Category: microbiology

Microbiology Case Study: An 18 Year Old with Gastrointestinal Bleeding

An 18 year old female with no significant past medical history experienced multiple episodes of gastrointestinal bleeding over the course of a few weeks. The most recent bout included a bloody episode that filled the toilet, for which she provided a picture for the clinician. She denies any other associated symptoms including epigastric pain, nausea, vomiting, fever, or chills. Her travel history is unknown.

Review of her history reveals an unremarkable family and social history. She has never had an incident similar to this in the past and no other family members have ever complained of similar symptoms. Review of systems was unremarkable and within normal limits. Physical exam was unremarkable. A rectal exam was performed and was noted to have brown stool that was guaiac (occult blood) positive. Non bleeding internal hemorrhoids were noted. There were no external hemorrhoids present.

Labs drawn including CBC were within normal ranges with the exception of absolute eosinophils which were at the upper limit of normal range at 0.6 x 103/µL [normal range= 0.0 – 0.6 103/µL].

The patient had an esophagogastroduodenoscopy (EGD) to further investigate the gastrointestinal bleed. The exam was otherwise normal with exception of the ascending colon where they noted a worm on the surface of the mucosa (Image 1-2). The worm was collected and transported to microbiology for examination (Image 3-4).

Image 1. View of a worm seen on the mucosal surface of the ascending colon.
Image 2. Another view of a worm seen on the mucosal surface of the ascending colon.
Image 3. Adult worm viewed under the dissecting microscope.
Image 4. Eggs viewed under the dissecting microscope.

Discussion

Examination of the worm and eggs revealed morphology consistent with Trichuris trichiura, or whipworm.

T. trichiura is most prevalent in warm, moist regions. The worldwide prevalence of infection is estimated to be roughly 800 million, mostly among poorer populations. Infection from T. trichiura is spread via fecal-oral route and caused by ingesting embryonated eggs. This occurs when contaminated dirt is ingested or by consumption of vegetables or fruits that have not been carefully cooked, washed or peeled.

The male and female worms both have the long whip-like structures at the anterior end. T. trichiura worms are 30-50 mm in length and the average life span is 1 year but they can live up to 10 years. The females have a straight and thick head while the males have a curly ended head. The males are typically longer the females. The eggs classically have barreled shaped, brown eggs with thick shells that measure 50-55 µm long by 22-24 µm wide. At each pole is lucent mucoid plug. The can also vary in size as noted in Image 5.

The adult female T. trichiura produces 1,000-7,000 eggs per day. The life cycle begins as unembryonated eggs passed in feces into soil (Figure 1). It takes approximately 21 days in the soil for an unembryonated egg to go through the process of embryonation to become the infective form of the parasite. Once ingested, the embryonated eggs hatch in the human intestine.

Image 5. T. trichiura eggs (CDC DPDx website)
Figure 1. Lifecycle of T. trichiura (CDC, DPDx)

Clinically, symptoms vary depending on the worm biomass present with most infections being asymptomatic. Symptoms include cramping, weight loss, growth restriction in children, bloody stool, and anemia. It can also result in Trichuris dysentery syndrome, which is more common in children. Recurrent rectal prolapse has also been reported. Lab findings include peripheral eosinophilia. T. trichiura is treated with Albendazole for 5-7 days +/- Ivermectin. Our patient was then prescribed albendazole and is being followed in GI clinic.

References

  1. Centers for Disease Control and Prevention. “Laboratory Identification of Parasites of Public Health Concern: Trichuriasis”. https://www.cdc.gov/dpdx/trichuriasis/index.html
  2. Procop, G. W., Church, D. L., Hall, G. S., Janda, W. M., Koneman, E. W., Schreckenberger, P. C., & Woods, G. L. (2017). Koneman’s color atlas and textbook of diagnostic microbiology (Seventh edition.). Philadelphia: Wolters Kluwer Health.

-Sharif Nasr, MD, 4th year anatomic and clinical pathology resident at University of Chicago (NorthShore). Dr. Nasr has an interest in GI pathology.

