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

Image generated by author.

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 assays4-5, 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

  1. 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
  2. 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
  3. 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.
  4. 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.
  5. 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.
  6. 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
  7. 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 1 and 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 species3,9

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

  1. 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.
  2. Prout J, Glymph R. Fusobacterium mortiferum septicemia. Clinical Microbiology Newsletter. 1985;7(4):29. doi: 10.1016/s0196-4399(85)80052-0.
  3. 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.
  4. 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.
  5. 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.
  6. Han YW. Fusobacterium nucleatum: A commensal-turned pathogen. Current Opinion in Microbiology. 2015;23:141. doi: 10.1016/j.mib.2014.11.013.
  7. Alon‐maimon T, Mandelboim OO, Bachrach G. Fusobacterium nucleatum and cancer. Periodontology 2000. 2000;89(1):166. doi: 10.1111/prd.12426.
  8. 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.
  9. 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.
  10. 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

  1. 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
  2. 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
  3. 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 4th 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.3 Signs 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.5 However, unfortunately, the numbers of vibriosis are in increasing trend due to rising sea surface temperature.10 Considering 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

  1. 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.
  2. 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.
  3. 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.
  4. https://www.oregon.gov/oha/PH/DiseasesConditions/CommunicableDisease/ReportingCommunicableDisease/ReportingGuidelines/Documents/vibrio.pdf
  5. 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.
  6. 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.
  7. 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
  8. 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.
  9. 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.
  10. 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.

-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.

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.

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.

Please Don’t Tell Me I Died of Cardiac Arrest

Ask any forensic pathologist what their professional pet peeve is and many of them will likely say “bad death certificates” (right after needing to scratch one’s nose in the middle of an autopsy). Despite the importance of death certificates to public health statistics, studies repeatedly demonstrate an unacceptably high error rate. Death certification isn’t taught in medical schools, and physicians usually learn on the fly. The media often perpetuates these errors, which is why you’ll see news headlines declaring a celebrity died of “cardiac arrest.” However, death certification is a relatively simple concept which can be easily grasped with a little instruction.

Cause of death is “that which in a continuous sequence, unbroken by an efficient intervening cause, results in death and without which death would not have occurred”. Put more simply, it is the etiologically specific disease or injury which triggers the chain of events leading to death. There’s no time limit; a cause can take years (as in breast cancer) or seconds (as in a gunshot wound). Conversely, mechanism of death describes the biochemical and biophysical processes by which the cause exerts its lethal effects. Mechanisms are non-specific and often happen in everyone who is dying (for example, hypoxia, metabolic acidosis, kidney failure). It’s easy to see why doctors list mechanisms on the death certificate—usually in a critically ill patient we’re focused on treating these mechanisms, by providing oxygen, replenishing electrolytes, and performing dialysis until kidney function has returned.

The most common example of this is “cardiac arrest.” Everyone who is dead is in cardiac arrest, by definition—what caused the cardiac arrest is what we really need to know. Putting only a mechanism on a death certificate doesn’t help families understand why their loved one died or inform them of their own potential medical risks, and it provides no useful information to public health prevention efforts.

Finally, manner of death describes the circumstances surrounding death. There are typically five options – natural, accidental, suicidal, homicidal, or undetermined. The most common manner of death error is ignoring fall-related injuries in the elderly or debilitated. A ground-level fall with femoral neck fracture can lead to death in a susceptible individual by blood loss, deconditioning, pneumonia, decubitus ulcers, or thromboembolism. Falls are not a “natural” event – they are potentially preventable, and especially in a vulnerable population may be a warning sign for neglect or abuse. For this reason, we categorize these deaths as accidental.

The nuances around death certification demonstrate one of my favorite roles as a forensic pathologist—public health informaticist. Accurate categorization of deaths allows us to track mortality data and intervene (for example, by notifying communities of a new potent fentanyl analog, or identifying trends in suicide). A death certificate of “cardiac arrest” is therefore frustratingly vague, and our patients and their families deserve a better answer. An academic autopsy program may find it worthwhile to do a quality assurance review of hospital death certificates to identify systemic errors or deficiencies. The CDC offers a free online tutorial (at https://www.cdc.gov/nchs/nvss/training.htm), which is an excellent resource for physicians or family members who want to learn more about this process.

Causes of DeathMechanisms of Death
Atherosclerotic cardiovascular diseaseCardiac ischemia
Type II Diabetes MellitusAcute renal failure
Blunt force injuriesExsanguination
Aspiration pneumonia due to cerebral infarctSepsis

Causes vs Mechanisms of Death: Notice that the causes are all etiologically specific diseases or injuries. The mechanisms are non-specific and lead the reader to ask “…due to what?”. For example, cardiac ischemia can be due to atherosclerosis, vasospasm, or blood loss from trauma.

