Microbiology Case Study: A Female with Diabetes and Renal Disease

Case history

A middle-aged female with a past medical history of diabetes and end stage renal disease resulting in kidney transplant presented for evaluation of right hip and knee pain for the previous two months. An MRI of the hip revealed a large effusion with evidence of septic arthritis, myositis in the surrounding muscle, and osteomyelitis of the hip. Blood cultures remained negative for the duration of her presentation. The patient underwent a joint aspiration, and synovial fluid was sent to the microbiology laboratory for culture. Due to subsequent culture positivity and the extent of the involvement of the surrounding anatomy, the patient was started on ceftriaxone and underwent a total joint replacement. Her symptoms improved post-procedure, and post-operative vertebral MRI and TTE revealed no evidence of osteomyelitis or endocarditis. The patient was discharged on post-operative day six with continued IV ceftriaxone for an additional 5 weeks.

Laboratory identification

The synovial fluid received in the microbiology laboratory was plated onto blood, chocolate, and MacConkey agars. No organisms were visible on direct Gram stain, but the culture revealed scant growth of alpha-hemolytic colonies on blood and chocolate plates. These colonies were comprised of faintly staining gram positive rods (Image 1). The organism was catalase negative. Given the characteristic appearance by Gram stain, the organism was inoculated to a triple sugar iron (TSI) slant where it demonstrated H2S production. A definitive identification of Erysipelothrix rhusiopathiae was achieved by MALDI-TOF MS.

Image 1. Synovial fluid culture sent to the microbiology laboratory. E. rhusiopathiae colonies growing on Sheep’s blood agar are denoted by black arrowheads. Characteristic Gram stain of the E. rhusiopathiae colonies from the plate revealing poorly staining gram positive rods. TSI slant from the colonies demonstrating H2S production.

Discussion

Erysipelothrix rhusiopathiae is a facultatively aerobic, non-spore forming, gram positive pathogen that is a resident of the digestive and respiratory tracts of mammals, bird, fish, and pigs.1 It is the etiological agent of Swine Erysipelas, causing either an acute septicemia, cutaneous disease, endocarditis, or chronic arthritis in pigs. Human infections with E. rhusiopathiae are usually due to exposure to infected animals or contaminated animal products or environments. Certain occupations with frequent animal exposure are at increased risk for infection (including fishermen, veterinarians, farmers, and butchers). Infection requires entry into the skin through cutaneous abrasions, which can be caused by sharp hooks, fish scales, teeth, and other occupational tools or hazards that damage epithelial barriers.1,2

Human E. rhusiopathiae infection can manifest as three distinct forms. An acute, localized cellulitis named eryspieloid (not to be confused with streptococcal erysipelas) is the most common manifestation. This usually impacts the hands, fingers, or other parts of the upper extremities that have contact with animals or animal products.3 A generalized cutaneous form more often associated with systemic symptoms including fever, joint aches, lymphadenitis, lymphadenopathy, and arthritis can also occur. Finally, septicemia frequently associated with endocarditis is a third manifestation. E. rhusiopathiae endocarditis is often subacute, with a tropism for native valves (particularly the aortic valve). Due to its indolent nature, this presentation often requires valve replacement at the time of diagnosis and is associated with increased mortality.1,4 While cases of non-severe eryspieloid may self-resolve, ampicillin or penicillin are the treatments of choice for cutaneous and systemic infections. Cephalosporins and fluoroquinolones are also efficient alternative agents.3 Importantly, the organism is intrinsically resistant to vancomycin, thus accurate and timely identification is critical to ensure appropriate intervention (Image 2). Susceptibility testing is generally not performed but may be useful in the setting of penicillin allergy.

Image 2. E. rhusiopathiae is intrinsically resistant to vancomycin. E. rhusiopathiae exhibits elevated MICs to vancomycin. Penicillin is the treatment of choice.

Laboratory identification of E. rhusiopathiae can be challenging.  Erysiepelothrix can easily decolorize during gram staining and can be mistaken as gram negative due to lack of stain retention. Additionally, the cells can exhibit variable morphologies including pairs, chains, and filaments. Colonies can also exhibit variable morphotypes when grown on routine media, including both rough and smooth forms.2An environmental exposure to animals was investigated in this patient’s case to possibly serve as the source of infection. While a direct link cannot be definitively proven, it was revealed that the patient owned a large fish tank which she regularly cleaned which could have been a potential source of infection. 

References

  1. Wang Q, Chang BJ, Riley TV. 2010. Erysipelothrix rhusiopathiae. Veterinary Microbiology 140:405-417.
  2. Clark AE. 2015. The Occupational Opportunist: an Update on Erysipelothrix rhusiopathiae Infection, Disease Pathogenesis, and Microbiology. Clinical Microbiology Newsletter 37:143-151.
  3. Veraldi S, Girgenti V, Dassoni F, Gianotti R. 2009. Erysipeloid: a review. Clinical and Experimental Dermatology 34:859-862.
  4. Brooke CJ, Riley TV. 1999. Erysipelothrix rhusiopathiae: bacteriology, epidemiology and clinical manifestations of an occupational pathogen. Journal of Medical Microbiology 48:789-799.

-Timothy J. Kirtek, M.D., originally from Grand Blanc, Michigan, graduated from American University of the Caribbean School of Medicine located on the island of Sint Maarten. There, he conducted research on tropical arboviruses including Dengue, Chikungunya, and Zika viruses. He then returned to Michigan to complete his clinical training and, upon graduation from medical school, moved to Dallas, Texas where he is currently an Anatomic and Clinical Pathology resident physician at UT Southwestern.

-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.

Microbiology Case Study: A Young Adult in Septic Shock

A 23 year old female with a previous medical history of endocarditis, hepatitis C, IV drug use, and aortic insufficiency status post emergent aortic valve replacement, presented to the ER in septic shock. After one week of hospitalization, she left against medical advice, and did not complete her prescribed course of antimicrobials.

One month later, she returned to the ER with tachypnea, lactic acidosis, and altered mental status, secondary to septic shock and she was admitted to the ICU. She was started on broad spectrum antibiotics based on the cultures from her previous hospitalization. Within one day, blood cultures from her central line were positive for growth of Serratia marcescens. Echocardiogram demonstrated prosthetic valve endocarditis with severe aortic regurgitation. Previous imaging had shown scattered septic emboli throughout her viscera, extremities, and now, MRI/MRA revealed cerebral lesions as well.

Ten and twelve days into her current hospitalization, blood and heart valve tissue cultures (respectively) were both positive for growth of the below-pictured organism. What is this causative organism?

Image 1. Central line blood culture.
Image 2. Heart valve tissue culture.

