Microbiology Case Study: 58 Year Old Man with Fatigue and Chills

Case History:

Two weeks after returning from a camping vacation in Cape Cod, a 58 year old man presented to the emergency room with six days of fatigue, fever, chills, arthralgia, myalgia, mild right upper quadrant pain, and a frontal headache. Clinical workup revealed worsening leukopenia, thrombocytopenia, and elevated transaminases when compared to preliminary testing done by the patient’s primary care provider at the onset of his symptoms. His preliminary workup was also negative for Lyme antibody, EBV and CMV IgM, and viral hepatitis markers. At no point did the patient notice a skin rash or a tick anywhere on his person.

Differential Diagnosis:

  • Lyme Disease
  • Anaplasmosis
  • Ehrlichiosis
  • Babesiosis
  • Rocky Mountain Spotted Fever
  • Viral Meningitis
  • Bacterial Meningitis
  • Leptospirosis
Blood smear showing granulocyte with intracytoplasmic morulae.
Blood smear showing granulocyte with intracytoplasmic morulae.
Blood smear showing granulocyte with intracytoplasmic morulae.
Blood smear showing granulocyte with intracytoplasmic morulae.

Laboratory Identification:

Anaplasma phagocytophilium was initially identified by PCR. Retrospectively, the blood smears originally examined for Babesia by both hematology and parasitology were reviewed. Both slides showed multiple granulocytes with intracytoplasmic morulae.

 

Discussion

Anaplasma phagocytophilium is the bacterium responsible for the tick-borne disease known as human granulocytic anaplasmosis. Anaplasma is transmitted to humans primarily through the bite of an infected Ixodes scapularis, the same species of tick which transmits Borrelia burgdorferi (Lyme disease) and Babesia spp. (human babesiosis). Anaplasmosis, Lyme disease, and babesiosis therefore share roughly the same geographical distribution in the United States with northeastern and upper midwestern states reporting the most cases.

Anaplasmosis most commonly presents about 1-2 weeks after a tick bite with the sudden onset of a variety of non-specific symptoms including fever, chills, headache, malaise, myalgia, nausea, and abdominal pain. Anaplasmosis, unlike other tick-borne diseases, rarely causes a rash. Routine blood tests may show thrombocytopenia, leukopenia, or elevated liver enzymes in some patients. Severe clinical presentations, more common in immunosuppressed patients, may include difficulty breathing, hemorrhage, renal failure or neurological problems. Anaplasmosis is estimated to be fatal in less than 1% of cases.

A routine blood smear is the quickest method for establishing an early presumptive diagnosis. Microscopic examination of the smear may reveal microcolonies of Anaplasma known as morulae within the cytoplasm of infected granulocytes. Ehrlichia, in contrast, will preferentially target and form morulae within monocytes. Because not all patients with anaplasmosis have visible morulae, this test is diagnostically insensitive and should be followed by further testing.

Confirmatory serologic testing for anaplasmosis includes an indirect immunofluorescence assay using an Anaplasma phagocytophilum antigen. For the highest sensitivity, this test should be performed on paired serum samples collected at least 2 weeks apart with the first sample taken as early in the disease as possible. A positive test will demonstrate a four-fold rise in antibody titers. Although it is a very sensitive detection method when run with paired samples, the lengthy testing time is less than ideal for patients requiring hospitalization for their disease.

A PCR assay on a sample of whole blood, although only available at a few reference laboratories, is the most efficient and accurate way to detect Anaplasma during the acute phase of the illness. The sample used for PCR testing should be taken before the initiation of antibiotic therapy as it causes the sensitivity of this test to rapidly decline.

Doxycycline is the first line treatment for adults and children of all ages with anaplasmosis as recommended by both the CDC and the AAP Committee on Infectious Diseases. Patients should be treated for at least 3 days after the fever subsides. Standard duration of treatment is 7 to 14 days. Therapy should be initiated immediately when there is a high clinical suspicion of anaplasmosis. A physician should never wait for the results of confirmatory testing to begin treatment. Most patients see improvement within 24-48 hours of treatment and non-response to doxycycline may indicate a different disease process.

