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.

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.

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.

Microbiology Case: Immunocompromised Patient with Altered Mental Status

Case Presentation

Patient is a 45 year old Vietnamese male who presented initially to the Emergency Room with altered mental status at home. Patient presented with hypotension and hypothermia and was admitted to the ICU. Past medical history is significant for HIV although the patient has not be on antiretroviral therapy (ART), syphilis, and active Pneumocystis infection. His CD4 count was 15 on arrival, and he was placed on multiple prophylactics for prevention of opportunistic infections. Blood and cerebrospinal fluid (CSF) were submitted for cultures. Encapsulated yeast were seen on the CSF which was positive for Cryptococcus neoformans on a rapid multiplex-PCR panel (BioFire Film Array Meningitis/Encephalitis panel) followed by isolation of the yeast in culture and identification using the MALDI-TOF. Yeast was also found in the blood cultures, also identified as Cryptococcus using a rapid blood culture identification panel (BioFire Film Array Blood Culture Identification Panel 2.0) which subsequently grew out C. neoformans, also identified using MALDI-TOF.

Discussion

Cryptococcus species areencapsulated yeast cells with a natural habitat in the soil. Promotion of organism replication happens in alkaline pH environments with higher nitrogen concentrations. For example, soil contaminated with turkey, chicken, bat, or pigeon droppings can contribute to this growing environment. Yeast cells can become airborne with soil disruption, and contribute to increased risk of infection to immunocompromised hosts with certain activities. Aside from pulmonary infections, meningoencephalitis is another common manifestation of infection.1 Patients may have neurological deficits and increased intracranial pressure. A wide spectrum of symptoms have been reported including fever, malaise, headache, neck stiffness, photophobia, nausea, vomiting and sometimes rarely a cough, dyspnea, and skin rashes. Generally speaking, Cryptococcus neoformans is usually associated with infections in immunocompromised patients while Cryptococcus gatti is associated with infections in immunocompetent patients.2 Positive blood cultures with Cryptococcus is typically representative of disseminated infection.

The major virulence factor is the capsule which plays a role in preventing phagocytosis and providing an adherence mechanism to mucosal linings. Not all strains produce capsules, but the colony on growth medium could be mucoid (image 1). The capsules of Cryptococcus may group to one another, almost forming a ‘honeycomb’ matrix with the polysaccharide capsule separating the forms from each other. Additionally, Cryptococcus produce a melanin pigment, which is considered a virulence factor because it protects the yeast from oxidant-induced stressors. As such, the Fontana-Masson stain used in histopathology will be positive due to the melanin production of the organism. Cryptococcus neoformans is responsible for most human infections, and Cryptocococcal infections are considered to be opportunistic, with immunocompromised populations being at highest risk.3

Image 1. Visible capsule stained with Giemsa on the CSF specimen is highly indicative of Cryptococcus (top left). Budding yeast stained with Gram-stain observed in blood cultures (top right). Mucoid colony growth of Cryptococcus neoformans on Chocolate agar, Sheep Blood agar, and cream-white colonies on Sabouraud dextrose agar (bottom).

Microscopically, Cryptococcus is an irregularly sized (4-10µm), round, encapsulated yeast. It can also appear as a budding yeast.3 Direct staining of the CSF specimen can be done using India ink which will form a “halo” around the yeast cells as the ink stains the capsule. Cream-colored, sometimes mucoid, colonies will appear in agar plates in 3-7 days. Aside from PCR and MALDI-TOF, differentiation between Cryptococcal neoformans and Cryptococcal gatti can be possible using canavanine, glycine, bromothymol blue agar. Growth of Cryptococcus gatti will turn the agar blue. Detection of cryptococcal antigen through immunodiagnostic tests of the serum and the cerebrospinal fluid can also provide a diagnosis of the infection. CSF parameters of infected individuals typically show low white blood cell count, low glucose, and elevated protein but up to 30% of the cases have also reported normal CSF parameters.4 Histopathology staining using mucicarmine is specific for the presence of Cryptococcus. Radiograph imaging of the brain have also been shown to be helpful.

Rapid detection of Cryptococcal infections and other opportunistic infections are imperative to improving patient outcomes. Mortality from cryptococcal meningitis in the “meningitis belt” of Sub-Saharan Africa approaches 75%, with an 89% incidence rate.5 A combination of factors including higher HIV carriage rate, lack of available preventative care, and dry seasons with dry winds and cold nights lend to this region’s higher incidence rates. Moreover, lack of cheaper and reliable testing methods for detection and possible initiation of prophylactic medications are contributors of higher mortality rate. Recent studies investigate how the efficacy of rapid antigen assays like lateral flow assays might have a role in filling some of these care gaps in an efficient and cost-effective way, but further study is required.5 Mainstays of treatment for cryptococcal infections include amphotericin B, flucytosine, and fluconazole.2 Monitoring intracranial pressure and keeping it under check plays an important role in reducing the mortality associated with cryptococcal meningitis.6 Lumbar puncture is the recommended option for management of intracranial pressure and either a ventricular drain or ventricular peritoneal shunt is used in patients who require frequent lumbar punctures.