-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 70 Year Old Male with Multiple Myeloma

Case History

The patient is a 70 year old male who was diagnosed with Kappa free light chain multiple myeloma. He was initially seen after he had a fall in the woods and underwent imaging which showed multiple lytic lesions and blood work showing monoclonal proteins and thrombocytopenia. He was found to have a lesion on his right scapula for which he received radiation. Bone marrow biopsy was performed which showed 60% plasma cells. To date he has completed radiation therapy, 5 cycles of chemotherapy, and is in the process of collecting stem cells for autologous stem cell transplant. Routine fungal culture of the stem cell collection grew a single tan white dry appearing colony on potato flake agar. A Gram stain of the organism revealed gram positive cocci mixed with filamentous structures.

Laboratory Identification

Image 1. Single tan white dry colony on potato flake agar.
Image 2. Modified acid fast stain (left) and Gram stain (right).
Image 3. Filamentous branching on Gram stain.

Based on the colony morphology and Gram stain results the organism was suspected to be in the Streptomyces genus. Identification with MALDI-TOF was attempted and did not yield a result as this bacteria is not in the data base.

Discussion

Streptomyces is a genus of gram positive aerobic saprophytic bacteria that grows in various environments, and has a filamentous form similar to fungi (1). The morphologic differentiation of Streptomyces involves identification of complex multicellular architecture with germinating spores that form hyphae, and multinuclear aerial mycelium, which forms septa at regular intervals, creating a chain of uninucleated spores (2,3). They are able to metabolize many different compounds including sugars, alcohols, amino acids, and aromatic compounds by producing extracellular hydrolytic enzymes (helping with degradation of organic matter). Their metabolic diversity is due to their extremely large genome which has hundreds of transcription factors that control gene expression, allowing them to respond to specific needs (3).

Streptomyces is also considered to be one of the most medically important bacteria because of its ability to produce bioactive secondary metabolites. These metabolites are used in the creation of antifungals, antivirals, antitumoral, anti-hypertensives, and many antibiotics and immunosuppressives. They are responsible for 2/3 of all the worlds naturally occurring antibiotics (1).

Streptomyces is usually considered a laboratory contaminant though they can cause infections in immunocompromised patients and are chiefly responsible for granulomatous lesions in skin also known as actinomycotic mycetomas (1,2). Invasive pulmonary disease has been seen in HIV patients, splenectomized patients with sarcoid, and rarely in immunocompetent hosts (1). More rare presentations include brain abscesses can be seen in patients with cerebral trauma, peritoneal infections have been shown to occur in patients undergoing multiple pericenteses, and bacteremia in patients with indwelling catheters (1). Infection with Streptomyces is not common so susceptibility data is limited. Available data shows that organisms were consistently susceptible to amikacin; frequently susceptible to imipenem, clarithromycin or erythromycin, minocycline, and trimethoprim-sulfamethoxazole; and infrequently susceptible to ciprofloxacin and ampicillin (4).

Our patient had not received the stem cell unit that this grew from, so another aliquot was requested. The second aliquot did not grow any organisms, so the Streptomyces was considered a contaminant.

References

  1. Procop, Gary W., et al. Konemans Color Atlas and Textbook of Diagnostic Microbiology. 7th ed., Wolters Kluwer Health, 2017.
  2. Tille, Patricia M. Bailey & Scotts Diagnostic Microbiology. 13th ed., Elsevier, 2014.
  3. Chater KF. Recent advances in understanding Streptomyces. F1000Res. 2016;5:2795. Published 2016 Nov 30. doi:10.12688/f1000research.9534.1
  4. Mona Kapadia, Kenneth V.I. Rolston, Xiang Y. Han, Invasive Streptomyces Infections: Six Cases and Literature Review, American Journal of Clinical Pathology, Volume 127, Issue 4, April 2007, Pages 619–624, https://doi.org/10.1309/QJEBXP0BCGR54L15