MALDI-ToF-MS identified the yeast from the blood culture and heart valve as Trichosporon asahii. It is a yeast-like basidiomycete. It is commonly found in soil, but is also a normal colonizer of mucous membranes of the GI and respiratory epithelium, and skin. It may also infect hair shafts and is the causative agent in “white piedra”. It is involved in several opportunistic infections in the immunosuppressed. Of all Trichosporon species, T. asahii is the most common cause of disseminated infection, especially in those with hematologic malignancies (leukemia, multiple myeloma, aplastic anemia, lymphoma), solid tumors, AIDS, and solid tumors. In immunocompetent patients, Trichosporon may cause infections including endophthalmitis following cataract surgery, endocarditis, following prosthetic heart valve replacement (as seen in this patient), and peritonitis in IV drug abusers or those receiving continuous ambulatory peritoneal dialysis (CAPD).

Trichosporon colonies are powdery, cream-colored, and with age, may develop surface wrinkles. On cornmeal Tween 80, yeast can either grow alone or in short chains. True and pseudohyphae may be seen. Barrel-shaped arthroconidia are typically present. Variable growth is seen on media containing cycloheximide. It may also cause Cryptococcal antigen agglutination tests to be falsely positive.

Diagnosis is typically via blood culture.

Combination therapy with amphotericin B and an -azole drugs seems to be the most successful treatment option.

Resources

  1. Brandt, ME, Lockhart, SR. Recent developments with Candida and other opportunistic yeasts. Curr Fungal Infect Rep. 6(3); 170-177. 2012.
  2. Dimorphic Systemic Mycoses | Mycology | University of Adelaide Accessed 10/22/21.
  3. Love, G. Mycology Benchtop Reference Guide. College of American Pathologists. P20. 2013.
  4. Maves, RC. Trichosporon Infections. Emedicine.medscape.com. Updated Feb 12, 2018. Accessed 10/18/21.
  5. Procop, GW, Church, DL, Hall, GS et al. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. 7th Edition. P 1366-1369. Wolter’s Kluwer Health. 2017.
  6. Ramos, JM, Cuenca-Estrella, M, Gutierrez, F, et al. Clinical case of endocarditis due to Trichosporon inkin and antifungal susceptibility profile of the organism. J Clin Microbiology. 42(5):2341-4. 2004.
  7. Trichosporon | Mycology | University of Adelaide Accessed 10/22/21.

-Jenny Pfeiffer, MD is a 1st year Anatomic and Clinical Pathology resident at the University of Vermont Medical Center.

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

Microbiology Case Study: An Elderly Male Presents with Chest Tightness

Case History

An elderly male with a complex past medical history presented to the Emergency Department with the primary complaint of chest tightness for 2 days. He denied symptoms of diaphoresis, nausea, shortness of breath, palpitations, light-headedness, productive cough, dyspnea, chest pain, fevers, chills, or hemoptysis. He had no known sick contacts or recent travel. A computer tomography (CT) scan of the thorax showed a right hilar mass (Image 1). He underwent a bronchoscopy and right hilar transbronchial needle aspiration (TBNA) and bronchoalveolar lavage (BAL) were collected. The pathology report indicated abnormal lymphocytic proliferation, concerning for a mature small B-cell lymphoproliferative disorder.

Image 1. CT scan of the thorax showing the right hilar mass.

The BAL was submitted for acid-fast bacteria (AFB) culture, Gram stain, aerobic bacterial culture, and fungal culture. The AFB culture, Gram stain, and bacterial culture were all negative. However, 3 tan-yellow creamy colonies of a yeast grew on the sabouraud dextrose agar (SAB) plate in fungal culture after 7 days (Image 2). An India ink stain was performed (Image 3). MALDI-TOF confirmed the identification as Cryptococcus neoformans.

Image 2. Fungal growth on the SAB plate observed after 7 days.
Image 3. India ink staining of the fungus.

Discussion

Cryptococcus neoformans is an encapsulated pathogenic yeast, which is typically associated with bird droppings and contaminated soil.1,2 In immunocompromised patients, it can lead to severe opportunistic infections such as meningitis or disseminated disease. C. neoformans can cause life-threatening fungal infections in these patients, especially those with T-cell mediated immunodeficiency.3,4 The three main virulence factors include the complex capsule, melanin production, and ability to grow at human body temperature.5,6 Signs of pulmonary infection include cough, production of mucoid sputum, pleuritic chest pain, low-grade fever, dyspnea, weight loss, and malaise.

Fungal culture is one of the primary methods of Cryptococcus identification. Upon microscopic examination, Cryptococcus appears as a single bud and a narrow neck between parent and daughter cell and measures 4 – 10 uM.7 It has a fragile cell wall and a polysaccharide capsule that can vary from a wide halo to a nearly undetectable zone around the cells. Colonies can exhibit a wide range of color (i.e. cream, tan, pink, or yellow) and typically grow within one week of inoculation.8,9 India ink smear is a rapid method that allows direct visualization cryptococcal capsule, but is infrequently used now. Certain non-specific histological stains (including Periodic Acid-Schiff and May-Grünwald-Giemsa) can be used to detect fungi directly in fixed specimens. Fontana-Masson is a silver stain used for detecting melanin and has a high sensitivity for cryptococcosis.10  Other useful stains include hematoxylin-eosin, which reveals the clear halo, and mucicarmine and alcian blue, which target the polysaccharide capsule.11 Cryptococcal serology and cryptococcal antigen testing can be used for blood or CSF infections. Radiographic findings (especially in asymptomatic and immunocompetent patients) include patchy pneumonitis, granulomas (typically 2-7 cm), and miliary disease similar to tuberculosis.8 Treatment will vary depending on location of infection and host immune status. In some cases, pulmonary Cryptococcus may not be treated. Some clinical considerations include:

  • CSF chemistry parameters are normal
  • CSF culture, cryptococcal antigen, India ink preparation, and serology results are negative
  • Urine culture results are negative
  • Pulmonary lesion is small and stable/shrinking
  • No predisposing conditions for disseminated disease6

If treatment is required, fluconazole, itraconazole, or amphotericin B with or without flucytosine can be used depending on severity of infection.12

Cryptococcus gattii is another species of Cryptococcus. It differs from C. neoformans in that it typically infects both immunocompromised and immunocompetent patients. Canavine glycine bromothymol blue (CBG) agar can be used to differentiate C. gattii from C. neoformans: C. gattii is able to grow in the presence of canavine, turning the agar blue, while C. neoformans does not, leaving the media color unchanged.13,14

