Microbiology Case Study: A 55 Year Old with Fever, Chills, and a Cough

Clinical History

A 55 year old patient with past medical history of stage IV non-Hodgkin’s lymphoma on rituximab and Campylobacter jejuni bacteremia 1 year prior presented to the Emergency Department on the orders of their primary care provider, after outpatient blood cultures grew gram negative bacilli resembling Campylobacter species. Their symptoms included a 1-2 month history of fatigue and weakness and a 3 week history of intermittent fevers and chills with developing productive cough, sinus pressure, sore throat, progressive dyspnea on exertion, nausea, and decreased appetite.

Laboratory Findings

Initial (outpatient) blood culture was positive in the aerobic bottle at 60.1 hours, with the initial gram stain showing no organisms. The bottle was placed back in the analyzer and flagged positive again, at which point a second gram stain was performed, which again showed no organisms. An acridine orange stain was performed (Image 1), revealing multiple spiral/”gull shaped” rods. A third gram stain (Image 2) with more time allowed for safranin staining revealed faint gram negative rods. MALDI-TOF MS was attempted with no identification. The culture growth was sent to a reference laboratory and was identified via sequencing as Helicobacter species. The organism was not viable for susceptibility testing.

Image 1. Acridine orange stain of blood culture sample: this stain causes the nucleic acids to fluoresce orange, highlighting the bacteria against the background of blood.
Image 2. Gram stain after allowing extra time for safranin staining, showing few gram negative rods.
Image 3. A replating on blood agar showed difficult to discern, thin spreading colonies.

Two sets of subsequent blood cultures also grew gram negative bacilli at 65 and 67 hours. The blood culture broth from one of these cultures was also sent to the reference lab, but again did not have viable growth for susceptibility testing.

Discussion

The genus Helicobacter includes 35 species, consisting of gram negative spiral bacilli, previously considered to be part of the Campylobacter genus. Pathogenic species are classically associated with the gastrointestinal tract as they are able to survive in the harsh acidic conditions of the human stomach. The most common clinically relevant species is H. pylori, which is associated with gastric ulcers as well as other inflammatory processes in the stomach and duodenum. In prior reports, bacteremia caused by Helicobacter species is typically associated with some other underlying disease process, such as malignancy, immunocompromised state, or disruption of the GI mucosal barrier (1, 2, 3, 4, 5, 6).

Helicobacter spp. are similar in morphology to Campylobacter spp. on a gram stain; given the patient’s prior history of C. jejuni bacteremia, it was not unreasonable for the gram smear to initially be called consistent with Campylobacter spp. However, the clinical course and antibiotic susceptibility profiles of Helicobacter and Campylobacter bacteremia cases can differ in important ways. Further, susceptibilities can differ between different species of Helicobacter. There are no established guidelines for the treatment of Helicobacter spp. bacteremia and breakpoints for antibiotic susceptibility testing for some Helicobacter species have not been established. (7)

The patient in this case was discharged on a course of azithromycin with clinical improvement: at that time, the sequencing result revealing Helicobacter had not yet been received, and the clinical team was acting on the belief that the organism in the patient’s blood was a recurrence of the previous Campylobacter infection. On a follow up outpatient appointment with Infectious Disease, wherein sequencing results were available, tetracycline was prescribed due to concern about the possibility of resistance or relapsing infection.

References

  1. Abidi, Maheen Z., et al. “Helicobacter Canis Bacteremia in a Patient with Fever of Unknown Origin.” Journal of Clinical Microbiology, vol. 51, no. 3, 2013, pp. 1046–1048.
  2. Araoka, Hideki, et al. “Clinical Characteristics of Bacteremia Caused by Helicobacter Cinaedi and Time Required for Blood Cultures To Become Positive.” Journal of Clinical Microbiology, vol. 52, no. 7, 2014, pp. 2745–2745.
  3. De Luca, et al. “Helicobacter Pylori Bacteremia: An Unusual Finding.” Infectious Disease Reports, vol. 8, no. 3, 2016, pp. 74–75.
  4. Han, Xiang Y., et al. “Helicobacter Pylori Bacteremia with Sepsis Syndrome.” Journal of Clinical Microbiology, vol. 48, no. 12, 2010, pp. 4661–4663.
  5. Imataki, Osamu, et al. “Enteral Malakoplakia Prior to Helicobacter Cinaedi Bacteremia.” American Journal of Gastroenterology, vol. 112, no. 1, 2017, pp. 187–188.
  6. Saito, Sho, et al. “Helicobacter Fennelliae Bacteremia: Three Case Reports and Literature Review.” Medicine, vol. 95, no. 18, 2016, p. e3556.
  7. Yamamoto, Kei, et al. “Comparison of the Clinical and Microbiological Characteristics of Campylobacter and Helicobacter Bacteremia: the Importance of Time to Blood Culture Positivity Using the BACTEC Blood Culture Systems.” BMC Research Notes, vol. 10, no. 1, 2017, pp. 1–6.