References

  1. Park BJ, Wannemuehler KA, Marston BJ, Govender N, Pappas PG, Chiller TM. Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS. 2009 Feb 20;23(4):525-30.
  2. Cox, Gary M, Perfect, John R. Cryptococcus neoformans meningoencephalitis in patients with HIV: Treatment and prevention. June 9, 2021, UptoDate. https://www.uptodate.com/contents/cryptococcus-neoformans-meningoencephalitis-in-patients-with-hiv-treatment-and-prevention?search=cryptococcal%20meningitis%20treatment&source=search_result&selectedTitle=1~83&usage_type=default&display_rank=1. Accessed 10/7/2022
  3. Winn, Washington C. Jr. et al. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, 6th Edition. 2006. Lippincott Williams and Wilkins.
  4. Garlipp CR, Rossi CL, Bottini PV. Cerebrospinal fluid profiles in acquired immunodeficiency syndrome with and without neurocryptococcosis. Rev Inst Med Trop Sao Paulo. 1997 Nov-Dec;39(6):323-5.
  5. Okolie CE, Essien UC. Optimizing Laboratory Diagnostic Services for Infectious Meningitis in the Meningitis Belt of sub-Saharan Africa. ACS Infect Dis. 2019 Dec 13;5(12):1980-1986. doi: 10.1021/acsinfecdis.9b00340. Epub 2019 Nov 18. PMID: 31738509.
  6. Rolfes MA, Hullsiek KH, Rhein J, Nabeta HW, Taseera K, Schutz C, Musubire A, Rajasingham R, Williams DA, Thienemann F, Muzoora C, Meintjes G, Meya DB, Boulware DR. The effect of therapeutic lumbar punctures on acute mortality from cryptococcal meningitis. Clin Infect Dis. 2014 Dec 01;59(11):1607-14.

-Dr. Katelyn Swanson is a currently a PGY-1 pathology resident at George Washington University. She completed a clinical laboratory science program at Franciscan Health in Indianapolis, IN, and received her MLS (ASCP) certification before attending and graduating medical school from Lake Erie College of Osteopathic Medicine at Seton Hill. She completed a transitional year internship at Walter Reed National Military Medical Center and one General Medical Officer billet with the Navy before starting pathology residency. She is still exploring her research interests.

-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.

Microbiology Case Study: What’s with the Rash?

Case presentation

A 79 year old female with a past medical history of COPD, hypertension, diabetes, and eczema presented to the emergency department with a localized rash on the right knee (Figure 1). The rash began after gardening and persisted for three weeks.

The patient reported some itching, warmth, and tenderness but denied nausea, vomiting, fever, and diarrhea. Her vital signs were BP 175/76| Pulse 91 | Temp 98.5 °F (36.9 °C) (Oral) | Resp 20 | SpO2 96%. The remainder of her physical exam was notable: right knee skin rash. There was no induration or fluctuance or drainage. She exhibited a full range of knee motion; there was no palpable knee joint effusion (Figure 1).

Lab CBC results were unremarkable. X-Ray knee AP and lateral – right showed soft tissue prominence anterior to the patella, which suggests prepatellar edema and a fluid collection. Lyme antibody screening was negative. Two sets of blood culture bottles were sent to the microbiology laboratory. After 24 hours of incubation, aerobic bottles were positive with the organism shown in: Gram stain (Figure 2), culture growth showing alpha-hemolytic colonies (Figure 3), H2S production on the TSA agar slant (Figure 4). 

Identification by Matrix-assisted laser desorption ionization Time of flight (MALDI-ToF) revealed Erysipelothrix rusiopathiae at a score above 2.0. 

Discussion

Erysipelothrix is a non-spore-forming, catalase-negative, facultative gram positive bacillus. It is not acid-fast or motile. It is distributed worldwide and is primarily considered an animal pathogen responsible for causing erysipelas that may affect a wide range of animals. Erysipelothrix is ubiquitous in soil, food scraps, and water contaminated by infected animals.1 It can survive in the soil for several weeks. In pig feces, the survival period of this bacterium ranges from 1 to 5 months.