References

  1. Hagen, F., Khayhan, K., Theelen, B., Kolecka, A., Polacheck, I., Sionov, E., Falk, R., Parnmen, S., Lumbsch, H. T., and Boekhout, T. Recognition of seven species in the Cryptococcus gattii/Cryptococcus neoformans species complex. Fungal Genet Biol. 2015; 78: 16-48. 
  2. Lortholary, O., Nunez, H., Brauner, M. W., and Dromer, F. Pulmonary cryptococcosis. Semin Respir Crit Care Med. 2004; 25: 145–57.
  3. Lanternier, F., Cypowyj, S., Picard, C., Bustamante, J., Lortholary, O., Casanova, J. L., and Puel, A.  Primary immunodeficiencies underlying fungal infections.  Curr Opin Pediatr. 2013; 25: 736–47. 
  4. National Organization for Rare Disorders (NORD). Cryptococcosis. Available from: https://rarediseases.org/rare-diseases/cryptococcosis/ Last updated 2007; cited 2021 October 8.
  5. Idnurm, A., Bahn, Y.-S., Nielsen, K., Lin, X., Fraser, J. A., and Heitman, J. Deciphering the model pathogenic fungus Cryptococcus neoformans. Nat Rev Microbiol. 2005; 3(1): 753-64.
  6. Vandeputte, P., Ferrari, S., and Coste, A. T. Antifungal Resistance and New Strategies to Control Fungal Infections. Int J Microbiol. 2012: 713687.
  7. Guarner, J. and Brandt, M. E. Histopathologic Diagnosis of Fungal Infections in the 21st Century. Clin Microbiol Rev. 2011; 24(4): 247-80.
  8. Borman, A. M. and Johnson, E. M. (2020).  Candida, Cryptococcus, and Other Yeasts of Medical Importance. Manual of Clinical Microbiology, 12th Edition. Washington, DC: ASM Press. 2056-86.
  9. Coelho, C., Bocca, A. L., and Casadevall, A. The tools for virulence of Cryptococcus neoformans. Adv Appl Microbiol. 2014; 87: 1-41.
  10. Bishop, J. A., Nelson, A. M., Merz, W. G., Askin, F. B., and Riedel, S. Evaluation of the detection of melanin by the Fontana-Masson silver stain in tissue with a wide range of organisms including Cryptococcus. Hum Pathol. 2012; 43(6): 898-903.
  11. Guery, R., Lanternier, F., Pilmis, B., and Lortholary, O. Cryptococcus neoformans (Cryptococcosis). Antimicrobe. Available at: http://www.antimicrobe.org/new/f04.asp. Last updated: 2014; cited 2021 October 8.
  12. Perfect, J. R., Dismukes, W. E., Dromer, F., Goldman, D. L., Graybill, J. R., Hamill, R. J., Harrison, T. S., Larsen, R. A., Lortholary, O., Nguyen, M.-H., Pappas, P. G., Powderly, W. G., Singh, N., Sobel, J. D., and Sorrell, T. C. Clinical Practice Guidelines for the Management of Cryptococcal Disease: 2010 Update by the Infectious Diseases Society of America. Clinical Infectious Diseases. 2010; 50(3): 291-322.
  13. Larone, D. (2011). Medically Important Fungi. Washington, DC: ASM Press.
  14. Klein, K. R., Hall, L., Demi, S., Rysavy, J. M., Wohlfiel, S. L., and Wengenack, N. L. Identification of Cryptococcus gattii by use of L-canavanine glycine bromothymol blue medium and DNA sequencing. J Clin Microbiol. 2009; 47: 3669-72.

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

-Paige M.K. Larkin, PhD, D(ABMM), M(ASCP)CM is the Director of Molecular Microbiology and Associate Director of Clinical Microbiology at NorthShore University HealthSystem in Evanston, IL. Her interests include mycology, mycobacteriology, point-of-care testing, and molecular diagnostics, especially next generation sequencing.

Microbiology Case Study: An Adult Male with Left Hand Pain

Case History

An adult male presented to the Emergency Department with edema and pain in his left hand. The patient stated that he was bitten by his cat 24 hours prior to admission. Bloodwork was drawn and patient was found to have mild leukocytosis (11.2 x103 cells/uL [reference range, 4-10 x103 cells/uL]) with an elevated neutrophil percentage (76.8% [reference range, 40-70%]). Debridement was performed in the operating room and purulent drainage was send to the lab for aerobic and anaerobic bacterial culture. Gram stain of the purulent drainage showed 3+ white blood cells and mixed bacteria. Mixed aerobic flora grew out in culture, but the predominate isolate was Pasteurella multocida, which grew on 5% sheep blood and Chocolate agar plates (Figure 1) as identified using MALDI-TOF MS.

Figure 1. Growth of Pasteurella multocida on Chocolate Agar after 48hrs incubation at 37°C, 5% CO2.
Figure 2. Gram stain of Pasteurella multocida showing gram negative coccobacilli.

Pasteurella multocida

Pasteurella multocida is one of the most common causes of animal bite-related bacterial infections among patients who present to the Emergency Department in the United States.1 Immunocompetent patients with P. multocida infection typically present with cellulitis at the site of physical injury. These infections usually have a rapid course that can develop within 3 – 48 hours following injury. While most commonly associated with animal bites, immunocompromised patients can acquire Pasteurella simply through animal contact. These rare cases can present as more severe infections involving the respiratory tract and invasive infections including meningitis and endocarditis.

P. multocida is a small, non-motile, facultative anaerobe, which contains an outer polysaccharide capsule. The outer polysaccharide capsule is used to further classify serogroup with most human infections caused by serogroups A and D.2 Further classification into sub-serogroups is determined by the composition of the outer lipopolysaccharide (LPS), which further separates isolates into 16 serovars.3 The bacterium stains as a Gram negative coccobacillus (Figure 2). The recommended growth medium to grow Pasteurella multocida is 5% sheep’s blood agar.4 Of note, P. multocida does not grow on MacConkey agar, unlike most other Gram negative organisms, which can aid in identification of the organism. Most strains of the bacterium test positive for oxidase, catalase, and indole.2

While P. multocida is typically susceptible to penicillin,5 due to the polymicrobial nature of animal bites, broad-spectrum anaerobic and oral flora coverage is recommended for animal bite-related infections. Often, amoxicillin-clavulanate (Augmentin) is used, which will cover the most common organisms including Pasteurella species. Antibiotics which should be avoided for therapy include cephalexin, dicloxacillin, and erythromycin as they have been shown to have inadequate activity against the bacterium.6

References

  1. Martin TCS, Abdelmalek J, Yee B, Lavergne S, Ritter M. Pasteurella multocida line infection: a case report and review of literature. BMC Infect Dis. 2018 Aug 23;18(1):420.
  2. Wilson BA, Ho M. Pasteurella multocida: from zoonosis to cellular microbiology. Clin Microbiol Rev. 2013 Jul;26(3):631-55.
  3. Harper M, Boyce JD, Adler B. The key surface components of Pasteurella multocida: capsule and lipopolysaccharide. Curr Top Microbiol Immunol. 2012;361:39-51.
  4. Lariviere S, Leblanc L, Mittal KR, Martineau GP. Comparison of isolation methods for the recovery of Bordetella bronchiseptica and Pasteurella multocida from the nasal cavities of piglets. J Clin Microbiol. 1993 Feb; 31(2):364-7.
  5. Hasan J, Hug M. Pasteurella Multocida. [Updated 2021 May 24]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK557629/
  6. Stevens DL, Bisno AL, Chambers HF, Dellinger EP, Goldstein EJ, Gorbach SL, Hirschmann JV, Kaplan SL, Montoya JG, Wade JC. Practice guidelines for the diagnosis and management of skin and soft tissue infections: 2014 update by the infectious diseases society of America. Clin Infect Dis. 2014 Jul 15;59(2):147-59.