-Tom Koster, DO 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 60 Year Old Male Status-Post Orthotopic Liver Transplant with Headache and Word-Finding Difficulty

Case History

A 60 year old male with a past medical history of ulcerative colitis requiring total proctocolectomy and immunomodulatory therapy followed by an anti-Tumor Necrosis Factor α blocker for the last two years and primary sclerosing cholangitis with subsequent decompensated cirrhosis that ultimately required an orthotopic liver transplant on tacrolimus and prednisone for immunosuppression presents 17 days post-transplant with worsening headache for two weeks with associated word finding difficulty and expressive aphasia.

Laboratory and Diagnostic Findings

Brain magnetic resonance imaging demonstrated, a “Heterogeneous, partially hemorrhagic and centrally necrotic mass within the posterior left temporal lobe…infectious etiologies such as pyogenic/non-pyogenic abscesses to include fungal organisms, are highest on the differential” (Image 1). At the time of admission, his complete blood count demonstrated a leukocytosis (16.48×109 cells/L), anemia (hemoglobin of 7.8 g/dL, hematocrit of 24.8%) and a normal platelet count (367×109 cells/L). The automated differential showed 82% neutrophils, 10% lymphocytes, 6% monocytes, 1% eosinophils, and 1% basophils. A lumbar puncture was performed to obtain cerebral spinal fluid (CSF) and the analysis showed a glucose of 60 mg/dL, protein of 34 mg/dL, nucleated cell count of <1, and 6 red blood cells (completely normal CSF indices). Broad spectrum antimicrobials (Vancomycin, Piperacillin/Tazobactam, Metronidazole and Micafungin) were initiated. A 1,3-β-D-glucan test had a result of >500 pg/mL in both serum and CSF. Galactomannan, Histoplasma urine antigen, Cryptococcus antigen and other fungal testing were negative. Antifungal therapy was changed to voriconazole. Craniotomy was determined to be the best course of action and the patient was taken to surgery for debridement and pathologic evaluation.

Frozen section evaluation during the time of surgery showed granulomatous inflammation. Septate hyphae were observed on the fungal smear. Following surgery, amphotericin was added. Histologic evaluation of the tissue submitted from surgery showed pyogranulomatous inflammation with pigmented, spore-like structures present in multinucleated giant cells on hematoxylin and eosin (H&E) stain (Image 2). Grocott’s methenamine silver (GMS) stain also highlighted short segments of septate hyphae (Image 3).

Cultures from the surgical debridement grew a mould with central pigmentation (Image 4). Direct microscopic examination of the mould revealed thick-walled, oblong conidia with 3-5 cells, and uniformly pigmented hyphae (Image 5). A germ tube test showed germ tubes originating from both ends of the conidia consistent with Bipolaris species.

Image 1. T1-weighted (left) and T2-weighted (right) magnetic resonance imaging of the brain demonstrating a left temporal lobe mass.
Image 2. Hematoxylin and Eosin stained photomicrographs showing pyogranulomatous inflammation with giant cell formation and circular structures within them (left) (40x objective magnification). The right shows gold-brown pigmented structures within granulomatous inflammation (40x objective magnification).
Image 3. Grocott’s methenamine silver stain highlighting short segments of irregular septate hyphae in the brain debridement specimen (10x objective magnification).
Image 4. Mature wooly brown-black colony on potato dextrose agar.
Image 5. Photomicrograph of a lactophenol blue tape prep of the mature fungal colony. Pigmented hyphae and short 3-4 cell conidia are readily identified (40x objective magnification). This specimen also tested germ tube positive (not shown), indicating that this dematiaceous fungus is Bipolaris spp.

The patient’s mental status significantly improved following surgical debridement, 2 weeks of liposomal Amphotericin B, as well as long term treatment with voriconazole. The voriconazole was later switched to posaconazole due to concerns for fluoride toxicity. He completed a year of posaconazole with significant improvement of the abscess observed on imaging and resolution of headaches with no other visual problems. He continued to recover cognitive function with some residual difficulty with reading, comprehension and speech that eventually resolved.

Discussion

Phaeohyphomycosis refers to infections caused by dematiaceous fungi that exist in a variety of forms when seen in tissues and commonly involves skin, soft tissue and nasal sinuses.1 In rare cases, central nervous system (CNS) involvement has been reported. CNS phaeohyphomycosis is predominantly seen in immunosuppressed patients; however, cases involving immunocompetent individuals do exist.2 In one case series from Houston, Texas, five of seven cases of cerebral mycosis were caused by a dematiaceous mould.3 Interestingly, the patient presented in this case came to medical attention around the Dallas-Fort Worth area of Texas.

Cladophialophora bantiana is the most common dematiaceous fungus associated with CNS phaeohyphomycosis, but rare cases of Bipolaris species have been reported previously in literature.4-6

We report a case of CNS phaeohyphomycosis by Bipolaris species following orthotopic liver transplant with an excellent patient outcome. This case is unusual, in part, because the typical hospital course of a patient with phaeohyphomycosis is generally dismal.7 The stories of successful treatment often involve complete debridement of discrete lesions.7-8 In our case, the patient underwent surgical debridement and treatment initially with liposomal Amphotericin B and later transitioned to long term therapy with newer azole antifungals.