-Casey Rankins, DO, 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 40 Year Old Woman with Fever, Chills, and Leg Pain

Clinical History

A 40 year old African American female with a history of sickle cell disease presented to an outpatient clinic with fever, chills, and leg and back pain consistent with a sickle cell crisis. Her past medical history was also significant for asthma and seizures. She rated her pain as 10 out of 10, her vitals showed a temperature of 101.0°F, and she was also tachycardic and hypotensive. Her white blood cell count was 23.0 TH/cm2, hemoglobin 8.4 g/dL, hematocrit 26.0%, and platelets 619,000 TH/cm2. In clinic, she received pain medications and a fluid bolus, two sets of blood cultures were collected, and she was transferred to the emergency department for further work up.

Laboratory Identification

Image 1. Gram stain from a positive blood culture bottle showing small, gram positive budding yeast (1000x oil immersion).
Image 2. A mucoid, salmon-colored yeast grew on Sabouraud dextrose and chocolate agars.

Blood culture bottles were positive after approximately two days on the automated instrument. The Gram stain showed small, gram positive budding yeast (Image 1). The BioFire FilmArray for blood culture identification was negative for Candida albicans, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis. At this time, she was started on micafungin for antifungal therapy. A mucoid, salmon colored yeast grew on both Sabouraud dextrose and chocolate agars (Image 2) and was identified by Vitek 2 as Rhodotorula spp.

Discussion

Rhodotorula spp. are basidiomycetous yeasts that make up the normal microbiota on moist skin and can be found in bathtubs and on shower curtains. Rhodotorula spp. are usually considered contaminants, but can rarely cause fungemia in patients with central lines, endocarditis, peritonitis, and meningitis, especially in those that are immunocompromised. R. mucilaginosa, R. glutinis, and R. minuta are the species commonly associated with human disease. 

In the laboratory, Rhodotorula spp. grow as a mucoid, salmon colored yeast within 1-3 days of incubation. On Gram stain or lactophenol cotton blue prep, the yeast is small and round to oval with multilateral budding. Pseudohyphae are not usually present. Rhodotorula spp. produce urease and fail to ferment carbohydrates. R. mucilaginosa is negative for nitrate assimilation. Identification can also be confirmed by commercial kits, automated systems, and MALDI-TOF mass spectrometry. Rhodotorula spp. are intrinsically resistant to echinocandins and fluconazole.

In the case of our patient, she was switched to intravenous amphotericin B after the identification of Rhodotorula spp. was made. Reference laboratory testing identified the isolate as R. mucilaginosa with high minimum inhibitory concentrations (MIC) to fluconazole and echinocandins. Amphotericin had an MIC of 0.5 µg/ml. She successfully completed a 14 day course with close monitoring of creatinine, electrolytes, and platelet count. Repeat blood cultures were negative and no other focuses of infection were found on CT scans, transthoracic echocardiogram, and ophthalmology exam.

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

Microbiology Case Study: A 35 Year Old Female with Post-Op Drainage

Case History

A 35 year old female with a history of BRCA-positive breast cancer (status-post right radical mastectomy February 2018) underwent prophylactic left mastectomy and revision of right mastectomy. She received prophylactic clindamycin and cefazolin. She was discharged on post-op day 5 with bacitracin and 3 weeks of cefadroxil. She initially healed well. On post-op day 43 she noted drainage from the left incision site. At presentation she was afebrile.  There was a 3.0 x 2.0 cm area of induration and erythema on the right lateral aspect of her abdominal incision with seropurulent drainage. Incision and drainage was performed in office and a swab of the fluid was sent to microbiology. The initial gram stain and cultures were negative for bacteria. The patient was placed on sulfamethoxazole-trimethoprim and levofloxacin. At follow up 7 days later, another abscess was medial to the prior site was incised and drained. A swab of the fluid was sent to microbiology for bacterial and fungal cultures.

