Microbiology Case Study: A 58 Year Old Female with Abdominal Pain

Clinical History

A 58 year old female with no significant past medical history presented her primary care physician with chief complaint of abdominal pain. She reported continued vague abdominal symptoms for the past two months, with intermittent diarrhea and increased flatulence. No recent travel history or significant exposures were identified. An ultrasound of the right upper quadrant was unremarkable and no gallstones were present. The patient was scheduled for a screening colonoscopy. A stool specimen was submitted to the microbiology laboratory for stool culture and ova & parasite exam.  

Laboratory Identification

Image 1. Trichrome stained fecal smear illustrating a binucleated trophozoite with fragmented karyosomal material from a stool ova & parasite exam.
Image 2. Additional trichrome fecal smear image highlighting both uninucleate and binucleate trophozoites that range in size from 5 to 15 um.

The findings from the ova and parasite exam were consistent with Dientamoeba fragilis, an intestinal flagellate. The stool culture was negative for Salmonella, Shigella, and Escherichia coli 0157:H7. Stool enzyme immunoassays were negative for Campylobacter spp.and Shiga toxin 1 and 2.

Discussion

Dientamoeba fragilis is an intestinal flagellate with worldwide distribution and causes asymptomatic and symptomatic infections, predominantly in small children. Symptoms of infection may include intermittent diarrhea, abdominal pain, anorexia, weight loss, and flatulence.  While the pathogenesis is not completely understood, transmission is thought to occur via the fecal oral route and it is hypothesized that the trophozoites are transmitted via the eggs of nematodes, Enterobius vermicularis and Ascaris lumbricoides, due to a higher incidence of co-infections between these organisms than expected.

In the laboratory, the diagnosis of D. fragilis is made by ova and parasite exam. The trophozoite resembles amebae and is typically 9-12 µm. Most trophozoites are binucleate with finely granular cytoplasm and the within the nuclei there are 4-8 fragments of karyosomal granules (Figure 1). Due to the fact that 30-40% of D. fragilis trophozoites are uninucleate (Figure 2) and they lack external flagella, they must be differentiated from Endolimax nana and Entamoeba hartmanni, which are both non-pathogenic amebae. Historically, no cyst phase was known for D. fragilis; however, recent studies have identified precyst forms or putative cysts. Permanently trichrome stained slides are essential to diagnosing D. fragilis infection, as the organism is hard to detect in concentrated smears.

Since our patient was symptomatic, she was treated with iodoquinol, the drug of choice for D. fragilis infections. Her symptoms resolved and colonoscopy did not reveal additional pathology.  

-Debbie Walley, MD, is a 4th year Anatomic and Clinical Pathology chief resident at the University of Mississippi Medical Center. 

-Lisa Stempak, MD, is an Assistant Professor of Pathology at the University of Mississippi Medical Center in Jackson, MS. She is certified by the American Board of Pathology in Anatomic and Clinical Pathology as well as Medical Microbiology. She is the Director of Clinical Pathology as well as the Microbiology and Serology Laboratories. Her interests include infectious disease histology, process and quality improvement, and resident education.

Microbiology Case Study: A 15 Year Old Male with Endocarditis

Case History

A 15 year old male with a past medical history significant for Tetralogy of Fallot (congenital heart defect), multiple valve replacements, chronic kidney disease, and prior Bartonella endocarditis. He presented with a “flu-like” illness including muscle aches, fevers, fatigue, and night sweats. His symptoms slowly dissipated after about three days. However, he had labs drawn including multiple blood culture sets which were all positive for growth.

Laboratory Findings

Gram stain showed gram positive bacilli and culture plates grew two morphologies of slow growing gray, granular and opaque colonies.This organism was identified by MALDI-TOF as Corynebacterium pseudodiphtheriticum.

Image 1. Gram stain with gram positive bacilli .
Image 2. Culture with small, grayish colonies with granular appearance and opaque centers (growth at day 2).

Discussion

The genus Corynebacterium comprises a collection of irregular-formed, rod-shaped or coccoid bacteria that are non-motile, catalase-positive, and non-spore-forming.

