clin Microbiol Rev. 1996 Jan;9(1):55-71. doi: 10.1128/CMR.9.1.55. PMID: 8665477; PMCID: PMC172882.
Han YW. Fusobacterium nucleatum: A commensal-turned pathogen. Current Opinion in Microbiology. 2015;23:141. doi: 10.1016/j.mib.2014.11.013.
Alon‐maimon T, Mandelboim OO, Bachrach G. Fusobacterium nucleatum and cancer. Periodontology 2000. 2000;89(1):166. doi: 10.1111/prd.12426.
Umaña A, Sanders BE, Yoo CC, Casasanta MA, Udayasuryan B, Verbridge SS, Slade DJ. Utilizing Whole Fusobacterium Genomes To Identify, Correct, and Characterize Potential Virulence Protein Families. J Bacteriol. 2019 Nov 5;201(23):e00273-19. doi: 10.1128/JB.00273-19. PMID: 31501282; PMCID: PMC6832068.
Garcia-Carretero R, Lopez-Lomba M, Carrasco-Fernandez B, Duran-Valle MT. Clinical features and outcomes of fusobacterium species infections in a ten-year follow-up. The Journal of Critical Care Medicine. 2017;3(4):141. doi: 10.1515/jccm-2017-0029.
Brennan CA, Garrett WS. Fusobacterium nucleatum — symbiont, opportunist and oncobacterium. Nat Rev Microbiol. 2018;17(3):156. doi: 10.1038/s41579-018-0129-6.

-Dr. Hayk Simonyan was born and raised in Yerevan, Armenia. He attended Yerevan State Medical University after Mkhitar Heratsi where he received his doctorate degree. He did his research at The George Washington University. His studies were focused on transcription factor activation in the SFO-PVN axis that leads to cardio-metabolic changes mediated by obesity, oxidative stress, and angiotensin-II. One of his other projects included collaboration with the National Cancer Institute, working on alternative treatment for glioblastoma multiforme. His academic interests include surgical pathology and molecular. In his spare time, Hayk enjoys spending time with family, playing soccer, tennis, and skiing. Hayk is pursuing AP/CP training.

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

Histio Makes History

An 81 year old female presented to the head and neck clinic after being diagnosed with cutaneous T cell lymphoma of the posterior mid-parietal scalp at an outside institution. She was initially treated with Brentuximab every three weeks but developed significant toxicities. The patient’s previous “T cell lymphoma” material was reviewed at our institution and the immunophenotypic report described the neoplastic cells as being positive for CD45, CD2, CD4, BCL6+, CD3 (subset), and CD123 (scattered), while negative for CD7, CD8, CD20, CD30, CD56, EBER ISH, PAX5, and lysozyme. Immunohistochemical slides were not provided for review. Flow cytometric analysis determined that there was no immunophenotypic evidence of a clonal T cell population in the patient’s peripheral blood.

A second scalp biopsy was performed at another outside institution, and the findings were similar to the parietal scalp; however, there were atypical pleomorphic cells which displayed irregular contours, hyperchromasia, and multiple nucleoli. The atypical cells were predominantly positive for CD4 and diffuse positivity for CD1a. These same pleomorphic cells were negative for CD3, CD8, CD20, CD30, ALK1, BCL6, CD56, EBER, AE1/AE3, SOX10, Desmin, PAX5, MUM1, CD5, and Cam 5.2.

The smears contained large, highly pleomorphic cells with irregular, elongated, and multilobated nuclei, frequent nuclear grooves and folds, fine chromatin, prominent nucleoli, and variable amounts of pale, eosinophilic cytoplasm, alt.

The outside tissue block on the original scalp biopsy was requested, and our pathology department performed additional immunostains. The neoplastic cells of interest were positive for CD1a, S100, CD68 (a small subset), and negative for lysozyme, CD21, CD30, and CD3. Ki67 proliferation index was interpreted at approximately 60%. An unstained FFPE tissue section was sent to a reference laboratory, and the neoplastic cells were strongly positive for Langerin.

