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.

Microbiology Case Study: A Novel Anaerobic Pathogen Causing Septic Arthritis

Clinical history

A 65 year old man with diabetes mellitus type 2 presented to the emergency department (ED) for left hip pain. He has a remote history of avascular necrosis of bilateral hips of unknown etiology for which he received a bilateral total hip arthroplasty and subsequent multiple revisions due to hardware failure several years ago. He initially presented to an urgent care clinic a few months prior for “noise with movement” of the left hip and mild lower back pain. Plain radiographs of the left hip in comparison to his prior imaging were unremarkable and he was subsequently discharged. Repeat imaging at a follow-up visit at the orthopedic clinic showed mild superior migration of the femoral head bilaterally secondary to periprosthetic osteolysis of the joint headliner. He was scheduled for surgery however presented to the ED prior to his scheduled appointment with severe crushing left hip pain and restricted joint mobilization. He denied fevers, chills, night sweats, or any other recent infections. The left hip was aspirated yielding 10cc of dark black fluid and a stat gram stain was ordered.

Laboratory identification

The stat gram stain showed many polymononuclear cells with moderate gram positive bacilli in a background of dark inorganic material (Image 1). Following 48 hours of incubation, there was anaerobic growth on the kanamycin and vancomycin (KV) and schaedler agar plates. A Gram stain of the broth showed gram positive bacilli arranged singly and in chains with some decolorization (Image 2). The KV and schaedler plates showed moderate growth of a single organism consisting of small glossy tan colored colonies (Images 3-4). No aerobic growth was observed on the blood, MacConkey, Columbia Naladixic Acid (CNA), or chocolate agar plates. Mass spectrometry (MALDI-TOF) identified the pathogenic organism as Clostridium innocuum.

Image 1. Synovial fluid Gram stain of the left hip showed moderate gram positive bacilli and many polymononuclear cells in a background dark inorganic debris (100x oil immersion).
Image 2. Gram stain from a positive broth culture showed gram positive bacilli arranged singly and in chains with some decolorization (100x oil immersion).
Image 3. Anaerobic growth on the schaedler agar showed growth of a single organism consisting of small round glossy tan colored colonies.
Image 4. Anaerobic growth on the kanamycin and vancomycin (KV) agar showed growth of a single organism consisting of small glossy tan colored colonies.

Discussion

Bacterial joint infections are more common in prosthetic joints as compared to native joints with a prevalence of 1-2% following hip arthroplasty (1). Most cases of bacterial septic arthritis are due to staphylococci (40 percent), streptococci (28 percent) or gram negative bacilli (19 percent) organisms (2). Joint infections secondary to anaerobes are less likely and account for 2-3% of all cases (3). A review of the literature shows less than 50 documented cases of septic arthritis due to Clostridium species. Amongst these cases Clostridium perfringens is the most commonly isolated pathogen (4). To date there are no documented cases of joint infections secondary to Clostridium innocuum species.

Clostridium innocuum is a non-motile, anaerobic, gram positive organism that reproduces by sporulation. These organisms are normally found as a part of the usual human gut flora and are rarely human pathogens. The name “innocuum” is derived from the term “innocuous” to convey the innocence of these organisms as they do not produce clostridial exotoxins. A review of the literature shows fewer than 20 reported cases of Clostridium innocuum infections with most reported cases being described in immunocompromised patients such as those with diabetes mellitus, chronic hepatitis, acquired immune deficiency syndrome (AIDS), leukemia, and organ transplantation (5-6). Clinically patients can present with a spectrum of symptoms which include fever of unknown origin, diarrhea/constipation, and non-specific respiratory symptoms. In almost all cases bacteremia ensued. Most cases were associated with a traumatic penetrating injury with few reported cases due to hematogenous spread (5-6).