Corynebacterium pseudodiphtheriticum (previously designated as Corynebacterium hofmannii) is a nonlipophilic, nonfermentive, urease- and nitrate-positive Corynebacterium species.1 C. pseudodiphtheriticum is part of the usual oropharyngeal bacterial flora, including the nares and throat. It appears to play a role in preventing colonization of oropharyngeal epithelia by pathogenic bacteria.

Most commonly, C. pseduodiptheriticum is a pathogen of the respiratory tract with cases of nosocomial and community-acquired pneumonia, bronchitis, tracheitis, pharyngitis, and rhinosinusitis. Endocarditis is the second most common infection site, although very rare. Cases of urinary tract and wound infections have also been reported.

Treatment is usually with penicillin alone or in combination with aminoglycosides. Antibiotic susceptibility profiling of C. pseudodiphtheriticum isolates showed that resistance to oxacillin, erythromycin, clindamycin, and macrolides are common.1

References

  1. Burkovski A. Corynebacterium pseudodiphtheriticum: Putative probiotic, opportunistic infector, emerging pathogen. Virulence. 2015;6(7):673–674. doi:10.1080/21505594.2015.1067747

-Nicole Mendelson, 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: A 24 Year Old Male with No Past Medical History Returning from Guatemala with Fevers, Myalgia, and Cough

Case History

A 24 year old male with no past medical history presented with fevers, myalgia, and cough following return from a 1-week trip to Guatemala where he spent significant time within caves. The patient described his cough as persistent, non-productive, and associated with mild shortness of breath at rest that significantly worsens with activity. In the emergency department, the patient was afebrile with a WBC of 10.2, Transaminitis, and chest X-ray showed diffuse reticular pattern. He underwent a bronchoscopy and BAL washout.

Laboratory Findings

Histoplasmosis Urine Antigen test came back positive.

Image 1. Fungal culture with white/tan, fluffy mold (growth at day 7).
Image 2. Scotch tape prep with tuberculate macroconidium. This mold was morphologically identified as Histoplasma capsulatum and sent to Mayo Laboratories for further confirmatory testing.

Discussion

Histoplasma capsulatum is an intracellular, thermally dimorphic fungus (grows as a yeast at body temperature/37°C in humans or culture media and as mold at 25°C in the environment/culture media). Histoplasma is found in soil, particularly in areas containing bird and bat droppings, such as caves. Within the United States Histoplasma in found in central and eastern states with a predominance in the Ohio and Mississippi River Valleys. This fungus is also found in parts of Central and South America, Africa, Asia, and Australia.

Infection with Histoplasma capsulatum causes significant morbidity and mortality worldwide. Upon inhalation of conidia, H. capsulatum transforms into the pathogenic yeast phase. This form replicates within macrophages that carry the yeast from the lungs to other organs. Histoplasmosis has three main forms:

  • Acute primary histoplasmosis which presents as a pneumonia with fever, cough, myalgia.
  • Chronic cavitary histoplasmosis which is characterized by pulmonary lesions that often resemble cavitary tuberculosis.
  • Progressive disseminated histoplamosis that spreads to infect many organs in immunocomprimised patients.

In the laboratory, culture of blood, tissue and respiratory specimens may be completed. In addition, a test for H. capsulatum antigen is sensitive and specific when simultaneous serum and urine specimens are tested. It is important to note that cross-reactivity with other fungi (Coccidioides immitis, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Penicillium marneffei) has been identified.

Growth on fungal culture shows white/tan, fluffy mold that turns to brown to buff with age. The organism may also produce wrinkled, moist, or heaped yeast-like colonies that are soft and cream when grown at 37°C on certain media. Scotch tape preparation of the mold form shows tuberculate macroconidia, a diagnostic structure of Histoplasma capsulatum. The mycelia are septate and produce microconidia and macroconidia. Yeast forms of Histoplasma capsulatum are small (2 to 4 μm) and reproduce by budding. These budding forms may be seen on histology specimens. A commercially available DNA probe can be performed on culture material to confirm identification.

Patients with mild-moderate histoplasmosis can often have resolution of their symptoms without treatment. Those with more moderate-severe disease require antifungal agents including amphotericin B or itraconazole.

-Nicole Mendelson, 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.