-Tristan R. Grams, is a PhD Candidate at the University of Florida in Gainesville, FL where he studies HSV-1 latency and characterizing SARS-CoV-2 antiviral agents.

-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: Fever and a Blister in a Young Female

A 25 year old female presented to the ED with a mild fever and a blister on her hand, which arose from a rat bite. She stated that she worked with different kinds of animals including rodents. The patient was discharged with clindamycin after two sets of blood cultures were drawn. Anaerobic bottles became positive 48 hours after incubation. Gram stain showed unusual gram negative bacilli (Figure 1). Rapid Multiplex PCR Blood culture identification (FilmArray BCID-2) panel was negative. Poor growth on Blood agar was observed after 48 hours of sub-culture. The organism was identified as Streptobacillus monoliformis by Bruker Biotyper MALDI-ToF.

S. monoliformis appears as pleomorphic gram negative rods, with its classic characteristics of “bulges” or swollen-rods in tangled chains and filaments (Image 1). Sodium polyanethol sulfonate (SPS) inhibits S. monoliformis growth. As aerobic blood culture bottles made by certain manufacturers contain SPS, the growth is mostly seen in anaerobic bottles devoid of SPS. In liquid broth, S. monoliformis grows as “puff balls.”

Since S. moniliformis is a fastidious organism, the Gram stain from direct smears, such as positive blood culture bottles, in the absence of growth on solid agar media provides an adequate preliminary diagnosis. In such circumstances, 16srRNA sequencing provides a definitive identification. When it grows on agar media, it takes from 2-3 days to as long as 7 days and may appear as “fried-egg” colonies.

Wild rodents as well as laboratory rats carry S. moniliformis in their upper respiratory tract. Cats or dogs preying on rodents also carry the organism. Hence, rat bite fever (RBF) typically occurs upon animal bites.

RBF is a systemic disease wherein patients present with fever, vomiting, headache, and muscle pain or joint swelling. While S. monoliformis is the only known cause of RBF reported in the United States, RBF in Asia is caused by Spirillum minus. Signs and symptoms of the RBF caused by these two organisms slightly differ in addition to the fact that S. minus cannot be cultured in vitro. Several cases of endocarditis and septic arthritis due to S. moniliformis have been reported in the United States.

The notion of rat bite fever as a rare infection is likely due to the fact that 1) S. moniliformis is not a reportable disease and 2) the challenges associated with recovery and identification of this organism from culture media. Thus its occurrence maybe underestimated in the past decades. However, the advancement in technology, such as 16srRNA sequencing and MALDI-ToF, has allowed better diagnosis of RBF in recent years. S. moniliformis is generally responsive to penicillin, cephalosporins, carbapenems, aztreonam, clindamycin, clarithromycin, and tetracycline while it may have intermediate susceptibility to aminoglycoside and fluroquinolones. Since there are no definitive minimal inhibitory concentration (MIC) breakpoint guidelines for this organism, clinical laboratories do not generally perform antimicrobial susceptibility testing.

Image 1. Gram stain of Streptobacillus moniliformis from A) positive blood culture bottle and B) subculture growth on blood agar plate (Arrow showing bulging gram negative rods).

References

  1. Elliott SP. 2007. Rat bite fever and Streptobacillus moniliformis. Clin Microbiol Rev 20:13–22.
  2. Manual of Clinical Microbiology. 11th Edition. 2018
  3. https://www.cdc.gov/rat-bite-fever/index.html
  4. Gaastra W, Boot R, Ho HT, Lipman LJ. Rat bite fever. Vet Microbiol. 2009 Jan 13;133(3):211-28. doi: 10.1016/j.vetmic.2008.09.079. Epub 2008 Oct 8. PMID: 19008054.

-Phyu M. Thwe, Ph.D., D(ABMM), MLS(ASCP)CM is Microbiology Technical Director at Allina Health Laboratory in Minneapolis, MN. 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 Male Patient with Fever, Chills, and Rigors

Case History

An 80 year old male from central Vermont with a history of coronary artery disease, hypertension, hyperlipidemia, hypothyroidism, polymyalgia rheumatica, and osteoarthritis status post bilateral knee replacements presented to the ED for fevers, chills, rigors, and fatigue complaining of a home temperature reading of 39.4C (103F). He explained that within the past week he visited a fair to enjoy petting farm animals and trying locally grown produce, meat, and dairy. In the ED he continued to complain of fever, chills, and fatigue, but did not endorse headaches, neck pain, chest pain, SOB, cough, abdominal pain, nausea, vomiting, or diarrhea. An open wound on his left arm was noted. Labs revealed mildly elevated lactic acid, hyponatremia, chronic anemia, but no leukocytosis.

Microbiology

Bacteremia was suspected, and blood cultures obtained. The next day, his blood cultures were positive. Before an extensive workup involving additional cultures and MALDI-TOF, a gram stain was performed and is shown in Image 1. Also, a preparation from a young broth culture demonstrated motility. Both findings indicate Listeria monocytogenes.

Gram positive rods seen in Gram stain from blood culture.

Discussion

Listeria monocytogenes is a gram positive rod. Features that aid in its diagnosis include a narrow zone of beta hemolysis, catalase positivity, tumbling motility, and that it is a facultative intracellular organism.  

Listeria monocytogenes has one to five peritrichous flagella and demonstrate a “tumbling motility” after incubation of a broth culture at room temperature from 8-24 hours. Listeria monocytogenes also demonstrate a “Christmas tree” or “umbrella” (shown below) pattern of motility when inoculated into a semi solid agar, or “motility medium” (Image 2). This pattern occurs because it grows best in a zone of reduced oxygen tension about 0.5cm below the surface that is also is not strictly anaerobic.1 It also has intracellular motility via polymerization of actin, referred to as “actin rockets,” and can even use this method to spread from one cell to another.