Laboratory identification of Clostridium innocuum can be challenging due to its variable gram staining morphology and atypical colony morphology on differing culture media. Most traditional phenotypic methods can only reliably identify these organisms to the genus level as a Clostridium species. However, using mass spectrometry (MALDI-TOF) these organisms can be identified to the species level. Rapid identification of Clostridium innocuum from the subset of Clostridium species is clinically important as these organisms are the only known Clostridium species with intrinsic resistance to vancomycin (7). Although they do not possess clostridial exotoxins, these organisms are thought to have a lipopolysaccharide-like virulence factor and have a mortality rate comparable to toxigenic Clostridium species (7). Due to resistance to vancomycin, metronidazole, piperacillin and ampicillin-sulbactam are the alternative recommended first-line treatment options.

For this patient, following the results of the gram smear the patient was started on IV vancomycin but due to an adverse allergic reaction was switched to intravenous pencillin G and oral ciprofloxacin. He was subsequently taken to the operating room for incision and drainage and left hip revision arthroplasty with cup exchange. Blood cultures were collected post-operatively and showed no growth, possibly due earlier antibiotic administration. Susceptibility studies from Mayo Laboratories showed pan susceptibility to penicillin, piperacillin-tazobactam, ertapenem, clindamycin, and metronidazole. The patient was subsequently switched to intravenous penicillin and continued to show clinical improvement during his remaining hospital course.

References

  1. Horowitz DL, Katzap E, Horowitz S, Barilla-labarca ML. Approach to septic arthritis. Am Fam Physician. 2011;84(6):653-60.
  2. Ryan MJ, Kavanagh R, Wall PG, Hazleman BL. Bacterial joint infections in England and Wales: analysis of bacterial isolates over a four year period. Br J Rheumatol. 1997;36(3):370-3.
  3. Shah NB, Tande AJ, Patel R, Berbari EF. Anaerobic prosthetic joint infection. Anaerobe. 2015;36:1-8.
  4. Gredlein CM, Silverman ML, Downey MS. Polymicrobial septic arthritis due to Clostridium species: case report and review. Clin Infect Dis. 2000;30(3):590-4.
  5. Leal J, Gregson DB, Ross T, Church DL, Laupland KB. Epidemiology of Clostridium species bacteremia in Calgary, Canada, 2000-2006. J Infect. 2008;57(3):198-203.
  6. Lee NY, Huang YT, Hsueh PR, Ko WC. Clostridium difficile bacteremia, Taiwan. Emerging Infect Dis. 2010;16(8):1204-10.
  7. Chia JH, Feng Y, Su LH, et al. Clostridium innocuum is a significant vancomycin-resistant pathogen for extraintestinal clostridial infection. Clin Microbiol Infect. 2017;23(8):560-566.

-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.

Microbiology Case Study: A Man with History of ALL Presents with Fever and Diarrhea

Clinical History

A man in his 40’s with a past medical history of acute lymphoblastic leukemia/lymphoma (in remission), multiple infections including bacteremia and pulmonary aspergillosis, presented to the hospital with fever and diarrhea. Over the course of his stay, he had worsening renal function and developed profound hypotension and shock, which prompted initiation of two vasopressors and high-dose steroids. Eventually he developed acute hypoxic respiratory failure, requiring intubation. Complete blood count demonstrated an absolute eosinophilia of 8.58 x109/L (reference range 0.04-0.62 x109/L). Imaging revealed bilateral pulmonary infiltrates and a pleural effusion. Respiratory culture with gram stain was ordered for his tracheal aspirate, which revealed few polymorphonuclear cells, many gram-negative rods, yeast, and larvae of Strongyloides stercoralis (Image 1A). Wet mounts of the tracheal aspirate revealed numerous larvae and a few eggs of S. stercoralis (Image 1B-C); many of the larvae were motile (Movie 1). Stool examination of ova and parasites (O & P) were positive for larvae. Given the burden of organisms and prior administration of steroids, he was diagnosed with severe strongyloidiasis, consistent with hyperinfection. Concurrent blood cultures grew Enterococcus faecalis and Stenotrophomonas maltophilia; the respiratory culture also grew S. maltophilia, and tracks from the migrating larvae were observed on respiratory culture bacterial media (Image 1D).

Image 1. Tracheal aspirate Gram stain with S. stercoralis larvae, 100x objective magnification (A). Wet mount of tracheal aspirate revealing larvae (B) and eggs (C), 40x objective magnification. Blood agar plate growing S. maltophilia in an abnormal pattern, indicating motile larvae tracking through the agar (D).

