Microbiology Case Study: 58 Year Old Male with Right Upper Quadrant Pain

Case:

A 58 year old male with a history of pica presented to the emergency room complaining of bilious, non-bloody emesis associated with right upper quadrant pain, fatigue, decreased oral intake, and fevers. Initial labs revealed a lactate of 7.4, a white blood cell count of 22K, alkaline phosphatase 456, AST 373, ALT 444, total bilirubin 5.8, and a troponin of 3.4. Imaging showed a distended, gangrenous gallbladder with compression of the biliary system. Aerobic and anaerobic blood cultures were drawn and the anaerobic culture flagged positive at 20 hours with the following gram stain and colony morphology.

 

Large Gram-positive boxcar shaped bacilli.
Large Gram-positive boxcar shaped bacilli.
Schaedler plate showing double zone of beta hemolysis.
Schaedler plate showing double zone of beta hemolysis.
Opacification of egg yolk agar around bacterial colonies.
Opacification of egg yolk agar around bacterial colonies.

Laboratory identification:

Clostridium perfringens was suspected due to the presence of large gram-positive boxcar shaped bacteria on the gram stain and obligate anaerobic growth with a double zone of hemolysis on the Schaedler plate. Opacification of the egg yolk agar demonstrated that the organism was lecithinase positive which is another characteristic of C. perfringens. Identification of the organism was confirmed by mass spectrometry.

Discussion:

Clostridium perfringens grows on the anaerobic blood agar plate as small gray to gray-yellow colonies with a glossy, dome- shaped appearance with a double zone of beta hemolysis. The organism is an obligate anaerobe and therefore the aerotolerance test is negative. Its biochemical characteristics including the following: lecithinase positive, lipase negative, and catalase negative.

C. perfringens is encountered in a number of clinical settings including wound infections, gas gangrene, bacteremia, septicemia, and food borne illness. The organism has a number of virulence factors including exotoxins and an enterotoxin. Although there are reports of resistance, penicillin is still recommended therapy in many cases.

C. perfringens is found in the biliary system and is associated with gangrenous cholecystitis.

 

-Lauren Pearson, D.O. is a 2nd year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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

 

Microbiology Case Study–A Very Sick 51 Year Old Man

Case history:

A 51 year old man with a medical history of liver abnormalities and long standing alcohol abuse presented with generalized weakness, hypoxemic respiratory failure, sputum production, significant hyperbilirubinemia, macrocytic anemia, and laboratory tests consistent with DIC. Chest X ray revealed a bronchopneumonia pattern. Sputum samples were sent for culture and blood cultures were obtained. Urine antigen legionella test was positive for Legionella pneumophila serogroup 1. The sputum was then plated on buffered charcoal yeast extract (BCYE) agar.

Laboratory identification:

Gram stain of colonies growing on BCYE revealed thin short to filamentous rod shaped organisms. The definitive diagnosis of Legionella pneumophila was made by isolating the organism on BCYE with confirmation on the MALDI-ToF. Colonies were speckled blue and

Legionella on BCYE agar plate
Legionella on BCYE agar plate
Gram negative, thin, short-to-filamentous rod shaped organisms
Gram negative, thin, short-to-filamentous rod shaped organisms

Discussion:

Legionella pneumophila was first identified and recognized during the outbreak that occurred during the American Legion Convention in Philadelphia in 1976. There are over 40 species of Legionella and 18 of those are human pathogens. Among those, L. pneumophila is an important cause of nosocomial and community-acquired pneumonia (CAP) and should be considered in the differential diagnosis in any patient who presents with pneumonia. Two clinical syndromes caused by this microorganism are: Legionnaires’ disease and Pontiac fever (acute, febrile, self-limited illness).

L. pneumophila is a thin faintly staining short to filamentous gram negative rod. Legionella is a fastidious organism and does not grow on standard media. Buffered charcoal yeast extract (BCYE) agar is the primary medium used for its isolation and it is the gold standard for its diagnosis although testing for the antigen in urine is more commonly performed. The urinary antigen assay only detects L. pneumophila serogroup 1 which cause 95-98% of community acquired Legionnaires’ disease.