Image 2. “Christmas tree” pattern when Listeria monocytogenes is inoculated into semi-solid agar.

Listeria monocytogenes prefers colder temperatures and is often acquired through consumption of un-pasteurized refrigerated dairy products, deli meats, or produce. Pregnant individuals are more likely to acquire infection as are the young, elderly, or the immunocompromised. Listeria monocytogenes can cause meningitis, usually in newborns or the elderly. Central nervous system or bloodstream infections are treated with ampicillin and gentamycin.2

References

  1. Allerberger F. Listeria: growth, phenotypic differentiation and molecular microbiology. FEMS Immunol Med Microbiol. 2003 Apr 1;35(3):183-9. doi: 10.1016/S0928-8244(02)00447-9. PMID: 12648835. (n.d.).
  2. Gelfand, M., Thompson, J., Geeta S. (2021). Treatment and prevention of Listeria monocytogenes infection. UpToDate. Retrieved September 2, 2021, from https://www-uptodate-com.ezproxy.uvm.edu/contents/treatment-and-prevention-of-listeria-monocytogenes-infe. (n.d.).

-Joe Teague is a Pathology Student Fellow and Brianna Waller, MD is a 3rd year Anatomic and Clinical Pathology Resident at the University of Vermont Medical Center.

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

Microbiology Case Study: A 67 Year Old Man Develops Severe Leg Pain

Case History

A 67 year old male presented with type II diabetes, hypertension, hyperlipidemia, obstructive sleep apnea, recurrent GI bleeds, and atrial fibrillation (status post ablation and on rivaroxaban). Given the history of recurrent GI bleeds, he was taken off rivaroxaban and underwent a left atrial appendage occluder device implant procedure. Several hours later, he developed severe leg pain and loss of lower extremity pulses. CT angiogram confirmed Watchman device embolization to the abdominal aorta. The patient received emergent surgical removal of the device. In the ICU, the patient developed worsening rhabdomyolysis, anuria, hypotension, ischemic bowel disease, and died within hours. An autopsy was requested by the next of kin, which revealed an unexpected finding of a 6 cm hilar-based lung mass.

Microbiology

Premortem and postmortem cultures were not collected. Fontana-Masson stain of a section from the hilar lung tissue reveals yeast of varying size with a lighter shade in the center and a thick capsule, though the capsule does not stain and can instead be appreciated as a “halo” (Image 1). Fontana Masson stain also reveals narrow based budding (Image 2). Both findings indicate Cryptococcus neoformans.

Image. 1. Fontana-Masson stain revealing yeast of varying size with a lighter shade in the center and a thick capsule.
Image 2. Fontana-Masson stain revealing narrow based budding

Discussion

Cryptococcus neoformans is a saprophytic yeast (5-10 µm) identified best by its thick polysaccharide, antiphagocytic capsule. It can be infectious when inhaled, often from soil or avian droppings.

While most immunocompetent individuals clear the pathogen, in the immunocompromised, it can form a primary focus in the lungs and then disseminate. It is often asymptomatic when localized to the lungs but can present as a cough or dyspnea. Dissemination to the brain presents as meningitis. Cryptococcal neoformans is the most common cause of fungal meningitis.

While C. neoformans most often presents as meningitis in the immunocompromised, a retrospective case analysis found diabetes mellitus II as a newly defined independent factor contributing to morbidity and mortality. This study analyzed cryptococcal infections in patients with DMII from 1997-2015. 57% of the DMII patients did not have any other underlying disease and 69% of patients who presented with pulmonary Cryptococcus neoformans experienced a misdiagnosis and treatment delays.1

Qualities that aid in the diagnosis include urease positivity, positive latex agglutination test due to its thick polysaccharide capsule, and characteristic features on mucicarmine red, methenamine silver, India ink, and Fontana-Masson stains.2

The Fontana-Masson silver (FMS) stain is a histochemical technique that oxidizes melanin and melanin-like pigments as it reduces silver.  FMS can be used to highlight the melanin-like pigment in Cryptococcus spp., including capsule-deficient variants because this pigment is cell-wall (and not capsule) associated. FMS is a very sensitive, but not completely specific stain, for Cryptococcus spp. as other yeasts and fungi can also produce melanin and melanin-like pigments.3 Though two C. neoformans yeast close together can resemble broad-based budding, Image 2 demonstrates the narrow-based budding.

While the India ink stain is often discussed as a popular stain for C. neoformans, it can only be performed on liquid samples (CSF, fluid samples) and cannot be performed on paraffin-embedded tissue samples. Of note, the India ink stain is a “negative stain”, resulting in the classic “halo” effect (image 3) because it is not picked up by the capsule of Cryptococcus spp. Because of this, it will miss capsule-deficient infections.4

Image 3. India Ink Stain showing “halo” effect of capsules.

Prognosis varies by the mechanism of immunosuppression. Acute mortality in in cryptococcal meningitis for HIV patients has improved dramatically with antifungals and ART, ranging from 6-16%. Poor prognostic indicators include abnormal mental status, a high yeast burden defined as CSF antigen titer > 1:1024 by latex agglutination or > 1:4000 by lateral flow assay, or a poor host response defined as CSF WBC count < 20/microL.5

Treatment of cryptococcal infections includes initial therapy with amphotericin B and flucytosine followed by long term fluconazole.6

References

  1. Boulware DR, Rolfes MA, Rajasingham R, von Hohenberg M, Qin Z, Taseera K, Schutz C, Kwizera R, Butler EK, Meintjes G, Muzoora C, Bischof JC, Meya DB. Multisite validation of cryptococcal antigen lateral flow assay and quantification by laser thermal contr. (n.d.).
  2. Li Y, Fang W, Jiang W, Hagen F, Liu J, Zhang L, Hong N, Zhu Y, Xu X, Lei X, Deng D, Xu J, Liao W, Boekhout T, Chen M, Pan W. Cryptococcosis in patients with diabetes mellitus II in mainland China: 1993-2015. Mycoses. 2017 Nov;60(11):706-713. doi: 10.1111/. (n.d.).
  3. McFadden, D., & Casadevall, A. (2001). Capsule and Melanin Synthesis in Cryptococcus neoformans. Medical Mycology, 39, 19-30.
  4. Perfect JR, Dismukes WE, Dromer F, Goldman DL, Graybill JR, Hamill RJ, Harrison TS, Larsen RA, Lortholary O, Nguyen MH, Pappas PG, Powderly WG, Singh N, Sobel JD, Sorrell TC. Clinical practice guidelines for the management of cryptococcal disease: 2010 up. (n.d.).
  5. Saag MS, Powderly WG, Cloud GA, Robinson P, Grieco MH, Sharkey PK, Thompson SE, Sugar AM, Tuazon CU, Fisher JF, et al. Comparison of amphotericin B with fluconazole in the treatment of acute AIDS-associated cryptococcal meningitis. The NIAID Mycoses Study. (n.d.).
  6. Winn, W. C., & Koneman, E. W. (2006). Koneman’s color atlas and textbook of diagnostic microbiology. Philadelphia: Lippincott Williams & Wilkins. (n.d.).