References

  1. Revankar SG, Sutton DA, & Rinaldi MG, (2004). Primary Central Nervous System Phaeohyphomycosis: A Review of 101 cases. CID, 38, 206-2016
  2. Filizzola MJ, Martinez F, & Rauf SJ, (2003). Phaeohyphomycosis of the central nervous system in immunocompetent hosts: report of a case and review of the literature. Int J Infec Dis, 7, 282-286
  3. Raparia K, Powell SZ, Cernoch P, Takei H, (2010). Cerebral mycosis: 7-year retrospective series in a tertiary center. Neuropathology, Jun; 30(3): 218-223.
  4. Frank T, Esquenazi Y, Nigo M, Wanger A, Portnoy B, & Shepard S, (2016). Disseminated Phaeohyphomycosis with Brain Abscess and Biliary Invasion Due to Bipolaris spp. In an Immunocompetent Patient. Annals of Clinical & Laboratory Science, 46(4). 
  5. McGinnis MR, Campbell G, Gourley WK, & Lucia HL, (1992). Phaeohyphomycosis Caused by Bipolaris spicifera, An Informative Case. Eur. J. Epidemiol, 8(3), 383-386
  6. Rosow L, Jiang JX, Deuel T, Lechpammer M, Zamani AA, Milner DA, Folkerth R, Marty FM, & Kesari S, (2011). Cerebral phaeohyphomycosis caused by Bipolaris spicifera after heart transplantation. Transpl Infect Dis, 13, 419-423.
  7. Dixon DM, Walsh TJ, Merz WG, McGinnis MR, (1989). Infections due to Xylohypha bantiana (Cladosporium trichoides). Rev Infect Dis, 11: 515-525.
  8. Gadgil N, Kupfermen M, Smitherman S, Fuller GN, Rao G, (2013). Curvularia brain abscess. J Clin Neurosci, Jan;20(1): 172-175.

-John Markantonis, DO is a second year Clinical Pathology resident at UT Southwestern in Dallas. He has interests in Medical Microbiology and Transfusion Medicine.

-Dominick Cavuoti, DO is a Professor at UT Southwestern in the Department of Pathology. He is multifaceted and splits his time as the Medical Director of the Parkland Hospital Clinical Microbiology Laboratory and Parkland Cytology attending among other administrative and educational activities.

-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: 18 Year Old Male with Shortness of Breath

Case History

A 18 year old male with no significant past medical history presented with 2-3 month cough, hemoptysis, fevers, night sweats and 15 pound unintentional weight loss. He originally started to feel mild shortness of breath with activity following his return from the Pacific Northwest. He was diagnosed with pneumonia and given antibiotics. He denied vaping, hookah use or any smoking. Of note, his maternal grandmother had pulmonary tuberculosis two years ago, but at that time he had a negative interferon-gamma release assay (IGRA). Since that time he had traveled extensively including Europe and Asia.

Upon admission, physical examination showed decreased breath sounds and coarse crackles in the right lower lung field. His blood test results were unremarkable. Chest X-ray and CT scan showed diffuse reticular pattern and a 4 cm cavitary lesion in his right lower lobe (Image 1). Brochoalveolar lavage fluid, sputum as well as blood were submitted to microbiology lab for bacterial, fungal and mycobacterial culture.

Image1. Chest X-ray and CT showed diffuse reticular pattern with a 4cm cavitary lesion in his right lower lobe.

Laboratory results and management

The patient’s Quanti FERON®-TB Gold test (an IGRA) was positive.Laboratory studies including fungal culture to look for endemic fungi and HIV were negative. Auramine-rhodamine (acid fast) staining of sputum smear showed 4+ acid fast bacilli (AFB). Mycobacterium tuberculosis was confirmed by both our laboratory developed TB-PCR assay and a commercial TB-PCR assay (Xpert MTB/RIF). DNA sequencing was performed by the Centers for Disease Control and Prevention (CDC) in order to predict antibiotic susceptibility profiles for first-line anti-tuberculosis drugs including Ethambutol, Isoniazid, Pyrazinamide, and Rifampin. Because the result showed drug-susceptible tuberculosis, the patient started the first-line anti-tuberculosis drugs. Four days after admission, the patient discharged home with follow up with the department of health. After six weeks incubation, the sputum culture grew Mycobacterium tuberculosis with a pan-susceptible antibiotic profile.

Image 2. Ziehl-Neelsen stain of acid fast bacilli (AFB) on sputum smear. Image from the CDC website.

Discussion

Mycobacterium tuberculosis (MTB) remains a global health problem. The continuing spread of drug-resistant tuberculosis is one of the most difficult challenges for MTB control. The CDC recommends rapid laboratory confirmation of MTB with using a nucleic acid amplification test (NAAT), followed early drug susceptibility testing. Our case is consistent with the previous reports that NAAT can identify MTB and DNA sequencing can determine the drug susceptibility within a few days. Our case can be summarized into two points.

First, early NAAT provided diagnosis MTB within 2 days after patient admission, enabling the timely initialization of infection control measures. Although culture remains the gold standard for laboratory confirmation of active TB infection, it can take 6-8 weeks to grow in a culture media since MTB is slow growing pathogen. Early laboratory confirmation of MTB with NAAT led rapid initiation of the patient treatment and transmission interruption.