Fecal Transplants in the News

An article posted today at The Atlantic discusses fecal transplants and FDA regulation. Dr. Colleen Kraft (co-author of a paper on fecal transplant protocols that appeared in Lab Medicine) is quoted in the article, and it’s worth a read.

Microbiology Case Study: A 30 Year Old Male with Fevers and Cough

Clinical History

A 30 year old African American male presented to the emergency department (ED) with fevers and cough. His past medical history was significant for type 1 diabetes & diabetic nephropathy requiring a kidney/pancreas transplant three years prior. He is compliant with his immunosuppressant regimen. He described the cough as non-productive and denied shortness of breath or chest pain. He denied sick contacts, recent travel, and has no pets. After hospital admission, he became septic, developed severe hypotension (70/30s), and was transferred to the intensive care unit (ICU). Chest x-ray showed multifocal consolidations in bilateral lung fields and a small pleural effusion consistent with pneumonia. He was empirically started on vancomycin, piperacillin-tazobactam, azithromycin, and micafungin. Infectious diseases was consulted and recommended a board variety of tests and cultures given the patient immunosuppressed status.

Laboratory Identification

The following results were obtained:

Sputum culture: normal respiratory flora, negative for fungi and acid fast bacilli

Streptococcus pneumoniae & Legionella pneumophila antigens: negative

Histoplasma & Blastomyces urinary antigens: negative

Fungitell: negative

Respiratory viral PCR panel: positive for adenovirus, coronavirus, and rhinovirus

Image 1. Cryptococcal lateral flow assay showing positive (left) and negative (right) results. The patient had a positive result from the serum with a titer of 1:20.

Given this positive serum result for Cryptococcus neoformans despite multiple negative sputum cultures, a bronchoalveolar lavage & lumbar puncture were performed and bacterial & fungal cultures were performed. 

Image 2. Discrete, mucoid, cream colored colonies of Cryptococcus neoformans growing on Sabouraud dextrose agar after the third week of incubation at 25°C from the bronchoalveolar lavage specimen.

Discussion

Cryptococcus neoformans is an encapsulated yeast that is most commonly acquired through inhalation and can infect & disseminate to multiple organ systems including the lungs, central nervous system, skin and bones, especially in immunocompromised patients such as those with HIV or organ transplant patients. The thick polysaccharide capsule gives colonies of C. neoformans a mucoid appearance, serves as a major virulence factor, and also plays an important part in various laboratory identification methods.

In the lab, C. neoformans will grow a variety of selective and non-selective agars including blood, chocolate, Sabouraud dextrose, and cornmeal agars as discrete, cream colored colonies (Image 2). On microscopic examination, the C. neoformans yeast are gram positive with narrow based budding and a thick capsule. The yeast vary in size from 2-20 µm and are evenly spaced from one another due to the capsule. C. neoformans is positive for both urease and phenoloxidase. Historically, India ink stain was performed on CSF specimens to highlight the capsule using direct microscopy. Grocott’s methenamine silver (GMS), mucicarmine, and Fontana-Masson histochemical stains are all positive for C. neoformans.

Cryptococcal antigen tests directed to the capsular polysaccharide can also be used to diagnosis C. neoformans infections from both serum and CSF specimens. Common methods include immunochromatographic lateral flow assays or particle agglutination. Advantages to these methods include increased sensitivity and the ability to provide semi-quantitative titer results which can be used to monitor the patient’s response to therapy. Rarely, false negative results can occur due to extremely high concentrations of the cryptococcal antigen. In order to combat the prozone effect, the sample should be diluted prior to repeating the test if there is a high suspicion of cryptococcal infection. False positive results may also occur when macroglobulins are present in the sample due to disease states such as rheumatoid arthritis or lupus. Use of pronase can prevent the interference of macroglobulins on serum test results. False positive test results have also be documented due to interferences from various collection devices such as anaerobic vials.

In the case of our patient, as C. neoformans is intrinsically resistant to echinocandins, he was switched from micafungin to fluconazole. He responded well and after completing the therapeutic course, he continued on a prophylactic dose of fluconazole. His cerebrospinal fluid culture showed no growth and the cryptococcal lateral flow assay was negative on the CSF specimen.