While the Brentuximab treatment initially appeared to have a positive impact on the overall disease burden, the PET CT following 3 cycles showed a mixed response, including resolution of cervical lymphadenopathy and identification of multiple new lung nodules and bulky mediastinal lymphadenopathy. Between that and numerous reported toxicities, the treatment protocol was discontinued. The patient was then referred to radiology for a CT-scan guided right lower lobe lung biopsy measuring 2.2 x 1.3 centimeters with an SUV or 29.6.

In the CT Scan suite, we received multiple FNA passes from the interventional radiologist and made air-dried and alcohol-fixed smears, rinsing the residual needle material into a tube of balanced salt solution for a cell block preparation. We determined our specimen was adequate for scant tumor cells, as depicted on the Diff-Quik smears below.

Images 1-2. Lung, right lower lob, CT-guided FNA. Diff-Quik stained smears.

In comparison to the material from the second scalp biopsy, the cells from the lung biopsy appeared identical. Our Pap-stained smears and H&E cell block sections also demonstrated the highly pleomorphic cells described above.

Images 3-6. Lung, Right Lower Lobe, CT-guided FNA. 3-4: Pap-stained smears, 5-6: H&E sections (5: 100x, 6: 400x).

Immunostains performed on the cell block slides with adequate controls show that the tumor cells are positive for CD1a, CD4, partially positive for CD45 and S100, negative for AE1/3, TTF-1, and p40.

Images 7-8. Lung, Right Lower Lobe, CT-guided FNA. Cell block section immunohistochemistry. 7: CD1a-positive; 8: partially S-100-positive.

Our pathologists felt the cells from the second scalp biopsy and the lung biopsy were representative of a Langerhans cell sarcoma, a form of malignant histiocytosis, rather than a T-cell lymphoma. It is possible that the first scalp biopsy’s diagnosis of T-cell lymphoma was due to sampling error and the pleomorphic cells of interest were missed. The Ki-67 proliferative index of 60% helped to distinguish between Langerhans cell histiocytosis and Langerhans cell sarcoma.

Molecular testing performed on the core biopsy was negative for a BRAF mutation and positive for an NF1 inactivating mutation. The tumor may then be sensitive to mTOR inhibitors and MAPK pathway inhibitors, such as MEK inhibitors. Appeals for a MEK inhibitor were denied by insurance, but fortunately, the tumor also demonstrated high PD-L1 expression at 90%, making this specific patient a candidate for pembrolizumab, which was fully covered by insurance.

____________________________________________________________________________________________

I can’t help but think about the disparities associated with cancer and the inaccessibility of potentially lifesaving or life-prolonging treatments. Sure, there may be viable alternatives, such as this case, but what if we had equal access to cutting edge, personalized therapies? What if the only therapy available was too costly to bear? Just because a cancer might be rare, such as Langerhans cell sarcoma, it doesn’t mean access to a proven effective therapy should also be rare. Even with drug assistance programs, so many patients face the harsh reality of tapping into their life savings to just to save their own life. When we became medical laboratory professionals, we promised to provide timely and accurate for all of our patients. Now, it’s time that pharmaceutical companies and our healthcare system as a whole work together to provide high quality, low-cost, readily accessible and personalized treatment options to every patient. They deserve that chance to overcome or at least manage their cancer.

-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 70 Year Old with Fevers, Rigors, and Dizziness

Case Description

A 70 year old female arrived in the hospital with chief complaints of 6 days of fever, rigors, weakness, headache, and dizziness; she has a history of asthma, type 2 diabetes, supraventricular tachycardia and exercise-induced ventricular tachycardia. The patient was also seen 5 days before the current visit for abdominal pain, nausea, and fever. The abdominal pain has gone, but she has had a loss of appetite. She admitted that she sleeps with her dog in bed during that visit. No scleral icterus, rash, cough, urinary tract burning, or neck stiffness was reported on any visits.

CT scan, CBC with differential, BMP, liver function panel, Coag, blood culture, and blood parasite tests were ordered. On the CBC, the cells below were flagged for review (Figure 1).

Figure 1. A Cellavision capture of morulae inside a neutrophil.