Discussion

Strongyloidiasis is a spectrum of clinical disease caused by the nematode Strongyloides stercoralis.1,2 Descriptions of acute infection have been described in other Lablogatory entries here,3,4 and the full lifecycle is described in detail on the CDC DPDx website.5

Severe strongyloidiasis includes the syndromes of hyperinfection and disseminated disease. Hyperinfection is when there is an elevated burden of the typical autoinfection cycle involving the lungs and GI-tract. Usually there is an antecedent immunosuppressive event, such as administration of corticosteroids. Within the GI-tract lumen, increased numbers of rhabditiform larvae transform into the infective filariform larvae, which traverse the GI mucosa, migrate to the lungs via bloodstream/lymphatics where they enter alveolar air spaces, then ascend the respiratory tract, and are coughed up by the host and swallowed to re-enter the GI tract. In the GI tract adult females lay eggs through parthenogenesis, which give rise to further rhabditiform larvae. In extreme cases of hyperinfection, adults can be found in the lungs, where they may also lay eggs. Finding eggs in respiratory specimens is unusual, and may be related to the burden of disease.6

Disseminated disease is when larvae can be found in any additional organs/organ systems, such as the central nervous system, kidneys, liver, adrenals, etc. Invasive sampling is not typically performed, and larvae can be observed at autopsy.

Laboratory diagnosis of S. stercoralis involves identification of rhabditiform larvae in stool O &P exam; the presence of adults or eggs in stool is rare. Rhabditiform larvae have short buccal cavities and an ovoid genital primordium structure midway through the body (Movie 2). O&P exams can be performed on other body fluids, such as sputum and CSF. Serology can be useful to identify past exposure, especially prior to initiating immunosuppressive therapeutics such as corticosteroids. A nonspecific finding can be observed, as in this case, in the complete blood cell count and differential. Relative and absolute eosinophilia can be found in patients with parasitic infections; therefore, it is reasonable to rule out parasitic infection in this subset of patients. In the case presented here, the absolute eosinophilia was likely due to a persistent S. stercoralis infection, since these nematodes can live in the human host for decades.

The treatment of choice for severe strongyloidiasis is oral ivermectin, though albendazole is an alternative therapy. In some instances, subcutaneous ivermectin administration may be used.7

Follow-up

Oral ivermectin was administered to treat the strongyloidiasis and antibiotics were administered to treat the bacterial infections. Over the coming days, serial tracheal aspirates continued to reveal many larvae and eggs, so therapy was escalated to subcutaneous ivermectin. Over the course of therapy, the patient developed a fungemia with Candida guilliermondii. Despite aggressive antimicrobial therapy and intensive care, the patient remained hypoxemic and hypotensive. The family decided to transition to comfort measures and the patient passed away.

References

  1. Maguire JH. Intestinal Nematodes (Roundworms), in Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases, B. Mandell, Dolin, Editor. 2010, Elsevier: Philadelphia, PA. p. 3577-3586.
  2. Parasitology, in Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, Procop et al., Editors. 2017, Lippincott Williams & Wilkins: China. p. 1452-1454.
  3. Kaur J, Stempak L. An 81 Year Old Female with Persistent Fevers. Lablogatory 2019 [cited 2019 11/5/2019]; Available from: https://labmedicineblog.com/2019/04/23/microbiology-case-study-an-81-year-old-female-with-persistent-fevers/.
  4. Mohammed M, Wojewoda C. A 47 Year Old Male with Abdominal Pain and Diarrhea. Lablogatory 2016 [cited 2019 11/5/2019]; Available from: https://labmedicineblog.com/2016/05/16/microbiology-case-study-a-47-year-old-male-with-abdominal-pain-and-diarrhea/.
  5. Centers for Disease Control. Strongyloidiasis. DPDx 2019 [cited 2019 11/5/2019]; Available from: https://www.cdc.gov/dpdx/strongyloidiasis/index.html.
  6. Keiser PB and Nutman TB. Strongyloides stercoralis in the Immunocompromised Population. Clin Microbiol Rev, 2004. 17(1): p. 208-17.
  7. Hurlimann E and Keiser J, A single dose of ivermectin is sufficient for strongyloidiasis. Lancet Infect Dis, 2019. 19(11): p. 1150-1151.