-Joe Teague is a Pathology Student Fellow and Brianna Waller, MD is a 3rd year Anatomic and Clinical Pathology Resident at the University of Vermont Medical Center.

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

Microbiology Case Study: A 28 Year Old Woman with Fever and Rash

Case History

A 28 year old female presented to the emergency department (ED) with fevers, chills, and rash. A maculopapular rash began two days prior to her presentation starting at her shins, then spreading to her abdomen, chest, and arms. On presentation she had left knee and right elbow pain and had pain with walking. The patient denied neck pain, headache, blurry vision. Her past medical history is significant for bacterial meningitis between the ages of 10-12. She does not recall the causative pathogen but does recall that her mother had to quarantine for a period of time. On physical exam, the patient was in no distress, had a low-grade fever (38.2 °C) with normal heart rate and blood pressure. Her left knee and right knee were swollen, warm, and painful to the touch with limited range of motion. She was found to have round, erythematous papules and plaques, some with central purpura primarily involving both legs as well as the back and arms (Figure 1).

Initial labs showed a WBC of 11.24/L, Hgb 10.2 g/dL, platelets of 172/L. Her acute inflammatory marker was elevated (CRP 24.7 mg/dL) and her sed rate was 30 mm/hr. One of two blood cultures on admission grew Neisseria meningitides. She was initially started on vancomycin and piperacillin-tazobactam and later transitioned to ceftriaxone to complete a 7 day course. Further blood cultures remained sterile. Dermatology was consulted for her skin lesions and a biopsy of a thigh lesion revealed leukocytoclastic vasculitis. Orthopedic surgery was consulted given her left knee and right elbow swelling. X-ray revealed trace effusions in both joints. A tap of both joints was unsuccessful. The patient developed acute renal insufficiency secondary to acute tubular necrosis during her hospitalization, which improved prior to discharge. Her fevers resolved and the joint swelling and pain improved prior to discharge. She had off and on headaches during her hospitalization but denied neck pain/stiffness and never required a lumbar puncture. She was discharged on prophylactic amoxicillin.

Two months after discharge she was seen in immunology clinic at which time she was tested for humoral and complement immune deficiencies. Her total complement (CH50) was found to be low at 15 U/mL while C3 and C4 were normal, suggesting a terminal complement deficiency. Her humoral immunity panel was normal.

Discussion

Meningococcemia without meningitis occurs in 20-30% of patients with invasive meningococcal disease.1 This patient showed evidence of invasive disease with a maculopapular rash, joint involvement, and renal injury. However, her disease never reached the level of septicemia or disseminated intravascular coagulation (DIC) likely because she presented early enough in the course of her infection. A prior history of bacterial meningitidis raised suspicion for a terminal complement deficiency that was later confirmed by a low CH50. Patients with terminal compliment (C5-C9) deficiency are 1,400-10,000x more likely to develop meningococcal disease and 40-50% of these individuals can have recurrent infection.2 Deficiencies in other components of the complement pathway such as C3 and C4, especially in association with systemic lupus erythematous (SLE), and properdin (a promoter of the alternative complement pathway) can lead to invasive meningococcal disease.3 Patients with anatomic or functional asplenia and patients on eculizumab therapy are also at increased risk.4

Neisseria meningitidis is a gram negative diplococcus and is an obligate human pathogen that is also a common human commensal found in the nasopharynx of 3-25% of the population. Meningococcal disease most commonly manifests as meningitis (40-65%) and meningococcemia (20-30%) followed by pneumonia (10%), septic arthritis (2%), and chronic meningococcemia.1 The major virulence factor associated with meningococcal disease is a capsular polysaccharide, which are classified into 13 serogroups (A-L, W-135, X, Y, Z).5 Serogroups A, B, C, W-135, X, and Y are associated with invasive disease and prevalence of each serogroup is geographically varied. Diagnosis is by visualizing gram-negative diplococci on gram-stain (see Figure) from CSF or other sterile body fluid and by culture of these sterile body fluids. It is worth noting that intravenous antibiotics can sterilize meningococci in the CSF within 3-4 hours after administration.6 Transmission is by large respiratory droplets and direct contact from those with carriage or infection. Disease, especially among high-risk patients with complement deficiency, is preventable with the meningococcal conjugate vaccine. Chemoprophylaxis with rifampin, ceftriaxone, ciprofloxacin, or azithromycin is recommended for close contacts regardless of vaccination status.

Figure 1: Maculopapular rash with central purpura of both legs.

References

  1. Stephens, D., Neisseria meningitidis. 9th ed. Principles and Practices of Infectious Diseases, ed. J. Bennett. Vol. 2. 2015.
  2. Ram, S., L.A. Lewis, and P.A. Rice, Infections of people with complement deficiencies and patients who have undergone splenectomy. Clin Microbiol Rev, 2010. 23(4): p. 740-80.
  3. Fijen CA, K.E., te Bulte MT, Daha MR, Dankert J, Assessment of complement deficiency in patients with meningococcal disease in The Netherlands. Clin Infect Dis, 1999. 28(1): p. 98-105.
  4. Lebel, E., et al., Post-eculizumab meningococcaemia in vaccinated patients. Clin Microbiol Infect, 2018. 24(1): p. 89-90.
  5. Chang, Q., Y.L. Tzeng, and D.S. Stephens, Meningococcal disease: changes in epidemiology and prevention. Clin Epidemiol, 2012. 4: p. 237-45.
  6. Crosswell, J.M., W.R. Nicholson, and D.R. Lennon, Rapid sterilisation of cerebrospinal fluid in meningococcal meningitis: Implications for treatment duration. J Paediatr Child Health, 2006. 42(4): p. 170-3.

-Denver Niles is the Medical Microbiology fellow at UT Southwestern Medical Center. Prior to his Medical Microbiology fellowship, he completed pediatric infectious disease training at Baylor College of Medicine/Texas Children’s Hospital.

-Dominick Cavuoti is a professor of Pathology at UT Southwestern Medical Center who specializes in Medical Microbiology, ID Pathology and Cytology.