Anaplasmosis, like many other tick-borne diseases, is a nationally reportable disease. All cases should be reported to local and state health departments as well as the CDC.

-Elaine Amoresano, MD, is a 1st 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: 29 Year Old in Preterm Labor at 30 Weeks Gestation

Case History:
A 29 year old woman presents to the hospital with contractions at 30 weeks gestation. This is her first pregnancy and it was previously uncomplicated. She did not experience loss of fluid or vaginal bleeding and did not have a history of recent illness or fever. A swab for group B Streptococcus (GBS) was collected and the patient was started on prophylactic penicillin. Clinical evaluation revealed evidence of acute infection with an elevated C-reactive protein and an increased white blood cell count with 97% neutrophils. Amniocentesis was performed and the amniotic fluid was sent to the laboratory for Gram Stain and culture.

Labor was allowed to progress and the infant was delivered vaginally. Cultures of cerebrospinal fluid and blood from the neonate were negative. The placenta was sent for histologic evaluation.

Gram stain shows Gram negative bacilli.
Gram stain shows Gram negative bacilli.
Tiny gray colonies on blood agar with a bleach-like odor.
Tiny gray colonies on blood agar with a bleach-like odor.
Small, pale yellow colonies on chocolate agar.
Small, pale yellow colonies on chocolate agar.

Laboratory Identification:
The laboratory workup revealed a gram negative bacillus with rounded ends that grew small grey to pale yellow colonies on blood and chocolate agars. The colonies had three regions; a raised central region, a refractile flat region, and an outer rougher spreading region. The colonies had a distinct bleach-like smell. There was no growth on MacConkey agar. The organisms were oxidase positive, catalase and indole negative. Mass spectrometry was utilized to identify the organism as Eikenella corrodens.

Discussion:
Eikenella corrodens is a component of normal mouth and upper respiratory tract flora. It is most notable for causing head and neck infections, periodontal disease, and as a significant player in “fight bite” infections. “Fight bite” results when a clenched fist hits another person’s mouth and the teeth cause lacerations to the hitter’s hand, which can subsequently lead to infection. Eikenella is implicated approximately 25% of the time in these types of infections. Only on very rare occasion is Eikenella known to cause gynecologic infections. Endometritis or cervicitis may infrequently be caused by colonization of an intrauterine contraceptive device (IUD) by Eikenella. And rarely, Eikenella is implicated as the isolated bacteria in cases of acute chorioamnionitis.

In the medical literature there are currently only 8 reported cases of chorioamnionitis caused by a pure Eikenella infection. As in our case, each of the women in the case reports had clinically silent infections and only presented with preterm labor. Most of the women were found to have elevated white blood cell counts in the absence of fever or alterations in other vital signs. In each case, the fetal membranes were intact. Two of the cases resulted in fatal infection of the neonates. Of note, three of the women were mentioned to be the recipients of oral intercourse throughout their pregnancies.

One of the reported cases involved a woman whose partner had a tongue piercing and it was noted that they engaged in daily oral sex during the pregnancy.  The authors speculated that the tongue piercing played a role in the development of chorioamnionitis by either ascending vaginal infection or hematogenous spread caused by trauma from the tongue ring.

It is not known if a similar history was present in this case. The patient was treated with ampicillin and gentamycin and discharged following delivery. She is currently doing well. The infant has had no signs of infection, but at the time of this writing he is being treated in the neonatal intensive care unit for sequelae of prematurity.

References:
Garnier F, Masson G, Bedu A, et al. Maternofetal infections due to Eikenella corrodens. J Med Microbiol 2009; 58, 273-275.

Jadhav A, Belfort M, Dildy G. Eikenella corrodens chorioamnionitis: modes of infection? Am J Obstet Gynecol 2009; 200, e4-5.

-Britni Bryant, MD 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 Two-Week-Old Infant with Fever and Fussiness

A two week old infant presents to the ED with fever and fussiness. She was born at 37 weeks gestation after an uncomplicated vaginal birth. Her mother reports she was in good health during her pregnancy and there are no family sick contacts since the infant was brought home from the hospital. In the ED, the infant was febrile and had a bulging fontanel. Blood and CSF were sent to the microbiology laboratory for culture. The following organism was isolated from blood and CSF specimens.