Laboratory Identification

Fungal cultures grew at 36 hours on potato-flake agar. Gram stain revealed gram-variable bacilli. Growth on 7H10 agar produced colonies at 72 hours, and Kinyoun staining was positive for acid-fast bacilli. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) at 72 hours identified Mycobacterium abscessus complex.

Image 1. Growth on 7H10 agar.
Image 2. Gram stain from 7H10 agar showing gram variable bacilli.
Image 3. Kinyoun stain showing acid-fast bacilli.

Discussion

M. abscessus complex is a group of rapidly-growing, nontuberculous mycobacteria. As such they are acid-fast bacilli that grow within 7 days when transferred from solid media to solid media. The subspecies are M. abscessus abscessus, M. abscessus massiliense, and M. abscessus bolletii. The complex is known to cause progressive pulmonary disease in patients with underlying structural lung diseases. It has been estimated to comprise up to 13% of all mycobacterial pulmonary infections. It has also been implicated in skin and soft tissue infections (SSTIs) following surgical procedures or environmental exposure (i.e. spas). SSTIs can also occur by seeding from disseminated disease. Rarer manifestations include central nervous system (CNS) and ocular involvement. Identification is by culture and molecular techniques. It is classically resistant to many drug classes with limited consensus on appropriate therapy. It can harbor the erm gene, which confers inducible erythromycin resistance. Clarithromycin, amikacin, and cefoxitin tend to have the lowest rates of resistance. Long-term multidrug regimens are recommended, based on susceptibility testing. Changes to initial therapy are usually required due to side effects or lack of efficacy. Surgical therapy is often required, when possible. Mortality post therapy is approximately 15%.

At two-week follow up, the wound had no purulent drainage or erythema. The plan was for prolonged three-drug therapy tailored to susceptibility data.

  1. Griffith DE. Rapidly growing mycobacterial infections: Mycobacteria abscessus, chelonae, and fortuitum. Von Reyn CF and B A, eds. UpToDate. Waltham, MA: UpToDate Inc. https://www.uptodate.com (Accessed on May 21, 2019.)
  2. Lee MR, Sheng WH, Hung CC, Yu CJ, Lee LN, Hsueh PR. Mycobacterium abscessus Complex Infections in Humans. Emerg Infect Dis. 2015;21(9):1638–1646.
  3. Novosad SA, Beekmann SE, Polgreen PM, et al. Treatment of Mycobacterium abscessus Infection. Emerging Infectious Diseases. 2016;22(3):511-514.

-Jonathan Wilcock, MD is a 1st year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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

Microbiology Case Study: A 55 Year Old Male with Altered Mental Status

Case History

A 55 year old male presented to the emergency department (ED) with altered mental status (AMS). His past medical history includes stage 4 pancreatic cancer with known invasion into the distal splenic vein. Currently undergoing chemotherapy, his last infusion was one week prior to presentation. On physical exam, the patient is a cachectic male with dry mucous membranes, scleral icterus, hypotension (79/37), hypothermia (35o C), tachypnea (Respiratory Rate of 20/min) and tachycardia (pulse up to 130s). Initial labs were ordered including blood cultures and were significant for hypoglycemia (40mg/dL), pancytopenia, mild liver function test abnormalities, an ammonia level of 80 µmol/L and lactate of 11.2 mmol/L. It was concluded that the AMS resulted as a consequence of hypoglycemia. However, there was also concern for intracranial pathology vs. stroke vs. metastatic disease. Brain imaging showed no obvious lesion or mass. The cause of the hypotension was uncertain but could be a result of volume depletion or sepsis. Empiric vancomycin and cefepime were initiated in the ED. However, the patient decompensated, developing mid-abdominal and periumbilical ecchymoses suspicious for a retroperitoneal hemorrhage. Despite aggressive therapy, he expired in the ED.

Laboratory Identification

An anaerobic blood culture bottle flagged positive at 5 hours incubation. A gram stain showed large, boxy, gram positive rods without spores (Image 1). Brucella blood agar was inoculated and incubated anaerobically.  Following overnight incubation, the surface of the plate showed a subtle film of growth covering the plate and detectable hemolysis (Image 2). No discreet colonies were identified. A catalase test was performed and was negative. Definitive identification of Clostridium septicum was obtained by MALDI-TOF.