Second, DNA sequencing guided us to start an optimal anti-tuberculosis treatment within 3 days because of the accurate prediction of susceptibility profiles for first-line anti-tuberculosis drugs. In comparison, the conventional susceptibility testing with the drug-containing medium requires 1 month to complete beyond initial growth and identification of the organism. Recent studies demonstrated high accuracy of genotypic predictions (>91.3 % sensitivity; >93.6% specificity). Our case supports a theory that DNA sequencing can help to determine which anti-tuberculosis drugs should be used for treatment.

Despite the notable advantage, NAAT has limitations. There is a relatively low positive predictive value in smear-negative pulmonary MTB. Recent meta-analysis found that overall sensitivity of NAAT to be 90.4% for diagnosis of pulmonary MTB. The sensitivity of the NAAT further defined to be lower in smear negative (75%) compared to smear positive (98%) pulmonary MTB. Additionally, cost effectiveness of NAAT and DNA sequencing is another concern. Further assessments of the benefit of NAAT and DNA sequencing utilization for smear negative MTB are needed.

In conclusion, our case demonstrated that NAAT and DNA sequencing was beneficial to reduce the time to initiation of an optimal MTB management.

References

  1. Prediction of Susceptibility to First-Line Tuberculosis Drugs by DNA Sequencing. N Engl J Med 2018; 379: 1403-15.
  2. Use of Nucleic Acid Amplification Tests in Tuberculosis Patients in California, 2010-2013. Open Forum Infect Dis. 2016 Oct; 3(4): ofw230.
  3. Assessment by Meta-Analysis of PCR for Diagnosis of Smear-Negative Pulmonary Tuberculosis. J Clin Microbiol. 2003 Jul; 41(7): 3233-3240.

-Sachie Ikegami MD, PhD is a 1st year anatomic and clinical pathology resident at University of Chicago (NorthShore). Sachie’s academic interests include neuropathology and molecular pathology. She is passionate about understanding how pathology informatics improving clinical practice. Outside of the lab, she enjoys jogging.

-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 Child with Acute Abdominal Pain

Clinical presentation

An elementary school age child presented to the pediatric emergency department with an acute onset of abdominal pain. According to the parents, the patient recently had an ear infection and completed a course of amoxicillin. They noted the patient was more tired than usual, but did not have a fever. They reported no recent sick contacts or travel. Past medical history was significant for constipation, but normal bowel movements were noted over the past few days. On physical exam, the abdomen was soft and non-distended with diffuse mild tenderness noted on the right side. No masses were noted.  Laboratory testing was unremarkable and the WBC count, liver & pancreas enzymes, and alpha fetal protein were within normal limits. An abdominal CT scan revealed a mass with central necrosis in the liver concerning for an abscess. The patient was started on ceftriaxone & metronidazole and underwent a surgical procedure to drain the lesion.   

Laboratory Identification

Image 1. Gram stain of the direct liver aspirate showed many gram positive cocci in pairs & chains and numerous white blood cells (oil immersion).
Image 2. Rare small, white non-hemolytic colonies grew on CDC agar after 42 hours of incubation at 35°C under anaerobic conditions.

No bacterial growth was observed on blood or chocolate agars incubated at 35°C in CO2. MALDI-TOF mass spectrometry identified the isolate as a viridans groups streptococci, Streptococcus intermedius. The organism was susceptible to penicillin, ceftriaxone, and vancomycin by broth microdilution. Blood cultures were not collected for this patient.

Discussion

Streptococcus intermedius is a viridans group streptococci that belongs to the S. anginosus group. The S. anginosus group also includes S. anginosus and S. constellatus. This group of viridans streptococci composes the normal flora of the oropharynx, urogenital, and gastrointestinal tracts. These organisms are known for causing peritonitis and abscesses, particularly in the brain, breast, liver, and oral cavity.

Similar to other streptococci, S. intermedius is a gram positive cocci that grows in chains and is catalase negative. The anginosus group are facultative anaerobes and grow as pinpoint colonies (<0.5 mm) on blood agar. This is in contrast to pyogenic, beta-hemolytic streptococci which are greater than 0.5 mm in size after the same incubation period. The anginosus group streptococci can exhibit a variety of hemolysis patterns, including alpha, beta, or gamma hemolysis. A distinct butterscotch or caramel odor is noted on examination. The anginosus group can possess Lancefield antigens A, C, F, G, or be non-groupable, so it is important not to misidentify them as other streptococci that also have these antigens.

Historically, further identification of viridans group streptococci was challenging; however, the advent of automated systems and MALDI-TOF mass spectrometry has been useful in providing species level identifications for more common isolates. Molecular sequencing methods using sodA gene can be helpful as well for the most reliable results. While penicillin resistance is becoming more frequent in viridians group streptococci, it is still rare in the S. anginosus group.  

In the case of our patient, an echocardiogram was performed and found to be negative for endocarditis. The patient’s symptoms improved and they were discharged home on ceftriaxone and metronidazole. A follow up CT scan to confirm resolution of the abscess was scheduled.  