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Gram stain of positive blood culture showing short, Gram-positive bacilli

 

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Organism growing on 5% sheep blood agar plate exhibiting a narrow zone of beta-hemolysis after 48 hours of incubation

 

The organism isolated was Listeria monocytogenes.

L. monocytogenes is a short, Gram-positive bacilli on Gram stain (Image 1). In the microbiology laboratory, L. monocytogenes grows on 5% sheep blood, chocolate, and colistin naladixic acid (CNA) agars. The bacteria form small colonies of 1-2 mm after 24 hours of incubation at 35-37⁰C. On sheep blood agar these colonies have a narrow zone of b-hemolysis, which is very subtle. To highlight the hemolysis, the blood agar plate must be held up to a light source (Image 2) or colonies can be removed to reveal hemolysis of the agar directly below the growing bacterial colony. L. monocytogenes is catalase, Voges-Proskauer, and methyl red positive but oxidase and urea negative. It is also CAMP test positive using a S. aureus streak and has tumbling leaf motility. L. monocytogenes is routinely identified in our laboratory by MALDI-TOF MS. In addition, many rapid diagnostic panels such as the Nanosphere Verigene Gram-positive Blood Culture Assay (Northbrook, IL, USA) and Biofire FilmArray Blood Culture Identification Panel (Salt Lake City, UT, USA) include a target for rapid identification of Listeria spp./L. monocytogenes from positive blood culture.

L. monocytogenes can grow at a wide range of temperatures between 0-50⁰C. Its ability to grow at 4⁰C allows the organism to persist and replicate under refrigerated conditions on food products such as meat, vegetables, raw milk, and cheeses. For the same reason, L. monocytogenes is also a concern for contamination of refrigerated blood products.

It is estimated that 1-5% of healthy adults are asymptomatically colonized with L. monocytogenes. The organism is most often contracted by consuming contaminated foods, which causes a mild gastrointestinal illness in otherwise healthy hosts. L. monocytogenes infection in pregnant women can present as a mild, self-limited, influenza-like illness Infection and 1/3 of women report no symptoms at all. The bacteria from infected mothers are able to cross the placenta, resulting in transmission to the fetus in utero or infants can be infected during the birthing process.

L. monocytogenes infections that occur within the first 7 days of life are characterized as early-onset. Patients often present with symptoms of preterm birth, pneumonia, and sepsis. Patients may also have an erythematous rash with small papules histologically described as “granulomatosis infantisepticum.” Late-onset infection occurs between 8-30 days of life with patients most often presenting with sepsis and meningitis. Both neonatal presentations are reported to have a high mortality rate—14-56% for early-onset and 25% for late-onset infection. Luckily, invasive neonatal L. monocytogenes infections have been declining over the past decades and are now a rare occurrence in newborns. These days, invasive listeriosis is more common in immunocompromised patients with defects in cell-mediated immunity. In 2013, the last year for which there is data, the CDC reported 633 cases of invasive listeriosis, but only 68 (11%) were pregnancy-associated. These infants had a 76% survival rate. More information about Listeria epidemiology in pregnant and non-pregnant populations can be found at the CDC website http://www.cdc.gov/listeria/pdf/listeria-annual-summary-2013-508c.pdf.

L. monocytogenes is intrinsically resistant to cephalosporins. Ampicillin is considered the “gold standard” for treatment. The addition of an aminoglycoside for synergy is often used in practice, but a retrospective cohort study showed it had no effect on reducing infant mortality. Trimethoprim-sulfamethoxazole, quinolones, or vancomycin can be used to treat penicillin allergic patients. Newer Gram-positive antibiotics including linezolid, daptomycin, and tigecycline have also shown clinical efficacy against L. monocytogenes infections.

 References:

Manual of Clinical Microbiology, 11th edition

Pediatric Red Book, 2015 Report of the Committee on Infectious Diseases, 30th edition

Centers for Disease Control and Prevention listeriosis website http://www.cdc.gov/listeria/

 

-Erin McElvania TeKippe, Ph.D., D(ABMM), is the Director of Clinical Microbiology at Children’s Medical Center in Dallas Texas and an Assistant Professor of Pathology and Pediatrics at University of Texas Southwestern Medical Center.