Image 1. Gram stain from the anaerobic blood culture bottle that flagged as positive following 5 hours of incubation. Boxy, large gram positive rods without spores are observed. Oil immersion photomicrograph (x100 objective).
Image 2. Brucella agar plate following 24 hours of incubation under anaerobic conditions at 35oC. Significant bacterial growth in a haze and detectable Beta hemolysis can be observed. No discreet colonies can be identified. Identification by MALDI-TOF was Clostridium septicum.

Discussion

Clostridium septicum is an anaerobic gram positive bacillus that can produce spores; however, spores are not frequently seen, especially in nutrient-rich environments. Spores, when present, are typically oval and located subterminally. Infection by C. septicum was once thought to be extremely rare, but improvements in anaerobic laboratory techniques have allowed for the discovery of the true potential of this agent. C. septicum is one of several bacteria that can cause myonecrosis (i.e., gas gangrene). Infections are typically seen in settings of immunodeficiency, trauma, surgery, malignancy, skin infections/burns, and septic abortions. The colon may promote the growth of C. septicum better than other anatomic sites due to its anaerobic conditions. As one of the more aggressive etiologies of gas gangrene, C. septicum infection progresses very rapidly, with a mortality rate of approximately 79% in adults, typically occurring within 48 hours of infection. Symptoms of infection include pain, described as a heaviness or pressure that is disproportionate to physical findings, tachycardia, and hypotension. Tissue necrosis then causes edema and ischemia resulting in metabolic acidosis, fever, and renal failure. The carbon dioxide and hydrogen produced during the growth of the organism move through tissue planes, causing their separation, producing features characteristic of palpable emphysema (i.e., crepitus). This also results in a magenta-bronze skin discoloration and bulla filled with a foul-smelling serosanguinous fluid.

Four toxins have been isolated from C. septicum: the lethal alpha toxin, DNase beta-toxin, hyaluronidase gamma toxin, and the thiol-activated/septicolysin delta toxin. Alpha toxin causes intravascular hemolysis and tissue necrosis and is well known as the primary virulence factor of C. septicum. 

C. septicum derived gas gangrene has shown strong correlations with increased levels of malignancy. Patients with C. septicum infections may have an occult colon cancer or a tumor that has metastasized to the colon. C. septicum bacteremia is also associated with typhlitis (defined as inflammation of the cecum that can extend proximally into the terminal ileum or distally into the ascending colon), which can develop in patients with hematologic malignancy receiving chemotherapy. Because the organism may be harbored in the gastrointestinal tract, the organism may gain access to the bloodstream through the ileocecal region.

Therapy includes antibiotics and surgical debridement (with occasional amputation). For antibiotic selection, typical anaerobic coverage includes piperacillin/tazobactam, ampicillin/sulbactam, metronidazole or meropenem. Vancomycin is also effective. Susceptibility testing is not typically performed; moreover, the CLSI makes an annual antibiogram which can be used as a guide. 

Key points

  • C. septicum often has swarming growth that covers the plate surface.
  • Spontaneous myonecrosis with C. septicum bacteremia can be an indicator of possible occult colonic malignancy.
  • C. septicum can be associated with typhlitis in neutropenic patients with hematologic malignancy undergoing chemotherapy.