-Charles Middleton, MD, is a first year Anatomic and Clinical Pathology resident at the University of Mississippi Medical Center.

 

-Lisa Stempak, MD, is an Assistant Professor of Pathology at the University of Mississippi Medical Center in Jackson, MS. She is certified by the American Board of Pathology in Anatomic and Clinical Pathology as well as Medical Microbiology. She is the Director of Clinical Pathology as well as the Microbiology and Serology Laboratories. Her interests include infectious disease histology, process and quality improvement, and resident education.

Microbiology Case Study: A 69 Year Old Male with Cardiac Symptoms

Case History

A 69 year old male with complicated past medical history of sarcoidosis, pulmonary nodules, atrial flutter, right septic arthritis, pulmonary embolism and coronary artery disease presented to the emergency department with worsening cardiac symptoms for the past few days. He denied any symptoms of fever, cough, dyspnea, or palpitations. He has no history of TB exposure, high-risk occupation or volunteer work. Chest x-ray, echocardiogram and computerized tomography (CT) scan were performed and he was diagnosed with constrictive pericarditis. Pericardiotomy was performed which showed thickened and calcified pericardium. On pericardial biopsy specimen, acute necrotizing and granulomatous pericarditis was identified (Image 1). Acid fast bacteria stain for mycobacteria was performed which showed numerous acid-fast bacilli (Image 2). In house validated testing for M. tuberculosis by PCR amplification of the IS6110 insertion sequence and nontuberculous mycobacteria species determination by heat shock protein 65 (hsp65) gene with melt curve analysis was performed. Testing was negative for M. tuberculosis. Nontuberculous mycobacteria testing was consistent with M. xenopi. For definitive diagnosis, culture was performed which grew M. xenopi (Image 3).

Image 1. H&E stain shows acute necrotizing granulomatous inflammation.
Image 2. Ziehl Neelsen stain shows numerous AFB positive bacilli.
Image 3. Lowenstein Jensen (LJ) media growing M. xenopi.

Discussion

M. Xenopi is a free-living nontuberculous mycobacterium (NTM). NTM are present in the environment, mainly in water, and are occasionally responsible for opportunistic infections in humans.1 With the availability of 16S ribosomal DNA sequencing and high-performance liquid chromatography (HPLC), polymerase chain reaction-restriction length polymorphism analysis (PRA), and multi-gene and whole-genome sequencing, the number of new species of nontuberculous mycobacteria has risen dramatically. There are about 180 different species of mycobacteria. The most common nontuberculous species causing human disease in the United States are the slowly growing species, Mycobacterium avium complex and M. kansasii. Less common human pathogens include the slowly growing species M. marinumM. xenopiM. simiaeM. malmoense, and M. ulcerans, and the rapidly growing species M. abscessus complex, M. fortuitum, and M. chelonae.2 NTM can cause pulmonary disease, osteomyelitis or disseminated disease in immunocompromised patients.

Microscopic examination after acid fast or fluorescent Auramine-Rhodamine staining and AFB culture using LJ and Middlebrook 7H10 media are the cornerstones of the identification of mycobacteria. All mycobacteria share the characteristic of “acid-fastness,” ie, after staining with carbol-fuchsin or auramine-rhodamine, they do not decolorize with acidified alcohol. Confirmation of the presence or absence of mycobacteria in clinical specimens requires culture, because of the relative insensitivity of direct microscopy.

The presented case highlights the importance of NTM causing infection in pericardium which is very rare. Special stains, molecular testing, and culture can aid in timely identification of the organism and aid in patient management.

References

  1. Tortoli E. Microbiological features and clinical relevance of new species of the genus Mycobacterium. Clin Microbiol Rev 2014; 27:727.
  2. Griffith DE, Epidemiology of nontuberculous mycobacterial infections. Reyn CF UpToDate. April 2017.
  3. Griffith DE, Microbiology of nontuberculous mycobacteria. Reyn CF UpToDate. Sept 2018.

-Amandeep Kaur, MD MBBS is a 2nd year anatomic and clinical pathology resident at University of Chicago (NorthShore). Academically, Amandeep has a particular interest in hematopathology.