Microbiology Case Study: 64 Year Old Male with Swollen Finger

A 64 year old male presented with one week of swelling in his right 4th finger. He was initially treated for a suspected bacterial infection, but did not respond to treatment and the finger was aspirated. The specimen was positive for fungal organisms.

Fungal plates grew the following:

Note the flat powdery/velvety colony growth.  It is common for the colonies to have a purple or lavender color with a white border.
Note the flat powdery/velvety colony growth. It is common for the colonies to have a purple or lavender color with a white border.

Scotch tape prep revealed the following morphology:

Note that the elongated Phialides, and taper to a long slender tube, resembling bowling pins.  Sometimes this morphology has also been referred to as "skeleton hands."
Note that the elongated phialides, and taper to a long slender tube, resembling bowling pins. Sometimes this morphology has also been referred to as “skeleton hands.”

Discussion:
Purpureocillium lilacinum (formerly Paecilomyces lilacinus) is a fungus that is found ubiquitously within our environment, but has rarely been associated with disease in humans. A review paper in 2004 found 119 reported cases that implicated P. lilacinum from 1964 to 2004. It has been most commonly associated with ocular infections, often linked to intraocular lens implantations. There is scarce data concerning its susceptibility. In the event of a cutaneous infection such as the one presented in this case, it is recommended that posaconazole be used as first line therapy. In the event of treatment failure, or intolerance, there is little data about which antifungals to treat with, though voriconazole has been shown to have successful in-vitro.

The patient in this scenario had a history of undifferentiated spondylarthropathy, and was on methotrexate for a monoarthropathy in same finger in which the fungal growth occurred. He also received a cortisone injection into the joint adjacent to, but not directly into the site of the infection approximately a month prior to presentation. It could be possible that the cortisone injection had allowed the fungus to be inoculated into the finger, but we may never be certain. The patient also was gardening prior to his infection, and that could have also possibly contributed to his fungal infection.

P. lilacinum is found readily in the environment, and should be considered on the differential of cutaneous infections. Though immunocompromised patients have historically been more susceptible, it has been reported in immunocompetent individuals and should be considered, especially in the event of failure of response to antibiotic treatment. More research needs to be done to better understand treatment regimens for this organism, though this is difficult as it has been difficult to test in animal models.

Reference:

Clinical manifestations, treatment and outcome of Paecilomyces lilacinus infections. F.J. Pastor and J. Guarro. Volume 12, Issue 10, pages 948–960, October 2006 DOI: 10.1111/j.1469-0691.2006.01481.x

-Rich Smith is a Pathology Student Fellow at 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 Male with Dysuria

A 22 year old male, treated for gonorrhea one month ago, presents with two days of dysuria and white penile discharge. He reported complete resolution of his symptoms with treatment. He has had sexual contact with 2 partners in the past month.

Cultures are taken and the following is seen on gram stain.

N. gonorrhoeae is a gram negative diplococci responsible for the disease of gonorrhea. Though most specimens for gonorrhea are received from sources suspected of having the disease, it should be noted that whenever dealing with a gram negative diplococci of unknown source, care should be taken, and the specimen should be handled under a hood for possible exposure to N. meningitidis.
N. gonorrhoeae is a gram negative diplococci responsible for the disease of gonorrhea. Though most specimens for gonorrhea are received from sources suspected of having the disease, it should be noted that whenever dealing with a gram negative diplococci of unknown source, care should be taken, and the specimen should be handled under a hood for possible exposure to N. meningitidis.

Seems like a clear cut case of gonorrhea, but given the patient’s history is this treatment failure, or likely reinfection?

The CDC has warned that rates of antibiotic resistant gonorrhea are on the rise. A once easily curable disease, is now becoming resistant to our mainstay treatment of ceftriaxone and azithromycin. Although cases have been reported, the majority of ‘failure of treatment’ scenarios are likely re-infection. Patients likely do not abstain until they are symptom free, or the partner may not be treated, leading to a reinfection of the patient. In the event of treatment failure, the CDC even recommends re-treating with the same ceftriaxone/azithromycin regiment. It is still recommended that a sample be taken for culture in the event that there is any concern over failure of treatment and possible antibiotic resistance.

