Microbiology Case Study: A 57 Year Old Man with Fever

Case History

A 57 year old male with a recent history of a left above the knee amputation developed a fever during the same admission of 101.1°F. His amputation had been complicated by poor wound healing, and he had a simultaneous right leg abscess that grew methicillin-sensitive Staphylococcus aureus. Examination of his wound showed serosanguinous drainage with no erythema or purulence. Blood cultures and a wound swab were sent for microbiological analysis.

Laboratory Findings Wound cultures grew methicillin-resistant Staphyloccocus aureus thought to represent colonization rather than true infection. Blood cultures flagged positive in one anaerobic bottle only at 30 hours. A gram smear showed gram-negative cocci (Image 1). Anaerobic blood plates grew pinpoint colonies (Image 2). MALDI-TOF identified the bacteria as a Veillonella species.

Image 1. Gram stain from anaerobic culture showing gram negative cocci.
Image 2. Growth on anaerobic blood plate.

Discussion

Veillonella species are gram negative cocci. They are lactate fermenting, obligate anaerobes and are considered normal flora of the intestines and oral mucosa. As such, they are usually regarded as a contaminant. They have, however, been implicated in osteomyelitis, prosthesis infections, and endocarditis. They are particularly associated with poor oral hygiene, chronic periodontitis, and smoking. They have important implications in dental disease due to their ability to form biofilms. They are frequently resistant to ampicillin and have also been noted to be resistant to tetracyclines in periodontal patients. Identification is done by molecular methods, typically MALDI-TOF. PCR has also been developed, but is not routinely used.

This was considered a contamination due the absence of symptoms and isolation in one bottle only. A follow up blood culture was negative. Routine wound care was resumed.

References

  1. Rovery C, Etienne A, Foucault C, Berger P, Brouqui P. Veillonella montpellierensis endocarditis. Emerg Infect Dis. 2005;11(7):1112–1114.
  2. Mashima I, Theodorea CF, Thaweboon B, Thaweboon S, Nakazawa F. Identification of Veillonella Species in the Tongue Biofilm by Using a Novel One-Step Polymerase Chain Reaction Method. PLoS One. 2016;11(6):e0157516. Published 2016 Jun 21.

-Jonathan Wilcock, 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 83 Year Old Male with Fever

Case History

The infectious disease service was consulted on an 83 year old male for fever.

His past medical history was significant for diabetes mellitus, anemia and renal insufficiency. He initially presented 3 weeks ago with chills, rigors and fever to 103 degrees Fahrenheit. For the past several months, the patient has had weight loss (10-20 pounds over an unspecified timeframe), fatigue and new iron deficiency anemia. A heart murmur was heard on physical exam. The patient was admitted for suspicion of sepsis and he was started on empiric antibiotics vancomycin and ceftriaxone. Three sets of blood cultures were drawn prior to initiation of antibiotics, which were all positive for gram positive cocci in pairs and chains. Transesophageal echocardiogram (TEE). TEE showed large vegetation on posterior mitral leaflet measuring 1cm x 1.8 cm, and a smaller mass on the anterior leaflet. A week after admission, a mitral valve replacement was performed followed and a portion of the valve was sent for culture (Figure 1).

Laboratory Identification

Image 1. Gram stain from mitral valve specimen showing a large accumulation of gram positive cocci in chains (100x magnification).
Image 2. Brown-Hopps stain of the surgically resected mitral valve tissue vegetation showing sheets of gram positive bacteria (40x magnification).

Discussion

The gram positive organism from blood and mitral valve culture was identified as Streptococcus mitis by MALDI-TOF mass spectrometry. S. mitis is a member of the Streptococcus genus. Streptococci have a number of features that aid in laboratory identification: they are Gram positive, catalase-negative, spherical/ovoid, with organisms that are usually found in chains. They are facultative anaerobes.

More specifically, S. mitis belongs to the viridans streptococci group which includes Streptococcus mutans, Streptococcus sanguis, and Streptococcus salivarius, among many others. The most common infection caused by viridans streptococci is bacterial endocarditis, as in the case of this patient. Other infections can include brain abscesses, liver abscesses, dental caries, and bacteremia.