 References

  1. Smith-Slatas CL, Bourque M, and Salazar JC (2006). Clostridium septicum infections in children: a case report and review of the literature. Pediatrics 117(4): e796-e805.
  2. Alpern, RJ and Dowell, VR (1969). “Clostridium septicum infections and malignancy”. JAMA. 209: 385–388.
  3. Ballard, J, Crabtree, J, Roe, BA, and Tweten, RK (1995). “The primary structure of Clostridium septicum alpha-toxin exhibits similarity with that of Aeromonas hydrophila aerolysin”. Infection and Immunity. 63 (1):340–344.
  4. Sidhu JS, Mandal A, Virk J, and Gayam V (2019). “Early detection of colon cancer following incidental finding of Clostridium septicum bacteremia”. J Investig Med High Impact Case Rep. Jan-Dec;7:2324709619832050.
  5. Srivastava I, Aldape MJ, Bryant AE, and Stevens DL. (2017). “Spontaneous C. septicum gas gangrene: A literature review” Anaerobe. Dec;48:165-171

–Xiang Xu, MD, PhD and Dominick Cavuoti, DO contributed to this case.

-Clare McCormick-Baw, MD, PhD is an Assistant Professor of Clinical Microbiology at UT Southwestern in Dallas, Texas. he has a passion for teaching about laboratory medicine in general and the best uses of the microbiology lab in particular.

Microbiology Case Study: A 64 Year Old Post-Chemotherapy Female

Case History

A 64 year old female with metastatic left breast cancer, status-post chemotherapy, presented for erythema, discomfort, and oozing from her port site for approximately one month. At presentation she was afebrile. Her port site exhibited erythema and fluctuance. Her most recent absolute neutrophil count was 1910/cmm. Her port was removed, and a tissue specimen was sent for microbiologic examination.

Laboratory Identification

Gram stain showed neutrophils without bacteria. Aerobic cultures grew a beaded gram positive rod on blood agar at 36 hours. Kinyoun stain was positive for acid fast bacilli. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) at that time identified Mycobacterium fortuitum group.

Image 1. Growth on 7H10 agar.
Image 2. Kinyoun stain showing acid fast bacilli.

Discussion

M. fortuitum is a group of rapid growing mycobacteria. Within the group is M. conceptionense, M. houstonense, and M. senegalense. The group comprises the second most-commonly isolated rapidly growing mycobacterial respiratory isolates in patients (after M. abscessus), generally those with underlying lung disease. Progressive pulmonary disease is generally not seen.  It has also been associated with skin and soft tissue infections (SSTIs), surgical wound infections, lymphadenitis, and catheter-related infections. It is seen in the environment and represents a common contaminant. Identification is by culture and molecular techniques. It is susceptible to many antibiotics (typically aminoglycoside, cefoxitin, imipenem, or levofloxacin). Therapy includes two agents based on susceptibility testing for 6 to 12 months. This is somewhat controversial in pulmonary disease as the clinical significance is not clear.

This patient is being treated through a peripheral IV. The chest port site at two weeks showed dehiscence of the wound with drainage. Susceptibilities are pending.

References

  1. Park S, Suh GY, Chung MP, Kim H, Kwon OJ, Lee KS, Lee NY, and Koh WJ. Clinical significance of Mycobacterium fortuitum isolated from respiratory specimens. Respiratory Medicine. March 2008;102(3):437-442.
  2. Sethi S, Arora S, Gupta V, Kumar S. Cutaneous Mycobacterium fortuitum Infection: Successfully Treated with Amikacin and Ofloxacin Combination. Indian J Dermatol. 2014;59(4):383–384.

-Jonathan Wilcock, MD is a 1st year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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

Microbiology Case Study: An 18 Year Old with Fever, Chills and Abdominal Pain

Clinical History

An 18 year old male presented to the emergency department (ED) with fever, chills, and generalized lower abdominal pain. He noted the fever began 6 days ago and had been intermittent since that time. He also reported nausea and vomiting with a decrease in appetite. The patient was from India and was treated for malaria 8 months ago, directly prior to arrival in the United States. He stated he received three days of intravenous medications with resolution of symptoms. In the ED, his vitals were blood pressure 129/75, heart rate 133, temperature 104.1°F, respirations 20, and 99% oxygen saturation on room air. On physical exam, patient had mild jaundice and scleral icterus and severe right lower quadrant pain on palpation. CT scan of the abdomen showed mesenteric adenitis, but no appendicitis. Initial laboratory testing showed a mild anemia and thrombocytopenia (hemoglobin 12.1 g/dL, hematocrit 35.9%, platelets 78,000 TH/cm2) and increased indirect bilirubin (2.67 mg/dL). The patient received piperacillin-tazobactam while blood and urine cultures as well as a malaria smear were pending.  