-Erin McElvania, PhD, D(ABMM), is the Director of Clinical Microbiology NorthShore University Health System in Evanston, Illinois. Follow Dr. McElvania on twitter @E-McElvania. 

Microbiology Case Study: A 72 Year Old Woman with Persistent Cough

Clinical History

The patient is a 72 year old female who overall has been fairly healthy. She has struggled with a cough for several years. A CT scan in 2015 showed some tree-in-bud changes in the lungs potentially consistent with an atypical mycobacterial infection. She had a positive methacholine challenge test and was diagnosed with asthma. Her cough has not improved with inhaled asthma treatments. The cough has been persistent and is at times productive of small amounts of whitish sputum. She has not noted any progressive shortness of breath. Over the last several months she has tried trials of both nasal corticosteroids, and treatment with Prilosec for gastroesophageal reflux. Both of these trials had no effect on her cough. At the end of November 2018, a CT scan of the chest was consistent with an atypical mycobacterial infection.

In January 2019, she came back from a skiing trip. She tried to ski but was unable to because of shortness of breath; she came home on the third day prematurely. During this time, she developed increased cough, fevers and chills. An x-ray was obtained by her primary care provider, which showed a right lower lobe infiltrate, and was placed on levofloxacin for 5 days. After completing the antibiotic, she is still very fatigued and still coughing. She presented to her pulmonologist in March 2019. She denied any fevers, chills or chest pain. Her cough has continued with intermittent sputum production. Her appetite and weight have been stable, along with bladder and bowel habits.

The patient’s past medical history is significant for arthritis, cataracts, depression, polymyalgia rheumatica, and sciatic nerve pain. Her past surgical history is only a tonseillectomy in her childhood. Her social and family history is that she runs an educational travel business, is currently divorced, has never been a smoker, and has no family history significant for recurrent infections.

Laboratory Findings

Induced sputum samples were obtained and inoculated on a 7H9 bottle that was incubated and continuously monitored for growth. Eight days later, the 7H9 bottle flagged positive for growth. A gram stain showed branching gram positive bacilli. The Kinyoun acid-fast stain was negative, but a modified Kinyoun showed rare positive staining cells. The bottle was subcultured onto chocolate agar, 7H11 agar, buffered charcoal yeast extract (BCYE) agar, and LJ slants. Two days later, all plates except the 7H11 plate showed growth of white, dry, crinkled colonies as depicted in Image 1. A gram stain of the colonies showed branching gram positive bacilli as shown in Image 2. Again, the Kinyoun stain of the colonies was negative but the modified Kinyoun again highlighted cells as seen in Image 3. A representative colony was sent for identification to Mayo laboratories a day later. MALDI-TOF performed by Mayo Laboratories revealed the bacteria to be Rothia aeria. An attempt was made to set up sensitivities but the organism was not able to grow out on the test medium.

Image 1. Isolated bacteria culture on chocolate agar.
Image 2. Gram stain, 1000x.
Image 3. Modified AFB stain, 1000x.

Discussion

Rothia aeria is a very rare pathogen with a current PubMed search yielding 20 references. It was first isolated from the MIR space station (1) and genomic sequencing was perform on the bacteria (2). It has been shown to be a normal oral flora (3), but also seems to be most associated with endocarditis. A few case reports have discussed sepsis, respiratory infections, and joint infections. Importantly, it has been documented to be confused with Nocardia species (4).

References

  1. Li, Y. et al. Rothia aeria sp. nov., Rhodococcus baikonurensis sp. nov., and Arthrobacter russicus sp. nov., isolated from air in the Russian space laboratory Mir. Int J Syst Evol Microbiol. 2004; 54(pt. 3): 827-835.
  2. Nambu, T. et al. Complete Genome Sequence of Rothia aeria Type strain JCM 11412, Isolated from Air in the Russian Space Laboratory Mir. Genome Announc. 2016 Dec 29; 4(6).
  3. von Graevenitz, A. et al. Coryneform bacteria in throat cultures of healthy individuals. J Clin Microbiol. 1998; 36: 2087-2088.
  4. Saraya, T. et al. Rothia aeria: a great mimicker of the Nocardia species. BMJ Case Rep. Published Online: November 18, 2014.

-Jeff Covington, MD, PhD is a 2nd 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.