-Lisa Stempak, MD is the System Director of Clinical Pathology at University Hospitals Cleveland Medical Center in Cleveland, Ohio. She is certified by the American Board of Pathology in Anatomic and Clinical Pathology as well as Medical Microbiology. Her interests include infectious disease histology, process and quality improvement, and resident education.

Microbiology Case Study: A 73 Year Old with Bacteremia Caused by an Unusual Pathogen

Case History

A 73 year old patient with a medical history significant for diabetes and diabetic foot ulcers presented to an outpatient orthopedic clinic complaining of right foot pain and fevers. Physical exam findings were significant for a right metatarsal ulceration that extended to the bone which prompted admission to a local hospital. Tissue from debridement of this ulcer was sent for routine bacterial culture and blood cultures were also collected. The patient was started on empiric antibiotics.

Laboratory Findings

The tissue culture gram stain showed mixed gram negative and gram positive bacteria. Two days after admission, an anaerobic blood culture bottle flagged positive with gram negative rods which could not be identified by Verigene nucleic acid detection test. It was plated on routine anaerobic and aerobic culture plates for further identification. Four days after admission, another blood culture set flagged positive with staphylococci which was identified on the Verigene as methicillin susceptible Staphylococcus aureus in the aerobic bottle. Seven days after admission, the gram negative organism grew and was identified by MALDI-TOF mass spectrometry as Campylobacter ureolyticus. The tissue culture grew mixed gram positive and negative bacteria including Staphylococcus aureus, Bacterodies fragilis group, and Trueperella bernardiae. The patient’s antibiotic therapy was tailored to cover the MSSA and Campylobacter and they were successfully discharged.

Image 1. Colony gram stain of Campylobacter ureolyticus isolated from the patient’s blood culture. The bacteria appear as thin S-shaped gram negative rods.

Discussion

The Campylobacter genus has 24 species of bacteria including C. jejuni and C. coli which are the most frequent cases of campylobacteriosis, a diarrheal illness which is described below. Other less frequent pathogenic species include C. fetus, C. upsaliensis, C. lari, and C. ureolyticus. Campylobacter species appear as a curved S-shaped spiral rods and are gram negative on gram stain, are nonspore forming, and motile, with the exception of C. ureolyticus, which is aflagellate. Because Campylobacter is difficult to culture, rapid detection tests have been developed including antigen detection tests, however, these lack specificity. Several FDA approved nucleic acid  amplification tests for Campylobacter exist, such as the BD MAX enteric bacterial panel that can detect C jejuni/C coli (speciation requires a reference lab).

Campylobacter grows best under microaerophilic conditions and at 42o C (closer to the body temperature of chickens). C. ureolyticus is unique as it grow anaerobically. Media that is selective and differential for Campylobacter, including charcoal cefoperazone deoxycholate agar (CCD) and charcoal based selective medium (CSM), is often used for stool specimens. Campylobacter appears as flat grey colonies that tend to spread along streak lines. Identification of Campylobacter includes a characteristic gram morphology, growth microaerophilically (expect for C. ureolyticus), and oxidase positivity.  C. jejuni are hippurate hydrolysis positive. C. coli are hippurate hydrolysis negative, however, there are C. jejuni that are hippurate hydrolysis negative, making this test non-specific.

Clinical and Laboratory Standards Institute (CLSI) recommends antibiotic susceptibility testing for C. jejuni and C. coli and includes testing for ciprofloxacin, erythromycin, and tetracycline resistance which requires microaerophilic conditions.

Campylobacter is mainly a zoonotic disease acquired from poultry, cattle, sheep, pigs, and domestic pets. C. ureolyticus, is thought to be transmitted most frequently form cattle, however, more research is needed in this area. A common cause of Campylobacter is consumption of undercooked meat, especially poultry due to the high prevalence of Campylobacter in US retail poultry. In 2015, 5,000 US retail poultry samples were tested for Campylobacter with 12% of samples testing positive; 24% of chicken breast samples tested positive and 0.2 % of ground turkey samples tested positive. The majority of isolates were C. jejuni and C. coli (65% and 34% respectively). In 2004, 60% of chicken samples tested positive in the US (1). Campylobacter most frequently infects young children ages 1-5 as well as adolescents and young adults and is most frequently seen between the months on June and August (1).

C. ureolyticus is a less studied species of Campylobacter, however there is evidence that this species can cause diarrheal disease and extra –intestinal infections. Some studies of fecal specimens from patients presenting with diarrhea illness in Ireland revealed 24% of Campylobacter positive stools were C. ureolyticus species (4). C. ureolyticus has also been isolated skin and soft tissue abscesses, however, C. ureolyticus is rarely the sole bacteria isolated, raising the questions of whether it a true soft tissue pathogen. The most frequent soft tissue site of infection is the perianal region (4).   