-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 Female in her 60s with Retro-orbital Headaches

Case History

The patient was a previously healthy female who presented with a five day history of retro-orbital headaches, lightheadedness, and intermittent falls. Her presentation was consistent with meningitis and further studies were pursued. Head computed tomography (CT), CT angiogram of the head and neck, brain magnetic resonance imaging (MRI), and electroencephalogram (EEG) were unremarkable. Analysis of the cerebrospinal fluid (CSF) demonstrated an elevated white blood cell count (605 white blood cells/µL) of which 88% were lymphocytes, 9% were monocytes, and 3% were neutrophils. CSF glucose was slightly decreased at 33 mg/dL and protein was elevated at 81 mg/dL. Additional tests requested on the CSF included herpes simplex virus (HSV), varicella zoster virus (VZV), West Nile virus (WNV), and Epstein-Barr virus (EBV). The CSF was positive for HSV-2 and negative for HSV-1, VZV, and EBV by PCR. WNV IgG and IgM were negative. Of note, the patient had two episodes of viral meningitis in the past of unknown etiology. The patient received a one week course of valacyclovir and was discharged. Per the patient, she continues to have fluctuating headaches and occasional lightheadedness. Follow-up imaging has been unremarkable.

Figure1. Results of the HSV-1 and HSV-2 PCR. HSV-2 (green) and internal control (purple) amplified. HSV-1 (red) was not detected.

Discussion

Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) are enveloped, double stranded DNA viruses that are members of the Herpesviridae family. They are common viruses that cause cold sores or fever blisters. HSV is a lifelong infection, and latent infection can cause reactivation. While both HSV-1 and HSV-2 can affect any area, HSV-1 is typically associated with non-genital sites whereas HSV-2 typically causes genital infections. In addition to herpetic gingivostomatitis, herpes labialis, and herpes genitalis, other associated clinical conditions include encephalitis, meningitis, keratitis, esophagitis, neonatal herpes, and disseminated primary infection. Most cases of HSV encephalitis have been linked to HSV-1 while HSV meningitis is typically caused by HSV-2. As seen in our patient, HSV-2 has been implicated in recurrent, aseptic, and self-limiting meningitis, also known as Mollaret meningitis.1 There are no specific treatment guidelines for HSV-2 meningitis with the main therapeutic strategy being symptom management. The utilization of acyclovir to manage uncomplicated HSV-2 management is controversial and there is no current consensus.2

Clinically, patients with meningitis typically present with acute onset of fever, headache, and neck stiffness. Other associated symptoms include malaise, rash, nausea, vomiting, sore throat, lymphadenopathy, and genitourinary symptoms. In order to differentiate between the infectious etiologies (i.e. viral, bacterial, tuberculous, or fungal) that cause meningitis, a lumbar puncture may be performed. For viral meningitis, CSF will usually show an elevated white count with predominantly mononuclear cells. The CSF:serum glucose ratio and protein levels are often elevated. The most common CSF viral pathogens in the non-immunosuppressed population are enteroviruses, HSV-1, HSV-2, and VZV, which can all be detected by real time polymerase chain reaction (RT-PCR) technology Molecular methods are faster, more sensitive, and more widely available that viral culture.3 Antibody tests are not recommended for HSV as ~70% of adults will be positive for HSV-1 and ~20-50% of adults will be positive for HSV-2.4

Given the broad range of infectious etiologies that can cause meningitis, there has been interest in the development of a multiplex molecular test. Currently, the FilmArray meningitis/encephalitis panel is the only one that has received FDA clearance. This panel includes 14 bacterial, fungal, and viral targets, including HSV-1 and HSV-2. However, this panel should be used cautiously as several studies have shown a high proportion of false negatives in the detection of HSV-1, HSV-2, and Crytococcus neoformans/gattii. It has been suggested that for HSV-1 and HSV-2, the multiplex panel does not work as well if the viral load is near the limit of detection of the assay or if the patient is having a reactivation of HSV. If there is a high clinical suspicion, particularly in neonates and immunosuppressed patients, an assay for detection of only HSV-1 and HSV-2 should be performed.5

References

  1. Koelle DM and Corey L. (2008) Herpes simplex: insights on pathogenesis and possible vaccines. Annual Review of Medicine, 59: 381-395.
  2. Bamberger DM. (2010) Diagnosis, initial management, and prevention of meningitis. American Family Physician, 82: 1491-1498.
  3. Logan SAE and MacMahon E. (2008) Viral meningitis. The BMJ, 336: 36-40.
  4. Schiffer JT, Corey L. (2020) Herpes simplex virus. Bennett’s Principles and Practice of Infectious Diseases, 9th edition.
  5. Tansarli GS and Chapin KC. (2020) Diagnostic test accuracy of BioFire FilmArray meningitis/encephalitis panel: a systematic review and meta-analysis. Clinical Microbiology and Infection, 26: 281-290.

-Melissa Tjota, MD, PhD is a Molecular Genetic Pathology fellow at the University of Chicago Medicine and NorthShore University HealthSystem. She completed her MD/PhD (Immunology) and AP/CP residency at the University of Chicago.

-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 Middle Aged Man with a Non-Healing Ulcer

Case History

A middle-aged man with osteogenesis imperfecta, poorly controlled HIV, and hepatitis C presented for outpatient management of an infected non-pressure ulcer on his ankle. The patient had no history of recent travel and was current on vaccinations. He was afebrile but reported increasing difficulty with ambulation due to pain from the lesion. Per the patient, the wound began as an itchy “bug bite” two weeks prior, which he had scratched, causing skin breakage. The ulcer became progressively larger with corresponding increases in pain, warmth and swelling. Specimens from the ulcer were collected for culture, and the patient started empiric doxycycline in addition to prophylactic amoxicillin/clavulanate he was already taking for management of a pre-existing ulcer on the opposite foot. The non-healing wound progressed to the size of a nickel with worsening pain. Due to these symptoms and the associated microbiological data, the patient was instructed to present to the emergency department where he was admitted for additional evaluation. Upon admission, the ulcer exhibited no surrounding erythema, but a slight exudate and pitting edema was noted (Image 1). An X-ray of the ankle was obtained which revealed soft tissue swelling, but no fracture or crepitus.

Image 1. Photograph of the ulcerated lesion when seen at hospital admission.

Microbiology

Specimens of the ulcerated lesion were submitted to the microbiology laboratory for routine bacterial culture. No growth was observed on MacConkey agar plates, while two beta-hemolytic morphotypes and one non-hemolytic morphotype were observed on blood agar. The two beta-hemolytic species were identified as Streptococcus dysgalactiae and Arcanobacterium haemolyticum by MALDI-TOF MS. The third, non-hemolytic organism was a catalase-positive, gram positive coryneform rod (Image 2A and B), and was identified by MALDI-TOF MS as Corynebacterium diphtheriae. This identification was subsequently confirmed by both a commercial reference laboratory and the US Centers for Disease Control and Prevention.