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

Microbiology Case Report: Left Upper Quadrant Abdominal Pain in a 39 Year Old Male

A 39 year old male presented to a hospital in Dallas, TX with left upper quadrant abdominal pain, nausea, decreased appetite, and a feeling of bloating. The abdominal pain was described as a gradual onset of pain over the course of 2 to 3 weeks. He had no known weight loss, night sweats, chills, diarrhea, or recent trauma. The patient was afebrile on exam with unremarkable vital signs and reported tenderness in the left upper quadrant on palpation of the abdomen. Of note, he was admitted to the hospital 6 weeks prior with abdominal discomfort and was found to have a splenic abscess on computed tomography (CT) scan of the abdomen. There was no surgical drainage of the abscess at that time, and he was treated with two weeks of antibiotics with initial improvement in symptoms. The patient had a past medical history of 3 previous episodes of acute sigmoid diverticulitis that were each treated with bowel rest and 14 days of empiric antibiotics. After the second episode of diverticulitis, the patient had a colonoscopy with findings of colitis and 2 polyps were removed that were negative for malignancy. Following the third episode of diverticulitis, the patient had a sigmoid and partial descending colectomy about 2 years prior to the current presentation.

On admission, a CT scan of the abdomen and pelvis revealed a 3.5 x 1.9 cm air and fluid collection of the inferior border of the spleen and 5.2 x 1.6 cm fluid collection of lateral spleen. The collections were noted to be increased compared to the prior imaging 6 weeks before. Blood cultures were without growth at 5 days. A transthoracic echocardiogram showed no significant valvular abnormalities or vegetations. On hospital day 5, the patient was taken to the operating room for a laparoscopic splenectomy and left diaphragm repair. Surgical findings included a large spleen with omental adhesions and a thick rind along the spleen, which was closely adherent to the diaphragm. A portion of the colon closely adherent to the spleen was also noted. Histopathologic examination showed multifocal splenic abscesses with surrounding fibrosis on hematoxylin and eosin (H&E) stain and granules with surrounding Splendore-Hoeppli material on higher magnification (Figure 1). On Grocott-Gomori methenamine silver (GMS) stain, the granule was seen to be composed of mixed bacterial morphologies with a predominance of filamentous rods typical of Actinomyces (Figure 2). Based on histopathological examination, a diagnosis of splenic actinomycosis was rendered.

Figure 1. Granule with surrounding Splendore-Hoeppli material (H&E 400x magnification).
Figure 2. Granule with mixed bacterial morphologies (GMS 100x magnification).

Discussion

Actinomycosis is a slowly progressive infection characterized by fibrotic mass-like lesions, abscesses, granules, progression across tissue planes, and the development of sinus tracts. The incidence of actinomycosis has declined in the U.S., which is thought to be due to better oral hygiene and the organism’s susceptibility to a wide range of antibiotics.4 The clinical manifestation of actinomycosis is classified by the anatomical site of infection. This includes oral-cervicofacial, thoracic, abdominopelvic, central nervous system, musculoskeletal, and disseminated forms of disease. Oral-cervicofacial disease is the most common form and classically develops with fevers and perimandibular soft tissue swelling that may have a firm or “woody” consistency on palpation.4 Abdominopelvic disease occurs in about 20% of cases with intra-abdominal manifestations usually due to appendicitis, inciting trauma, or previous surgical procedure and pelvic disease most often due to intra-uterine contraceptive devices.1 The clinical manifestations of actinomycosis are often difficult to correctly diagnose, and the presentation and imaging findings often mimic malignancy further complicating the assessment. Diagnosis relies on consideration of the disease process and diagnostic sampling for histopathology and microbiologic studies.

Although most actinomycotic lesions are polymicrobial, species of the genus Actinomyces are the predominant etiologic agents.2 Actinomyces are a group of gram positive filamentous facultatively anaerobic or microaerophilic bacteria that are normal flora of the gastrointestinal and genitourinary tracts. The organisms typically have true branching and may appear beaded due to irregular Gram staining. Importantly, Actinomyces spp. will be negative with modified acid-fast staining, which can be used to differentiate it from Nocardia spp. The bacteria are relatively slow growing on primary culture and mature colonies may have a variety of morphologies. The classic “molar tooth” appearance is characteristic of A. israelii.3 On histopathology, actinomycotic lesions have a surrounding area of fibrosis and central suppurative inflammation with granules. The granules consist of accumulations of organisms with club-shaped ends and filamentous rods seen on special staining.4 Optimal diagnosis would consist of visualization of these features on histopathology or other direct method. Isolation of the organism can be useful but should be taken in the context of the clinical picture as the mere isolation of Actinomyces in culture does not always imply actinomycosis.

Splenic involvement of actinomycosis is an uncommon cause of the intra-abdominal disease process. In our case, the most likely etiology for splenic actinomycosis was due to the recurrent episodes of acute sigmoid diverticulitis with breaches in the mucosal barrier and direct invasion into the spleen. The surgical management in this case was splenectomy to avoid splenic rupture. Medical management involves antibiotic therapy with high-dose penicillin as first-line therapy. The treatment duration has historically been to treat with parenteral penicillin for 2 to 6 weeks and then transition to oral penicillin or amoxicillin up to a year based on clinical response.

References

  1. Bennhoff D: Actinomycosis: diagnostic and therapeutic considerations and a review of 32 cases. Laryngoscope 1984; 94: pp. 1198-1217.
  2. Blaser MJ, Dolin R, Bennett JE. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. Ninth edition. Elsevier; 2020.
  3. Pfaller, M. A., Carroll, K. C., & Jorgensen, J. H. (2015). Manual of clinical microbiology (11th edition.). ASM Press.

-Zane Conrad, MD is a medical microbiology fellow at UT Southwestern Medical Center.

-Dominick Cavuoti, DO is a professor at UT Southwestern and practices Infectious disease pathology, medical microbiology and cytology.

-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.