Discussion

The round light purple dots pointed by the arrow in Figure 1 are morula indicative of Anaplasma phagocytophilum, formally named “human granulocytic anaplasmosis (HGA)”. Historically, Ehrlichia phagocytophila and Ehrlichia equi were recognized separately (Sexton & McClain, 2022). HGA is a tick-borne illness more commonly found in the northeast U.S., and the case number has continuously increased in recent years (Centers of, 2022). The tick bite is not painful, and the first symptom usually shows after about a week from the bite. Early diagnosis can be hard at the initial stage since laboratory serology tests often give negative results for the antibodies. It is essential to carefully review the clinical signs and symptoms, travel history, outdoor activity, and animal contacts (Centers of, 2022). PCR is the most sensitive and specific method of diagnosis. Blood smears can be made to confirm the parasite morphology, although patients can have leukopenia leading to decreased sensitivity.

Lab results showed critical hyponatremia (121 mmol/L) and thrombocytopenia (33 K/uL) in this case. The patient was admitted to the floor and prescribed 10 days of doxycycline.

Extreme hyponatremia related to anaplasmosis is not common, and the causing mechanism is unclear; however, all the reported cases fit the description of SIADH – syndrome of inappropriate secretion of antidiuretic hormone (Ladzinski et al., 2021).

References

Centers for Disease Control and Prevention. (2022, August 15). Epidemiology and statistics. Centers for Disease Control and Prevention. Retrieved 2022, from https://www.cdc.gov/anaplasmosis/stats/index.html
Ladzinski, A. T., Baker, M., Dunning, K., & Patel, P. P. (2021). Human granulocytic anaplasmosis presenting as subacute abdominal pain and hyponatremia. IDCases, 25. https://doi.org/10.1016/j.idcr.2021.e01183
Sexton, D. J., & McClain, M. T. (2022, March 21). Human ehrlichiosis and anaplasmosis. UpToDate. Retrieved 2022, from https://www.uptodate.com/contents/human-ehrlichiosis-and-anaplasmosis

-Sherry Xu is a Masters Student in the Department of Pathology and Laboratory Medicine at the University of Vermont Larner College of Medicine.

-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: An Elderly Adult Presenting with Foodborne Illness Related to Shellfish Consumption

Case History

An adult consumed shellfish at a restaurant. Approximately 12 hours after this dinner, the patient experienced the first signs of loose stools, fever, and abdominal cramping. The patient had watery diarrhea for the next three days with 8 bouts a day. The patient did not have a fever after the first day. The patient denied blood in stool or nausea or vomiting. The patient did not have a recent travel history and denied recent antibiotic use. On the 4^th day of symptoms, the patient was seen by their primary care provider. The physical exam was unremarkable except for dehydration. A stool and blood sample were obtained and aggressive hydration was recommended. Blood smear, complete blood panel, and basic metabolic panel resulted in normal. Shigella, Salmonella, Campylobacter, and Shiga-toxin-producing gene were not detected by PCR. The stool sample was set up for culture. Mucoid colonies were noticed after 12 hours on the blood agar plate. MALDI revealed Grimontia hollisae.

Discussion

The genera of Grimontia is one of the new members of the Vibrionaceae family. Grimontia hollisae, previously known as Vibrio hollisae, is currently the only known pathogenic species in the Grimontia genera. Vibrio hollisae was first described and named by Hickman et al. in 1982.¹ However, based on phylogenetic and phenotypical differences V. hollisae was placed into a novel genus, named Grimontia.² It is named after French microbiologist Patrick P. A. Grimont.

G. hollisae are halophilic, gram negative, oxidase-positive, indole-positive, ornithine-negative, and motile by a single polar flagellum.² One of the most important features of G. hollisae is its failure to grow on thiosulfate-citrate-bile salts-sucrose (TCBS) agar, the main phenotypical difference from vibrios.² However, it does grow well on sheep blood agar and marine agar.³ G. hollisae is generally transmitted via shellfish (mostly oysters, mussels, and prawns etc.).² However, it can also be transmitted through infected ocean water, and other foods that are cross-contaminated with the organism.⁴ To date, the person-to-person spread has not been documented.⁴