Image 2. Growth of the isolate of C. diphtheriae on a blood agar plate and associated Gram stain.  A) Non-hemolytic colonies observed after 24 hours incubation.  B) gram positive coryneform rods of C. diphtheriae.

Discussion

Corynebacterium diphtheriae is the etiological agent of diphtheria, a toxin-mediated disease classically associated with respiratory and cutaneous infections. C. diphtheriae is infrequently encountered in the United States due to a robust national vaccination program but remains endemic in other parts of the world. Respiratory manifestations include pharyngitis with dysphagia, lymphadenitis (often described as a “bull-neck” appearance), and the development of a characteristic white/grey pharyngeal pseudomembrane which can cause airway obstruction. The pathogenesis of C. diphtheriae is mediated by diphtheria toxin which inhibits host cell protein synthesis leading to cell death. Diphtheria toxin can also cause cardiac, nephrotic, and neurological sequalae due to dissemination. The diphtheria toxin gene (tox) is encoded on a bacteriophage which lysogenizes into the bacterial chromosome and is expressed in response to low iron concentrations. While C. diphtheriae is most frequently associated with diphtheria toxin production, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis can also express the toxin if infected with the bacteriophage.

Cutaneous C. diphtheriae infections manifest initially as a vesicle, eventually developing into a painful ulcerative lesion that may or may not have a pseudomembrane. These infections can be caused by either fully toxigenic strains, non-toxigenic strains lacking the tox gene, or non-toxigenic toxin gene bearing (NTTB) strains.1 NTTB strains are genotypically positive for the presence of the tox gene on the lysogenized phage, but do not express functional diphtheria toxin. This can be due to 1) mutation of truncation of the tox gene coding sequence, 2) promotor mutations, or 3) alterations in proteins regulating tox expression. NTTB strains are important from an epidemiological perspective in that they serve as an environmental reservoir for tox gene-harboring phage which could convert circulating non-toxigenic C. diphtheriae into toxin-producing organisms.1-3 This phenotype also presents additional diagnostic challenges as toxin gene expression must therefore be confirmed by more laborious phenotypic methods instead of genotypically (i.e. by PCR).

Recovery of C. diphtheriae in the routine setting is challenging as the organism morphologically resembles other coryneform rods usually representative of flora in cutaneous and respiratory specimens. While selective and differential medias are available (i.e. Cystine Tellurite Blood Agar or Tinsdale medium), they are not used routinely due to low incidence. As both toxigenic and non-toxigenic strains of C. diphtheriae circulate, this isolate was referred to the CDC’s Pertussis and Diphtheria laboratory for additional typing and toxin analysis. The organism was determined to belong to the mitis biotype and was positive for the tox gene by PCR. Toxin gene expression was then evaluated by the Elek Immunodiffusion test (Image 3). In this classical method, a filter paper strip saturated with anti-toxin is placed perpendicular to control and test strains of the organism on non-selective media. If the organism expresses diphtheria toxin, the toxin and the antisera form a complex and precipitate from solution. This phenomenon is visualized as precipitin lines in the agar after 24 hours of incubation at 37°C.4 Despite tox gene PCR-positivity, the Elek Immunodiffusion test revealed that this patient’s isolate did not express diphtheria toxin. Thus, this C. diphtheriae isolate was a representative example of a NTTB strain.

Image 3. Representation of the Elek Immunodiffusion assay for the detection of diphtheria toxin.  Filter paper soaked with antitoxin placed perpendicular to test and control isolates.  The presence of precipitin lines indicates diphtheria toxin gene expression.  Figure adapted from (3).

In contrast to respiratory presentations which have declined due to vaccination, cutaneous infections with C. diphtheriae have become more frequently recognized. Unlike respiratory diphtheria, cutaneous diphtheria was not reportable to the National Notifiable Diseases Surveillance System until the clinical criteria changed in 2019. Since that time, toxigenic isolates recovered from either respiratory or cutaneous sources are reportable.5 Additionally, the incorporation of MALDI-TOF MS into routine workflow has facilitated identification of the organism when isolated clinically and likely increased reporting.

Although immunization protects against clinical diphtheria, it does not prevent colonization by non-toxigenic C. diphtheriae including NTTB strains.2,3 Non-toxigenic C. diphtheriae causing cutaneous infections are often recovered with other pyogenic organisms including Staphylococcus aureus, beta-hemolytic streptococci, and A. haemolyticum6 as was observed in this case. Risk factors for cutaneous infections include a compromised immune system, eczema, travel to endemic regions, intravenous drug use, homelessness/unsanitary living conditions, and alcoholism.3,7  It is unclear how this patient was exposed, although he did have risk factors including immunosuppression and those associated with his osteogenesis imperfecta. The patient was started on erythromycin and ampicillin/sulbactam for subsequent management and has continued to improve when seen at follow-up. Subsequent cultures of the wound have remained negative for C. diphtheriae.

  1. Zakikhany K, Neal S, Efstratiou A. 2014. Emergence and molecular characterisation of non-toxigenic tox gene-bearing Corynebacterium diphtheriae biovar mitis in the United Kingdom, 2003–2012. Eurosurveillance 19:20819.
  2. Doyle CJ, Mazins A, Graham RMA, Fang N-X, Smith HV, Jennison AV. 2017. Sequence Analysis of Toxin Gene-Bearing Corynebacterium diphtheriae Strains, Australia. Emerging infectious diseases 23:105-107.
  3. Sharma NC, Efstratiou A, Mokrousov I, Mutreja A, Das B, Ramamurthy T. 2019. Diphtheria. Nature Reviews Disease Primers 5:81.
  4. Kates O, Starr K, Bourassa L, Burnham C-AD. 2020. The Brief Case: Nontoxigenic Corynebacterium diphtheriae in a Nonhealing Wound. Journal of Clinical Microbiology 58:e00506-00520.
  5. United States Centers for Disease Control and Prevention. 2019. Diphtheria (Corynebacterium diphtheriae) 2019 Case Definition – National Notifiable Diseases Surveillance System. https://ndc.services.cdc.gov/case-definitions/diphtheria-2019/. Accessed August 1st, 2021.
  6. Lowe CF, Bernard KA, Romney MG. 2011. Cutaneous Diphtheria in the Urban Poor Population of Vancouver, British Columbia, Canada: a 10-Year Review. Journal of Clinical Microbiology 49:2664-2666.
  7. Gubler J, Huber-Schneider C, Gruner E, Altwegg M. 1998. An Outbreak of Nontoxigenic Corynebacterium diphtheriae Infection: Single Bacterial Clone Causing Invasive Infection Among Swiss Drug Users. Clinical Infectious Diseases 27:1295-1298.

-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.