Campylobacter usually presents as a diarrheal illness, causing fever, diarrhea (can be bloody or non-bloody), and abdominal cramping with symptoms lasting days to weeks. The disease is usually self- limited, but in 10-15% of cases patients are admitted to hospitals (1). Generally, patients will clear campylobacter enteritis without the need for antibiotics. Indications for antibiotics include severe bloody diarrhea, relapsed cases, high fever, greater than 1 week course, and extraintestinal infections or immunocompromised status. Interestingly, presentations of C. jejuni/C. coli can mimic appendicitis and lead to unnecessary appendectomies. Extra-intestinal infections include bacteremia, septic arthritis, abscess formation, meningitis, peritonitis, prostatitis, urinary tract infections, and neonatal sepsis. Guilian-barre syndrome can be seen after C. jejuni infections, especially the heat stable serotypes HS19 and HS41, which is medicated by antibodies that develop against ganglioside-like epitopes in the bacterial cell wall LPS region which cross react with peripheral nerve gangliosides. C. jejuni/C. coli can also induce reactive arthritis and rarely have been implicated in inciting inflammatory bowel disease exacerbations and celiac disease (1-2).

In severe infections or extraintestinal infections, azithromycin is the preferred antibiotic as fluoroquinolone resistance is rising in the US. In 2014, 27% of C. jejuni and 36% of C. coli isolates were resistant to ciprofloxacin, and 2% of C. jejuni and 10% of C. coli isolates were resistant to azithromycin (1-2). In an Italian cohort of patients, greater than 60% of Campylobacter strains were ciprofloxacin or tetracycline resistant, while 29% of C. coli isolates were resistant to tetracycline, fluoroquinolones, and macrolides (3). Interestingly, use of these antibiotics in animal feed has been directly associated with the occurrence of antibiotic resistant Campylobacter stains (1-3). Antibiotic resistance and guidelines for the management of C. ureolyticus infections is largely unknown.

Overall, Campylobacter usually presents as a self-limiting diarrheal illness, however, less frequently extra-intestinal infections can occur such as in this patient’s case. The most common pathogenic species include C. jejuni and C. coli, while other Campylobacter species are seen less frequently. In this patient’s case, C. ureolyticus was isolated from the blood after the patient developed a right metatarsal ulcer. While we were unable to culture Campylobacter from the patient’s wound culture, this is the most likely source of their blood stream infection. 

References

  1. Whitehouse CA, Young S, Li C, Hsu CH, Martin G, Zhao S. Use of whole-genome sequencing for Campylobacter surveillance from NARMS retail poultry in the United States in 2015. Food Microbiol. 2018;73:122-128.
  2. Tack DM, Marder EP, Griffin PM, et al. Preliminary incidence and trends of infections with pathogens transmitted commonly through food – Foodborne Diseases Active Surveillance Network, 10 U.S. sites, 2015-2018. Am J Transplant. 2019;19(6):1859-1863.
  3. Garcia-Fernandez A, Dilonisi AM, Arena S, Iglesias-Torrens Y, et al. Human Campylobacteriosis in Italy: Emergence of Multi-Drug Resistance to Ciprofloxacin, Tetracycline, and Erythromycin. Front Microbiol. 2018 Aug 22;9:1906. doi: 10.3389/fmicb.2018.01906. eCollection 2018.
  4. O’donovan D, Corcoran GD, Lucey B, Sleator RD. Campylobacter ureolyticus: a portrait of the pathogen. Virulence. 2014;5(4):498-506.

-Liam Donnelly, MD 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.

A 54 Year Old with Vomiting and Diarrhea Followed by Pneumonia

A 54 year old male former smoker and alcohol user presented to the Emergency Department with a five day history of nausea, vomiting, diarrhea, weakness, fever with chills, breaking out in sweats, and abdominal discomfort. He denied recent sick contacts, travel or exposure to potentially contaminated foods. He had a past medical history that was significant for Chronic Obstructive Pulmonary Disease (COPD), mitral valve regurgitation and ST elevation myocardial infarction (STEMI). Some of his medications are inhaled Fluticasone, Advair Diskus, Furosemide and Spironolactone. He has also had a mitral valve replacement.

His initial laboratory tests revealed leukocytosis with neutrophilia, non-specific electrolyte derangements and negative stool tests for enteric bacterial pathogens. His symptoms progressed within the first 24 hours of admission, with a decrease in oxygen saturation (SPO2) and dyspnea so further investigations were carried out. Subsequently, a chest X-Ray was done, which showed pneumonia. The patient had a bronchoscopy and bronchoalveolar lavage (BAL) fluid was sent to the laboratory for aerobic, fungal, and acid fast bacilli culture, as well as Legionella spp. and Pneumocystis jiroveci direct fluorescent antigen testing.

Image 1. Results of Bronchoalveolar Lavage. A. Direct Fluorescence Antibody to Legionella antigens; B. Legionella pneumophilia colonies on Buffered Yeast Charcoal Extract agar plate (BCYE), showing convex, round colonies with entire edges.

Discussion

The presence of pneumonia and diarrhea in the patient raised suspicion for Legionnaires’ Disease, so the patient’s specimens including BAL fluid and bronchial washings were tested by direct fluorescent antigen (Image 1A) which confirmed Legionnaires’ Disease as the diagnosis.