-IJ Frame, MD, PhD, Microbiology Fellow, University of Texas Southwestern Dallas, Texas

-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: 77 Year Old Man With History of Travel to India

Case Report

A 77 year old male presented to the hospital with chest pain, lightheadedness, burning urination for the past few weeks. He has blood in his urine due to a previously diagnosed neoplasm. The patient moved from India to the United States in February, with a diagnosis of bladder cancer and a history of hypertension, congestive heart failure, coronary artery disease, and atrial fibrillation. In the hospital, abscesses on both right and left kidneys were found, and patient had nephrostomy tubes placed. Purulent discharge confirmed he had a severe urinary tract infection.

Laboratory Identification

The patient’s urine culture grew >100,000 colony forming units/milliliter (CFU/ml) of an oxidase-positive, non-lactose fermenting Gram-negative rod. On the blood agar plate, large gray, smooth, flat, mucoid, β-hemolytic colonies were found. Although bacteria growing on solid media should not be actively smelled, the organism emitted a grape or tortilla smell from the plate. The organism was identified as Pseudomonas aeruginosa by MALDI-TOF mass spectrometry. The isolate was plated onto Mueller Hinton agar for Kirby-Bauer disc diffusion antibiotic susceptibility testing (Image 1). A fluorescent green lawn of bacteria grew up to the edge of all discs, indicating high-level resistance to all antibiotics tested (Table 1). Modified carbapenem inactivation method (mCIM) testing was positive and Cepheid GeneXpert CarbaR PCR testing revealed that this P. aeruginosa isolate carried the New Delhi metallo-β-lactamase-1 carbapenemase (NDM-1).

Image 1. Kirby-Bauer disc diffusion was used for antimicrobial susceptibility testing. Note no zones around any of the antibiotic discs, indicating resistance to all antimicrobials tested.
Table 1. Antimicrobial susceptibility testing interpretations. All drugs tested were resistant to this P. aeruginosa isolate.

Discussion

The issue of super bugs is on the rise, with the fear of antibiotic resistance disseminating through more bacterial populations and species. Carbapenems are drugs that are very powerful broad-spectrum antibiotics, usually reserved as a last resort treatment for serious and resistant infections.1 β-lactamases are divided into four Ambler classes: A, B, C, and D. Class B differs from the others because it utilizes zinc as a metal cofactor for its catalytic activity. The others use a serine residue for their catalytic activity.2

NDM-1 is a class B β-lactamase. It was named after New Delhi, India when a Swedish resident presented with an extremely resistant infection after a trip to India in 2008. NDM-1 bacteria can now be found with high prevalence in India and China, and increasingly in other countries such as the UK and US.3,4 While the origination of the gene may not have been India, many of these infections are from people who have traveled to India or other Asian continents.5 Concerns about overprescribing and misuse of antibiotics in India are rising, where India is one of the biggest consumers of antibiotics in the world. One study even found striking evidence of this misuse, demonstrating that 2 out of 3 adults under 20 presented antibiotic resistance isolates to fluoroquinolones and/or cephalosporins.6,7,8

Image depicting the NDM-1 protein anchored in the outer membrane of the bacterium. (Taken from Bahr, Guillermo, et al. “Clinical Evolution of New Delhi Metallo-β-Lactamase (NDM) Optimizes Resistance under Zn(II) Deprivation.” Antimicrobial Agents and Chemotherapy, vol. 62, no. 1, 2017, doi:10.1128/aac.01849-17.)