Erysipelothrix can also cause zoonotic infections in humans, called erysipeloid. Most human infections are acquired through occupational exposure, such as fish handlers, veterinarians, and butchers, via direct injection of the organism through abrasion or injuries. Notably, the human disease of “erysipelas” is not caused by Erysipelothrix but by Streptococcus. 

Erysipeloid typically develops at the site of infection between 2 and 7 days after exposure. E. rusiopathiae infection can be categorized as 1) localized cutaneous erythematous 2) generalized cutaneous form due to traumatic injury and skin penetration of the organism, and 3) septicemic form.2 Skin infection can sometimes progress to bacteremia, most commonly associated with endocarditis3. The implication of endocarditis in the setting of E. rusipathiae infection is associated with increased mortality rate.2,3 

E. rusiopathiae can easily be grown on routine media, including blood and chocolate agar plates, in a clinical microbiology laboratory.1 The colonies appear as small alpha-hemolytic and can resemble alpha Streptococcus species. It can also be confused with Corynebacterium species due to the similarity in Gram stain characteristics. E. rusipathiae produces H2S on the triple iron sugar media (Figure 4), which is one of the distinguishing morphologies from other Gram-positive rods, such as Listeria or Bacillus species.1 It can be identified by Matrix-assisted laser desorption ionization Time of Flight (MALDI-ToF) directly from the positive blood culture broth (using Sepsityper Kit with Bruker MALDI-Biotyper (MBT)) or from isolated colonies. 

E. rusiopathiae is generally sensitive to penicillin. It is intrinsically resistant to vancomycin and aminoglycosides.4 CLSI (Clinical Laboratory of Standard Institution) M45 ED3 recommended ampicillin or penicillin for primary testing agents.4 While antimicrobial susceptibility testing is not warranted for every case of E. rusiopathiae, it is imperative that the organism be identified due to the critical nature of infection resulting in endocarditis. Since vancomycin is typically used for broad-spectrum coverage of gram positive organisms,4 early identification of this organism and notification of clinicians is helpful for appropriate antimicrobial management.

References

  1. Jorgensen et.al., Chapter 27. Manual of Clinical Microbiology. 11th Edition.

2. Principe L, Bracco S, Mauri C, Tonolo S, Pini B, Luzzaro F. Erysipelothrix Rhusiopathiae Bacteremia without Endocarditis: Rapid Identification from Positive Blood Culture by MALDI-TOF Mass Spectrometry. A Case Report and Literature Review. Infect Dis Rep. 2016 Mar 21;8(1):6368. doi: 10.4081/idr.2016.6368. PMID: 27103974; PMCID: PMC4815943.

3. Wang T, Khan D, Mobarakai N. Erysipelothrix rhusiopathiae endocarditis. IDCases. 2020 Sep 9;22:e00958. doi: 10.1016/j.idcr.2020.e00958. PMID: 32995274; PMCID: PMC7508995.

4. CLSI. Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria. 3rd ed. CLSI guideline M45. Wayne, PA: Clinical and Laboratory Standards Institute; 2016.

-Azal Al-Ani, MD is a third-year AP/CP pathology resident at Montefiore Medical Center, Bronx, NY. She completed her medical school at Al-Anbar Medical College, Iraq. Her interest includes hematopathology and dermatopathology

-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.

Microbiology Case Study: A 67 Year Old with Foot Pain

Case description

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

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

Image 1. Patient stool sample wet mount preparation.

Discussion

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

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

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

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

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

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

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

References

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

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

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

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

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

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

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

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

Microbiology Case Study: A 46 Year Old with Chest Pain

Case History

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

Laboratory Identification

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

Image 1. (Left) Modified acid-fast (MAF) staining revealing small, MAF-positive coccobacilli (black arrowheads).  (Right) Characteristic, mucoid salmon-colored colonies of the isolate on blood agar after 96 hours incubation. ​
Image 2. (Left) Transbronchial biopsy revealing areas of histiocyte aggregation and mononuclear infiltrate (H&E, 10X magnification).  (Middle) Representative image of expanded histiocytes with small, pale-staining round forms in a background of neutrophils (H&E, 40X magnification).  (Right) Representative image of histiocytes filled with coccoid and coccobacilliary forms (GMS, 40X magnification).​

Discussion

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

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

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

References

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

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

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

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

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

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


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

Microbiology Case Study: A 17 Year Old with Chest Pain

Case History

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

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

Discussion

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

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

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

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

References

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

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

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

Microbiology Case Study: Severely Immunocompromised Female with Respiratory Failure

Case History

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

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

Discussion

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

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

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

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

References

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

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

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