The microorganism is found in natural water supplies and soil. It is also found in recirculating and water supply systems, where it breeds rapidly in favorable conditions (temperature of 35°C, range 25-45, stagnant water and water containing organic debris which can provide nutrients for growth). Macrolides (azithromycin or clarithromycin) or fluoroquinolones (levofloxacin or moxifloxacin) are the standard antibiotic drugs used to treat Legionnaires’ disease in humans.

Kossivi Dantey, M.D. is a 4th year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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–Christi Wojewoda, MD, is certified by the American Board of Pathology in AP/CP and Medical Microbiology. She is currently the Director of Clinical Microbiology at the University of Vermont Medical Center and an Assistant Professor at the University of Vermont.

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Microbiology Case Study: 5 Year Old Male Immigrant with Rough Skin Lesions

Case:

A 5 year old boy who recently immigrated to the United States from Africa presented to his primary care physician with several rough, hypopigmented skin lesions and some hair loss on the scalp. The rash was treated with topical antifungal medication. The patient was treated with griseofulvin for tinea capitis and initially responded well. Six months later he developed pruritic macules on his body, for which clotrimazole was prescribed. The patient’s scalp at that time showed few lesions concerning for recalcitrant tinea capitis. He was prescribed weekly fluconazole and topical clotrimazole. Fungal cultures grew an organism with the following scotch tape prep and colony morphology.

Lactophenol analine blue tape preparation.
Lactophenol analine blue tape preparation.
Colony morphology with deep purple red pigmentation.
Colony morphology with deep purple red pigmentation.

 

Laboratory identification:

The scotch tape prep showed branched, tortuous hyphae and chlamydoconidia in chains but no microconidia or macroconidia were present. The colonies took over two weeks to grow and were initially cream-colored but later developed a port wine to deep violet color. Based on this information Trichophyton violaceum, Trichophyton rubrum, and Trichophyton soudanense were on the differential diagnosis. The specimen was sent to a referral lab for definitive speciation. The referral lab identified the organism as Trichophyton violaceum by MALDI-ToF. The organism was also sequenced for confirmation.

Discussion:

Trichophyton violaceum is a dermatophyte that can be recovered from hair, skin, and nails. The organism requires 14-18 days to grow. Its growth is enhanced by media containing thiamine, which helps differentiate it from other species within the Trichophyton genus. Infection typically causes “black dot” tinea capitis, tinea corporis, and onychomycosis. Infections with this particular Trichophyton species are seen primarily in persons living in Mediterranean region, the Middle East, and Africa. It is treated with oral griseofulvin.

 

-Lauren Pearson, D.O. is a 2nd year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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

 

Microbiology Case Study: 22 Year Old Female with Joint Pain

A 22 year old female with no significant past medical history presented with a fever, joint pain and a petechial rash. She endorsed having cold/flu symptoms for two weeks prior. The patient was admitted to the hospital where blood cultures were drawn and antibiotics were initiated. One set of blood cultures from the patient flagged positive at 14 hours of incubation with the following gram stain and colony morphology.

Gram stain showing Gram negative diplococci.
Gram stain showing Gram negative diplococci.
Chocolate agar plate with small gray, slightly mucoid colonies.  Photo courtesy of pixgood.com
Chocolate agar plate with small gray, slightly mucoid colonies.
Photo courtesy of pixgood.com

 

Laboratory identification:

The patient’s blood was cultured on aerobic blood agar and chocolate agar plates. The gram stain revealed gram negative diplococci. Medium sized, round, gray to white, slightly mucoid colonies grew on blood and chocolate agars. The organism was definitively identified as Neisseria meningitidis by VITEK-MS. Prior to adoption of mass spectrometry, biochemical tests were performed for further characterization of the organism. Neisseria meningitidis is catalase positive, ferments glucose and maltose but not lactose, is oxidase positive, and does not reduce nitrate.