Legionnaires’ Disease (LD), is a form of pneumonia caused by Legionella species, most commonly Legionella pneumophilia. Legionella spp.are motile, obligate aerobic, facultative intracellular and weakly gram negative rods. They are also nutritionally fastidious, requiring specific nutrients such as L-cysteine, and iron. They live in amoebas or in biofilms all over the world and are seen in high concentrations in warm waters plumbing systems, water heaters, warm water spas and cooling towers, and in very low concentrations in freely flowing cold water and biocide-treated waters.1 They are disseminated by devices that aerosolize water such as cooling units, hot tubs, water fountains and showers and cause disease when this contaminated aerosolized water is inhaled. The inhaled bacteria then enter the bacteria-killing macrophages in the lungs. Once in, they hijack the intracellular mechanism of the macrophages, feed off them, multiply within them, and then kill the macrophages, releasing more bacteria into the surrounding tissues.1

The incubation period of Legionella infections is 2 to 14 days, with a median of 4 days. In humans, Legionella spp. causes Legionellosis which comprises two separate diseases. These are Pontiac fever, a mild, self-limited flu-like illness, and LD, an atypical form of pneumonia which affects multiple organs. LD can range from mild to fatal in severity and about 12% of patients die from the disease.1 In most cases, LD begins with fever and symptoms of gastrointestinal infection including diarrhea and vomiting before patients develop respiratory symptoms such as cough and difficulty in breathing. However, LD also involves other organs/systems, causing renal failure and cardiogenic shock. LD occurs world-wide and all-year round but most cases occur between late fall and early spring.1

Although LD is relatively rare in the US, it is believed to be underdiagnosed due to failure to test for Legionella infection, poor sensitivity of test methods used to detect the disease, and failure to report all diagnosed cases.1 However, the rate of reported cases in the US has increased by about 5.5 times in the past 20 years to 7,500 reported cases in 20172, which may be partially attributed to increased and improved testing. 

LD is more likely to occur in people with a suppressed immune system – particularly those on high-dose corticosteroids like Fluticasone, people with chronic lung, heart or kidney diseases, people who smoke or smoked in the past, people who travel, especially overnight travel, people who have received solid organ transplants, and people who use certain medications such as anti-tumor necrosis factor drugs.1 LD is often fatal and survival depends on how severe the pneumonia when treatment starts, the presence or absence of other serious comorbidities, and how early specific treatment for the disease is commenced.1 Therefore, prompt diagnosis is very important to survive LD.

Unfortunately, LD patients often present with nonspecific symptoms as well as chest X-ray, biochemical and hematological laboratory tests results. Therefore microbiological investigations which identify Legionella spp. are crucial in the management of these cases. The most commonly used test method is the Urinary Antigen test which detects the most common cause of Legionnaires’ disease, L. pneumophila serogroup 1. But, it does not detect other potentially pathogenic Legionella species and serogroups. Legionella spp. can also be cultured and identified, but this requires the use of Buffered Charcoal Yeast Extract [BCYE] agar which provides the specific growth requirements of Legionella spp.Common specimens for culture include lower respiratory secretions such as BAL and bronchial washings, lung tissue and pleural fluid. Other methods used to diagnose LD include polymerase chain reaction (PCR), direct fluorescence antibody (DFA), and paired serology. However, the Centers for Disease Prevention and Control, CDC recommends testing with culture and urinary antigen test in combination.2

LD is treated with Azithromycin or Levofloxacin. 95 to 99% of cases can be cured if they are otherwise healthy but treatment is started early.1

References

  1. Edelstein, Paul H. and Lück, Christian. “Legionella.” In Manual of Clinical Microbiology, Eleventh Edition, pp. 887-904. American Society of Microbiology, 2015.
  2. Centers for Disease Control and Prevention. Legionella (Legionnaires’ Disease and Pontiac Fever). https://www.cdc.gov/legionella/clinicians.html. April 30, 2018

Adesola Akinyemi, M.D., MPH, is a first year anatomic and clinical pathology resident at University of Chicago (NorthShore). He is interested in most areas of pathology including surgical pathology, cytopathology and neuropathology–and is enjoying it all. He is also passionate about health outcomes improvement through systems thinking and design, and other aspects of healthcare management. Find him on Twitter: @AkinyemiDesola

-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 Unusual Morphologic Presentation of an Uncommon Dimorphic Fungi in Vermont

Clinical history

A 62 year old woman from the state of Vermont with a past medical history of anxiety, depression, and granulomatous dermatitis presented to her primary care physician for bilateral Achilles tendonitis, bilateral knee effusion, and bilateral conjunctivitis; all of which were self-limited. Additional laboratory work-up from the encounter revealed an incidental 7mm right medial lung nodule with associated unilateral hilar lymphadenopathy. The patient denied any history of using tobacco products but had a remote travel history to Arizona and seasonally visited California. An endobronchial ultrasound guided fine needle aspiration was performed and the on-site evaluation showed abundant necrosis with scattered refractile objects consistent with fungal organisms. Additional tissue was submitted for cytology and fungal cultures.