Laboratory Identification

The BinaxNOW lateral flow immunochromatographic assay for Plasmodium spp. was performed.

Image 1. The BinaxNOW assay was positive for malaria protein antigen, representing P. vivax, P. ovale, P. malariae, or a mix of these species.
Image 2. A thin smear showed amoeboid gametocytes in enlarged red blood cells as compared to uninfected cells (Giemsa stain, 100x oil immersion).
Image 3. A thin smear showed very rare trophozoites with thick chromatin bands and single, large chromatin dots (Giemsa stain, 100x oil immersion).

The positive BinaxNOW results and morphologic findings on smear review were most consistent with a P. vivax infection. The level of parasitemia was approximately 0.2%. Blood and urine cultures were negative.

Discussion

Malaria classically presents with fever and chills, weakness, headache, myalgias, nausea, and vomiting in patients who live in tropical and subtropical regions. The four most common species that infect humans through transmission by the female Anopheles mosquito include P. falciparum, P. vivax, P. ovale, and P. malariae. If malaria is not diagnosed and treated in a timely manner, complications including anemia, thrombocytopenia, renal failure, acute respiratory distress syndrome (ARDS), and cerebral malaria can result. P. falciparum is the most deadly species due to the parasite’s ability to cause high levels of parasitemia.  

In laboratories in the United States, malaria testing often times incorporates Plasmodium spp. antigen detection via the BinaxNOW assay and peripheral blood smears. While the performance of the BinaxNOW is acceptable, particularly for P. falciparum, thick and thin peripheral blood smears remain the gold standard for malaria diagnosis, especially when the parasitemia level is low. The thick blood smear allows for screening a large amount of blood for malarial parasites and the thin smear allows for species identification and assessment of parasitemia. Ideally, multiple blood smears obtained from different times of the day should be collected in order to exclude the diagnosis. The window prior to a febrile spike is the best time to obtain the specimen, as the number of circulating parasites is greatest.

Clinically, the most important distinction is between P. falciparum and all other species. A number of features including the morphology of the trophozoites, schizonts, and gametocytes, size of the infected red cells, the presence of multiply infected red blood cells, and the region that the patient lives in or traveled to are helpful in determining species level identification.

P. vivax infects enlarged, young red blood cells and multiple trophozoites may be present in one red blood cell. The trophozoites have thick, blue cytoplasm and usually one, large chromatin dot. The schizont can contain 12 to 24 merozoites and the gametocyte is large and oval in shape. Schuffner’s stippling and malarial pigment are common. It is important to correctly identify P. vivax and P. ovale as they have hypnozoite forms in the liver and patients can relapse unless they are treated with an additional medication to eradicate these forms.

In the case of our patient, he received chloroquine, the treatment of choice for P. vivax arising in India. Primaquine and tafenoquine are both options for eradication of the hypnozoite form in the liver. These medications can cause hemolytic anemia in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency so quantification of the enzyme is required prior to administering therapy. Our patient had normal G6PD levels and received tafenoquine as well. 

-Karla Perrizo, MD, is a clinical pathology resident at the University of Mississippi Medical Center.

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

Microbiology Case Study: A 57 Year Old Man with Fever

Case History

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

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

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

Discussion

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

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

References

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

-Jonathan Wilcock, MD is a 1st year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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

Microbiology Case Study: An 83 Year Old Male with Fever

Case History

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

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

Laboratory Identification

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

Discussion

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

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

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

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

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

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

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

Microbiology Case Study: An 8 Year Old Male with Left Knee Pain

Case History

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

Laboratory Findings

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

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

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

Discussion

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

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

References

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

-Jonathan Wilcock, MD is a 1st year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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