Discussion:

Neisseria meningitidis asymptomatically colonizes the oropharynx and nasopharynx of humans. It is transmitted by person-to-person spread of contaminated respiratory droplets. Infection causes a spectrum of disease including life-threatening meningitis. Bacteremia causes the characteristic petechial rash, thrombocytopenia, DIC, and shock. The organism may also cause conjunctivitis, pneumonia, and sinusitis. Its virulence factors include surface structures to facilitate attachment to and invasion of epithelial cells. Once the organism gains access to the vascular system, its survival is mediated by the polysaccharide capsule. Endotoxin release mediates many of the systemic manifestations of infection such as shock.

The differential diagnosis for this organism based on the gram stain and colony morphology includes Neisseria gonorrhoeae and Moraxella species. Different Neisseria species can be identified by the sugars they are able to ferment. For example, N. gonorrhoeae ferments only glucose, but N. meningitidis ferments both glucose and maltose.

There is a vaccine that is available for N. meningitidis that includes serogroups A, C, W-135, and Y. There are 12 different serogroups that can be distinguished based on the polysaccharide capsule. Our patient had been fully vaccinated. The isolate was sent to the state public health lab and it was reported back as non-typable and was sent to the CDC.

Treatment of N. meningitidis consists of supportive therapy for shock plus antimicrobial therapy with penicillin, ceftriaxone, or cefotaxime.

***Rare, fatal cases of meningococcal disease have been reported in laboratory staff. Any potential N. meningitidis should be worked with under a class II biological safety cabinet.

 

-Lauren Pearson, D.O. is a 2nd year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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

 

Microbiology Case Study–Joint Pain After Left Knee Arthroscopy

Case history:

A 51 year old male with a history of recent left knee arthroscopy presented with increasing joint pain, swelling, and reduced range of motion accompanied by subjective fevers. He was receiving physical therapy and admitted to soaking the knee in a hot tub on two occasions after the therapy sessions. An aspirate of synovial fluid obtained at the clinic showed the following colony morphology and Gram stain:

Spready, rough, bluish-green colonies on blood agar.
Spready, rough, bluish-green colonies on blood agar.
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Gram stain of the predominant colonies showing gram negative bacilli

Laboratory identification:

Pseudomonas aeruginosa is a non-spore forming, non-encapsulate, motile gram-negative bacillus. The bacteria typically form characteristic colonies with a metallic sheen and have a green-blue appearance due to production of soluble blue phenazine pigment (pyocyanin) and yellow-green pigment fluorescein (or pyoverdin). The organisms classically emit a grape-like odor in culture. They are oxidase positive. Lab diagnosis is based on colony morphology and the organism’s biochemical characteristics. P. aeruginosa is also the only clinically significant fluorescent pseudomonad that grows at 42oC. In our laboratory, the isolate was identified on the MALDI-TOF.

Discussion:

P. aeruginosa has been documented to infect any external site or organ. Community acquired infections are associated with otitis media, skin ulcers, corneal infection, and rashes secondary to contaminated hot tub water which is consistent with our patient’s history. Hospital acquired infections are typically related to catheters, bedsores, burns, and eye infections. People with cystic fibrosis are particularly susceptible to infection with this organism although asymptomatic colonization in these patients is also possible. Patients with extensive burns are also at risk for infection with Pseudomonas. Rarely, a septicemic infection characterized by black necrotic skin lesions known as ecthyma gangrenosum may happen.

The organism has two important virulence factors for pathogenesis- exotoxin A and exoenzyme S. It also produces various cytotoxic substances, all of which contribute to the local tissue destruction.

Pseudomonas infections can be treated with aminoglycosides, beta lactam, and fluoroquinolone antibiotics.

-Lauren Pearson, D.O. is a 2nd year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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

Microbiology Case Study: 92-year-old with Itchy Rash

A 92 year old female nursing home resident presented to her primary care physician with an itchy rash between her fingers and at her waist. A skin scrape revealed the following:

Sarcoptes scabiei  (the itch mite) from skin scraping.
Sarcoptes scabiei (the itch mite) from skin scraping.