The gene for NDM-1 is blaNDM-1 and has been found on both plasmid and chromosomal components of different bacteria. Due to its presence on plasmids, the gene can easily spread through bacterial populations and other bacterial species – as has already been documented in Enterobacteriaceae and A. baumannii.3 The β-lactamase that it codes for is a lipoprotein that is anchored in the outer membrane of the gram negative bacteria. Other metalo-β-lactamases (MBLs) are periplasmic proteins, which are more affected by changes in essential metal cofactors in their enzymatic function. Thus far, it has been found that there are 16 discovered variants of NDM. Some variants being more fit than NDM-1. It is hypothesized that these variants are being selected for in the clinical setting, with the protein being more stable and demonstrating higher affinities for zinc during time of metal-chelating (a process the immune system adapts to combat infections).9 Unfortunately, NDM-1 and its variants are resistant to almost all antibiotics. Usually the only option is colistin and tigecycline.3

The disturbing issue, and the big picture, is the capability of MDR organisms and their genes of disseminating. As previously mentioned, NDM-1 is capable of spreading to other species and within its population. Yet, a terrifying report has demonstrated blaNDM-1 detection in artic soil samples from 2013, 4 years after the first detection of the gene.10 This demonstrates the ability for antibiotic resistance to spread on a global scale, and how serious this battle truly is.

References

  1. “Carbapenem-Resistant Enterobacteriaceae (CRE) Infection.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 23 Feb. 2015, http://www.cdc.gov/hai/organisms/cre/cre-patientfaq.html.
  2. Walther-Rasmussen, Jan, and Niels Høiby. “Class A Carbapenemases.” Journal of Antimicrobial Chemotherapy, vol. 60, no. 3, 2007, pp. 470–482., doi:10.1093/jac/dkm226.
  3. Khan, Asad U., et al. “Structure, Genetics and Worldwide Spread of New Delhi Metallo-β-Lactamase (NDM): a Threat to Public Health.” BMC Microbiology, vol. 17, no. 1, 2017, doi:10.1186/s12866-017-1012-8.
  4. Mohapatra P. R. (2013). Metallo-β-lactamase 1–why blame New Delhi & India?. The Indian journal of medical research137(1), 213–215.
  5. Roos, Robert. “Canada Finds More Infections with NDM-1 Resistance Factor.” University of Minnesota, Center for Infectious Disease Research and Policy, 11 Nov. 2010, http://www.cidrap.umn.edu/news-perspective/2010/11/canada-finds-more-infections-ndm-1-resistance-factor.
  6. Gupta, M., Didwal, G., Bansal, S., Kaushal, K., Batra, N., Gautam, V., & Ray, P. (2019). Antibiotic-resistant Enterobacteriaceae in healthy gut flora: A report from north Indian semiurban community. The Indian journal of medical research, 149(2), 276–280. doi:10.4103/ijmr.IJMR_207_18
  7. Kotwani, Anita, and Kathleen Holloway. “Access to Antibiotics in New Delhi, India: Implications for Antibiotic Policy.” Journal of Pharmaceutical Policy and Practice, vol. 6, no. 1, 2013, doi:10.1186/2052-3211-6-6.
  8. Kotwani, Anita, et al. “Antibiotic-Prescribing Practices of Primary Care Prescribers for Acute Diarrhea in New Delhi, India.” Value in Health, vol. 15, no. 1, 2012, doi:10.1016/j.jval.2011.11.008.
  9. Bahr, Guillermo, et al. “Clinical Evolution of New Delhi Metallo-β-Lactamase (NDM) Optimizes Resistance under Zn(II) Deprivation.” Antimicrobial Agents and Chemotherapy, vol. 62, no. 1, 2017, doi:10.1128/aac.01849-17.
  10. Mccann, Clare M., et al. “Understanding Drivers of Antibiotic Resistance Genes in High Arctic Soil Ecosystems.” Environment International, vol. 125, 2019, pp. 497–504., doi:10.1016/j.envint.2019.01.034.

-Ben Dahlstrom is a recent graduate of the NorthShore University HealthSystem MLS program. He currently works as a molecular technologist for Northwestern University in their transplant lab, performing HLA typing on bone marrow and solid organ transplants. He graduated with a bachelors in Biology at the University of Illinois at Chicago (UIC) and concurrently from the UIC Honors College. He discovered his passion for the lab through his experience in healthcare. His interests include microbiology, molecular, immunology, and blood bank.

-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.