Patients with bacterial endocarditis have an infection of the heart valves or the endoecardial wall that leads to formations of vegetations. These vegetations are composed of thrombotic debris and organisms (Image 2), often associated with destruction of cardiac tissue. Its onset often involves severe symptoms including fever, chills, and weakness. Fever is the most consistent symptom of infective endocarditis, but it may be subtle or even absent in some cases, especially in older adults. Weight loss and flu-like symptoms may also be seen. Left-sided infective endocarditis, as in the case of our patient, will present with murmur in 90% of cases. In long-standing infective endocarditis, patients may present with Roth spots (retinal hemorrhages), Osler nodes (subcutaneous nodules in the digits), microthromboemboli (which appear as splinter hemorrhages under fingernails and toenails), and Janeway lesions (red nontender lesions on the palms or soles).

In the laboratory, the diagnosis of S. mitis and other viridians streptococci is often detected via blood culture as in the case of this patient. Once the blood culture bottle becomes positive, a Gram stain is performed, which shows Gram positive cocci in chains (Image 1). These features are helpful in differentiating Streptococcus from Staphylococcus (which appears as clusters instead of chains). Biochemical testing can be done to narrow down the species and identify S. mitis, which is optochin resistant (as opposed to S. pneumonia), acetoin negative (in contrast to most other viridans organisms), and urease negative (which differentiates it from S. vestibularis which is urease positive).

Surgical pathology can also aid in diagnosis by microscopically identifying vegetations on the affected valve (Image 2). Treatment of bacterial endocarditis is usually with penicillin or ceftriaxone, however susceptibility testing should be performed on S. mitis and other viridians streptococci because resistance can occur to penicillin. Blood cultures are followed until they are negative for 72 hours. In the case of our patient, his cultures became negative shortly after he started treatment. Susceptibility testing showed that the organism is sensitive to penicillin and ceftriaxone. The patient was continued on ceftriaxone and is clinically improving.

-Haytham Hasan, MD, is an Anatomic and Clinical Pathology resident at NorthShore Evanston Hospital (University of Chicago).

-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: An 8 Year Old Male with Left Knee Pain

Case History

An 8 year old male with no significant medical history presented with left knee pain and swelling for one week. Physical examination revealed a temperature of 101.2°F and a left swollen, tender knee with reduced range of motion. A joint aspirate was performed, and synovial fluid and blood were sent for microbiological analysis.

Laboratory Findings

Synovial fluid analysis showed increased neutrophils, a nucleated cell count of 90,840 cells/cmm, and no crystals.

Blood cultures were negative. Gram smear of the joint fluid showed many neutrophils and no bacteria. Fluid culture grew convex tan-yellow colonies on blood and chocolate plates at 48 hours (Image 1). Gram smear revealed gram-negative cocci (Image 2). The organism was identified by MALDI-TOF as Aggregatibacter aphrophilus. Antibiotic susceptibility testing showed susceptibility to augmentin, ampicillin, ceftriaxone, and levofloxacin.

Image 1. Growth on anaerobic chocolate plate.
Image 2. Gram stain from anaerobic culture showing gram negative cocci.

Discussion

Aggregatibacter aphrophilus is a gram negative coccobacillus that requires 5% CO2 and grows best on blood agar. It is oxidase negative and catalase negative. It is categorized as a HACEK organism, being a cause of culture-negative endocarditis. It is considered normal oral flora, and dental procedures can be a source of infection. Aggregatibacter endocarditis can cause a positive P-ANCA and be misdiagnosed as a vasculitis. It has also been reported as causes of sacroiliitis, bartholinitis, endophthalmitis, and brain abscesses. Treatment is generally ceftriaxone for 8 weeks. Identification is by biochemical methods or MALDI-TOF. Broad range PCR (br-PCR) has also been described, which targets a highly-conserved region of 16S rDNA, and then compares the sequences to database sequences.

The patient was given cefazolin, and his temperature downtrended. He was discharged prior to results but placed on oral augmentin. After susceptibility testing, infectious disease was consulted and he was placed on ceftriaxone for 8 weeks. He continued to improve and subsequent cultures were negative.

References

  1. Ratnayake L, Olver WJ, Fardon T. Aggregatibacter aphrophilus in a patient with recurrent empyema: a case report. J Med Case Rep. 2011;5:448. Published 2011 Sep 12. doi:10.1186/1752-1947-5-448
  2. Hirano K, Tokui T, Inagaki M, Fujii T, Maze Y, Toyoshima H. Aggregatibacter aphrophilus infective endocarditis confirmed by broad-range PCR diagnosis: A case report. Int J Surg Case Rep. 2017;31:150–153. doi:10.1016/j.ijscr.2017.01.041

-Jonathan Wilcock, 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 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.