Diagnosis of G. hollisae can be challenging since it does not grow on Vibrio-selective media (TCBS agar) or on MacConkey.⁵ However, the organism grows well on blood agar plate. Spot oxidase and indole tests may be helpful to rule-in a possible Vibrio or Grimontia species in suspicious cases.⁵ It is important that the stool sample should be collected as soon as possible in patients suspicious for vibrio gastroenteritis.⁵ Cary-Blair medium should be used as transport medium.⁵

The incubation period of G. hollisae is usually 12-24 hours (ranging between 4-96 hours).⁴ It primarily causes moderate to severe gastroenteritis.³Signs and symptoms of G. hollisae gastroenteritis include fever, abdominal cramping, watery diarrhea, nausea, and vomiting. Although it is mostly self-limited, it may also cause serious conditions such as hypovolemic shock, sepsis, hepatitis, and ileus.^{3, 6-8}Rarely, grossly bloody stool can be seen in severe cases.⁹ Treatment is mostly supportive, oral hydration is preferred over intravenous in tolerating patients.

G. hollisae disease, clinically, is still considered Vibriosis.⁴ Janda et al. showed that among the all other causes of Vibriosis, G. hollisae comprises only 1.2% of the cases.⁵ In 83% of these cases, the organism was isolated from the gastrointestinal system.⁵ Skin and soft tissue specimens were other resources where G. hollisae was isolated.⁵ In the same study, it has been shown that unlike V. cholerea, V. mimicus, and V parahaemolyticus, G. hollisae has never caused an epidemic, a pandemic, or an outbreak.⁵However, unfortunately, the numbers of vibriosis are in increasing trend due to rising sea surface temperature.¹⁰Considering the record high temperatures and heat waves in recent years, it is more than a lucky guess that we may see more and more Vibriosis cases in the next years, especially in the summer seasons. As microbiologists and healthcare workers we should be aware of these organisms, their capabilities, their limits, and how to prevent the spread of them.

References

Hickman FW, Farmer JJ 3rd, Hollis DG, Fanning GR, Steigerwalt AG, Weaver RE, Brenner DJ. Identification of Vibrio hollisae sp. nov. from patients with diarrhea. J Clin Microbiol. 1982 Mar;15(3):395-401. doi: 10.1128/jcm.15.3.395-401.1982. PMID: 7076812; PMCID: PMC272106.
Thompson FL, Hoste B, Vandemeulebroecke K, Swings J. Reclassification of Vibrio hollisae as Grimontia hollisae gen. nov., comb. nov. Int J Syst Evol Microbiol. 2003 Sep;53(Pt 5):1615-1617. doi: 10.1099/ijs.0.02660-0. PMID: 13130058.
Hinestrosa F, Madeira RG, Bourbeau PP. Severe gastroenteritis and hypovolemic shock caused by Grimontia (Vibrio) hollisae infection. J Clin Microbiol. 2007 Oct;45(10):3462-3. doi: 10.1128/JCM.01205-07. Epub 2007 Aug 17. PMID: 17704283; PMCID: PMC2045321.
https://www.oregon.gov/oha/PH/DiseasesConditions/CommunicableDisease/ReportingCommunicableDisease/ReportingGuidelines/Documents/vibrio.pdf
Janda JM, Newton AE, Bopp CA. Vibriosis. Clin Lab Med. 2015 Jun;35(2):273-88. doi: 10.1016/j.cll.2015.02.007. Epub 2015 Apr 9. PMID: 26004642.
Edouard S, Daumas A, Branger S, Durand JM, Raoult D, Fournier PE. Grimontia hollisae, a potential agent of gastroenteritis and bacteraemia in the Mediterranean area. Eur J Clin Microbiol Infect Dis. 2009 Jun;28(6):705-7. doi: 10.1007/s10096-008-0678-0. Epub 2008 Dec 17. PMID: 19089475.
Gromski MA, Relich RF, Siwiec RM. Grimontia hollisae: A Cause of Severe Ileus in a Seafood-Loving Traveler: 968. American Journal of Gastroenterology: October 2015 – Volume 110 – Issue – p S415-S416
Edouard S, Daumas A, Branger S, Durand JM, Raoult D, Fournier PE. Grimontia hollisae, a potential agent of gastroenteritis and bacteraemia in the Mediterranean area. Eur J Clin Microbiol Infect Dis. 2009 Jun;28(6):705-7. doi: 10.1007/s10096-008-0678-0. Epub 2008 Dec 17. PMID: 19089475.
Abbott SL, Janda JM. Severe gastroenteritis associated with Vibrio hollisae infection: report of two cases and review. Clin Infect Dis. 1994 Mar;18(3):310-2. doi: 10.1093/clinids/18.3.310. PMID: 8011809.
Baker-Austin C, Trinanes J, Gonzalez-Escalona N, Martinez-Urtaza J. Non-Cholera Vibrios: The Microbial Barometer of Climate Change. Trends Microbiol. 2017 Jan;25(1):76-84. doi: 10.1016/j.tim.2016.09.008. Epub 2016 Nov 12. PMID: 27843109.