Laboratory identification

Initial review of the cytology revealed abundant alveolar macrophages and inflammatory cells in a background of necrotic debris. The modified gomori methenamine-silver nitrate(GMS) stain highlighted several variably sized 8-15um broad-based budding yeast with a double contour cell wall (Image 1). Secondary review of the slides showed additional rare large 50-70um thick-walled spherules with 2-4um endospores (Image 2). Following 10 days of incubation there was growth of a fluffy white colony of mold on both the potato flake and mycosel agar plates (Images 3-4). The lactophenol cotton blue adhesive tape preparation highlighted large thick-walled barrel shaped arthroconidia with alternating empty cells (Image 5). A nucleic acid probe definitively identified the organism as Coccidiodes posadasii/immitis.

Image 1. Modified gomori methenamine-silver nitrate (GMS) stain highlighting broad-based budding yeast with a double contour cell wall measuring 12um in greatest dimension (100x oil immersion).
Image 2. Modified gomori methenamine-silver nitrate (GMS) stain highlighting a large 70um spherule with 2-4um endospores. (100x oil immersion).
Image 3. Aerobic growth of a white fluffy mold on potato flake agar plate following 10 days of incubation.
Image 4. Aerobic growth of a white fluffy mold on mycosel agar plate following 10 days of incubation.
Image 5. Lactophenol cotton blue adhesive tape preparation highlighted large thick-walled barrel shaped arthroconidia with alternating empty cells

Discussion

Coccidiodes immitis is a dimorphic fungus commonly found in the Southwestern United States and Central/South America. Immunocompetent patients can develop a self-limited acute pneumonia (Valley Fever) and the diagnosis is frequently under recognized. Immunocompromised patients however, such as those with acquired immunodeficiency syndrome (AIDS) can develop systemic disease with fungal dissemination to the bones, lungs, and skin (1). The estimated risk of exposure in endemic regions is approximately 3% per year with the greatest risk occurring during the dry season (5). Infection typically begins with inhalation of the arthroconidia which then mature in the lungs from barrel shaped cells to enlarging spherules up to 80um in diameter. Mature spherules consist of internal septations with 2-4um endospores which subsequently rupture releasing endospores into infected tissues which can mimic Histoplasma. When large round yeast forms are observed in tissue it is important to maintain a broad differential diagnosis which includes Coccidioides, Blastomyces, Paracoccidioides and in some instances Candida species. Although 8-15um broad-based budding yeast lacking endospores are typically associated with Blastomyces this can also represent germinating Coccidiodes endospores. Within the literature there are case reports describing Coccidioidomycosis infections with unusual in vivo morphologic forms consisting of juxtaposed immature spherules without endospores and germinating endospores (2). These elements on microscopic examination can easily be mistaken for budding cells of Blastomyces. Furthermore Blastomyces can rarely exhibit “giant” multinucleated yeast cells, mimicking Coccidioides (3).  Therefore, as highlighted by our case for accurate differentiation of these two fungi correlation of the patients travel history, fungal cultures, and cytology is recommended. 

In the case of our patient, although she was a resident of Vermont, a growing endemic hotspot for Blastomyces infections, she had additional travel history to Arizona and California increasing her risk for infection with Coccidiodes. Furthermore her urine antigen test for Blastomyces was negative and her fungal culture only grew Coccidiodes as confirmed by the lactophenol cotton blue preparation and nucleic acid probe. In addition, Blastomyces typically take several weeks longer than Coccidiodes to grow on culture and microscopically present with septate hyphae with short or long conidiophores with pear-shaped conidia at the tips of conidiophores (lollipops). Collectively her clinical history and laboratory work up was most suggestive of a Coccidioidomycosis. Although rare and less likely, it is also possible that she may have had a remote or concurrent Blastomyces infection which was unable to grow on culture and could not be definitively excluded.

Typically for immunocompetent hosts, asymptomatic Coccidioidomycosis (Valley fever) infections do not need require treatment. However in the context of this patient’s history of dermatologic and rheumatologic complaints, her systemic course warranted treatment with 6 months of itraconazole (4). Of note, itraconazole is also the treatment of choice for Blastomyces ­infection but would require a shorter 3 months duration of treatment.

References

  1. Saubolle MA, Mckellar PP, Sussland D. Epidemiologic, clinical, and diagnostic aspects of coccidioidomycosis. J Clin Microbiol. 2007;45(1):26-30.
  2. Kaufman L, Valero G, Padhye AA. Misleading manifestations of Coccidioides immitis in vivo. J Clin Microbiol. 1998;36(12):3721-3.
  3. Wu SJ, Valyi-nagy T, Engelhard HH, Do MA, Janda WM. Secondary intracerebral blastomycosis with giant yeast forms. Mycopathologia. 2005;160(3):253-7.
  4. Monaco WE, Batsis JA. A case of disseminated blastomycosis in Vermont. Diagn Microbiol Infect Dis. 2013;75(4):423-5.
  5. Dodge RR, Lebowitz MD, Barbee R, Burrows B. Estimates of C. immitis infection by skin test reactivity in an endemic community. Am J Public Health. 1985;75(8):863-5.

-Noman Javed, 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.