Laboratory identification:

It is critical that an appropriate specimen is collected for identification of the organism. A fresh unopened papule on the skin should be selected for skin scraping. A scalpel coated in mineral oil should be used to vigorously scrape the papule and transfer the scrapings to a glass slide. A well collected skin scraping draws blood.

Female mites are 330-450 microns long; males are slightly smaller at 200-240. The eggs are thin shelled and approximately 150 x 100 microns in size. It is also possible to see fecal pellets in scrape specimens.

Discussion:

Sarcoptes scabiei is transmitted by direct contact. The gravid female mite burrows into the epidermis leaving behind a trail of up to 40 eggs. The burrowing process is enhanced by the presence of suckers and specialized cutting surfaces on the organism. The larvae hatch in 3-4 days, leave the burrow, and reach adulthood in hair follicles. The typical patient presentation is intense pruritis, often in folds of skin, with possible secondary bacterial infection due to itching and excoriation.

Scabies is treated with aqueous solutions of malathion or permethrin.

-Lauren Pearson, D.O. is a 2nd year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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

You Never Know What You Might Find on Peripheral Smear Review

A 15 month patient was seen in the Pediatric Hematology-Oncology clinic in June 2014 for mild normocytic anemia.

Review of Systems

Negative 12 system review. No history of pallor, jaundice or high colored urine.

Ref. Range 6/14
WBC 6-17 K/uL 11.4
Hemoglobin 10.5-13.5 g/dL 8.9 (L)
Hematocrit 33-39 % 25.4 (L)
Platelets 150-400 K/uL 648 (H)
RBC 3.7-5.3 M/uL 3.57 (L)
MCV 70-86 fL 71.2
MCH 23-30 pg 24.9
MCHC 31-36 % 34.9
RDW 11.5-14.5 % 16.4 (H)

His serum iron profile was normal, serum lead levels were normal. Reticulocyte percentage and absolute reticulocyte count were also both not elevated.

Review of peripheral smear revealed moderate anisopoikilocytosis with presence of numerous elliptocytes.

he1

he2

Molecular studies demonstrated a heterozygous mutation in the EPB41 gene associated with HE.

Patient was diagnosed with Hereditary Elliptocytosis (HE).

He has been followed up at the hematology clinic for a year now. His follow up CBC results are as follows. He has reached his age appropriate milestones and continues to grow well.

Ref. Range 7/14 10/14 12/14 4/15
WBC 6-17 K/uL 10.3 11.3 7.4 10.0
Hemoglobin 10.5-13.5 g/dL 9.3 (L) 9.9 (L) 9.7 (L) 11.0
Hematocrit 33-39 % 26.5 (L) 28.7 (L) 28.1 (L) 33.6
Platelets 150-400 K/uL 599 (H) 570 (H) 403 (H) 447 (H)
RBC 3.7-5.3 M/uL 3.74 3.91 3.87 4.58
MCV 70-86 fL 70.8 73.3 72.6 73.5
MCH 23-30 pg 24.8 25.4 25.1 23.9
MCHC 31-36 % 35.0 34.7 34.5 32.6
RDW 11.5-14.5 % 16.9 (H) 17.7 (H) 18.2 (H) 18.0 (H)

Hereditary elliptocytosis (HE) is an inherited hemolytic anemia, secondary to red cell membrane defect more commonly assembly of spectrin, spectrin-ankyrin binding, protein 4.1 and glycophorin C with a clinical severity ranging from asymptomatic carriers to a severe hemolytic anemia. It is more common in individuals from African and Mediterranean decent – neither applies to our patient.It is inherited in an autosomal dominant pattern, typically individual who are heterozygous are asymptomatic while those who are homozygous or compound heterozygous have a mild to severe anemia. Occasional patients with more severe hemolysis may require splenectomy.

Regardless of the underlying molecular abnormality, most circulating red cells are elliptical or oval. They still have an area of central pallor, since there is no loss of the lipid bilayer (as seen in Hereditary spherocytosis).

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-Neerja Vajpayee, MD, is an Associate Professor of Pathology at the SUNY Upstate Medical University, Syracuse, NY. She enjoys teaching hematology to residents, fellows and laboratory technologists.