-Kadir Isidan, MS, MD is a pathology resident at University of Chicago (NorthShore). His academic interests include gastrointestinal pathology and cytopathology.

Feed the Safety Need

Ben was excited to bring the new analyzer into the laboratory until he discovered the manufacturer’s newest security feature. Anytime a user was to log into the analyzer’s computer to diagnose issues or to perform maintenance, a unique numeric passcode would have to be entered, and that code would be sent via text to the app that staff could download on their cellphones. John knew that the use of cell phones in the lab violated the personal electronic device policy.

Emily was proud of the work she had done to design the new outpatient collection draw area. It included a row of collection rooms each with their own computer for order entry. The central area outside the rooms had a phone and printer set up for an efficient workflow. However, every time she performed a site visit she noticed her staff were using cell phones in the patient collection rooms. When she asked why, they told her they often had to make calls to clarify orders, and that talking on the central phone meant discussing patient information in front of people seated in the waiting area.

When a basic need of a human being is not met, conflict is automatically set up in the mind, and humans will deal with that conflict with a workaround or possibly with aggression. Often laboratories and their procedures are designed without considering all of the potential needs of the staff who will work there. Conflict will arise, and policies will not be followed, and you may also wind up with unhappy employees.

When it comes to safety policies and procedures, it is important to educate why they must be followed. It is vital to discuss the possible outcomes of not using safe practices. That may mean exposures to chemicals and biohazards, and it may also mean injuries. It can take time to explain that the use of a smart watch with contaminated gloves can lead to infection and potentially severe illness at work and in the home.

While this understanding is important, it must be coupled with a system of practices that allows staff to easily follow the prescribed safe practices. It must be easy for staff to perform safe acts, there should be no hindrances in their way for that to happen. Otherwise, conflict will occur, and the set policies will not be followed. Staff may know the regulations, they may even understand the potential consequences of not following them, but they will not conform to the policies because of some software glitch or because some vital tool is missing in their environment.

When you notice a lab safety violation, or if a safety incident has occurred, the first thing to look for in the investigation is something in the system that may have caused it. Unless the incident occurred because of a blatant act by the employee, blame should never first be focused on the person. What departmental design flaw exists? What engineering control could have been in place? What PPE should have been readily available? What was the temperature and humidity in the department, etc.?

Upon further discussion with the vender, Ben learned that the manufacturer’s security code system could not be bypassed, but that the app could be downloaded onto an electronic tablet rather than a cell phone. Ben purchased a tablet that could be used in the lab and remain there so as not to create any infection control issues. The tablet was also used for lab safety and quality audits so that pictures of issues could be taken and that results of audits could be entered directly. It became a real time saver, and no cell phones were needed in the laboratory.

Upon review, Emily realized that access to phones in the new outpatient collection area needed to be better. There was no way to even call or help from a collection room should there be an adverse reaction to phlebotomy. Emily was able to acquire portable phones in the short term until she could get permanently-mounted telephones into each of the three blood collection rooms. Staff no longer needed to use cell phones in the biohazardous areas.

Humans have basic needs like food, shelter, and clothing. When those needs are not met, some may act in surprising ways to obtain them. The same holds true in the laboratory. There is a need to be safe, there is a need to follow safety regulations and policies, and unsafe behaviors will arise if it cannot be achieved. Feed the safety needs of your employees. Provide a safe working environment with good engineering controls, PPE, and polices that allow for workdays that have no safety conflict.