Historically, testing for N. gonorrhoeae consisted of culture and gram stain, but the CDC now recommends testing for gonorrhea with nucleic acid amplification testing (NAAT). This test has improved sensitivity, less subjectivity and a much faster turnaround time than gram staining and culturing. It cannot, however, be used for test of cure, as the NAAT tests for DNA, which still may be present, even if the organisms have been killed within the host, leading to a false positive result.

When a clinician suspects gonorrhea, he or she should take care with what site the specimen is being collected from, and what sample is specifically needed. This lab is validated for only certain types of specimens, and it is important to communicate with the clinicians to ensure the proper specimens are being submitted.

The CDC states that the optimal samples should be vaginal swabs for women, and first catch urine for men. Great care should be used when collecting the ‘first catch urine’ as this is a dirty catch, and that some tests have specific requirements about quantity. For instance, at this lab, we require no more than 30 ml of urine. Any more volume could dilute the specimen which could cause a false negative result.

If a culture is required in the event of treatment failure, it is best if the clinician innoculates the sample directly onto Thayer Martin agar (or chocolate agar if that is all that is available). Given that N. gonorrhoeae is such a labile organism, it need special media to grow, and great care must be taken with transport. This lab recomends inoculating onto the Jembec plates (Thayer Martin media with a CO2 pellet) allowing survival during transport.

N. gonorrhoeae inoculated directley onto the Jembec plate
N. gonorrhoeae inoculated directly onto the Jembec plate
Cultures can also be innoculated onto chocolate agar. Notice the smaller grey colonies, which is the N. gonorrhoeae.  Notice the other colonies on the plate, as chocolate agar is not a selective medium.
Cultures can also be innoculated onto chocolate agar. Notice the smaller grey colonies, which is the N. gonorrhoeae. Notice the other colonies on the plate, as chocolate agar is not a selective medium.

In conclusion, although the majority of cases of gonorrhea treatment failure are likely to be due to re-infection, antibiotic resistance is still of growing concern and clinicians should know when a culture is necessary for antibiotic sensitivity testing. N. gonorrhoeae requires nutrient supplementation for growth, either on Thayer Martin media, or chocolate agar. Care must be taken when sampling and transporting the specimen. Clinicians also need to be aware of the requirements for sample collection for NAATs, as it is not always clear as to why one can’t simply run a test on a sample greater than 30 ml, or a clean catch urine.

-Rich Smith is a Pathology Student Fellow at 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 Make the Diagnosis: A 68 Year Old with Epigastric Pain

A 68 year old male presents with chronic, gnawing epigastric pain which has been getting worse over the past year. An endoscopy is performed and a representative gastric biopsy section, stained with Giemsa stain, is shown here. What organism is responsible for this patient’s symptoms?

gastric biopsy Giemsa stain
A. Candida albicans
B. Helicobacter pylori
C. Bacillus cereus
D. Staphylococcus aureus
E. Campylobacter jejunum

The diagnosis in this case is Helicobacter pylori. Described by Warren and Marshall in 1983, Helicobacter pylori is now known to be the cause of most cases of chronic gastritis and peptic ulcer. Helicobacter is a tiny, corkscrew-shaped bacillus, and is easily missed on routine H and E-stained histologic sections. Giemsa staining, as seen in the image above, can be helpful, as can silver staining, as seen in this image:

H. pylori

Helicobacter does not invade the gastric mucosa, but produces its symptoms through continual stimulation of the host immune response. Treatment involves triple therapy (e.g., omeprazole, amoxicillin and clarithromycin), and prognosis is excellent. A small number of patients, however, develop gastric adenocarcinoma or MALT lymphoma.

Krafts

-Kristine Krafts, MD, is an Assistant Professor of Pathology at the University of Minnesota School of Medicine and School of Dentistry and the founder of the educational website Pathology Student.

Microbiology Case Study: Immunocompromised Boy with Skin Nodules

An elementary school aged boy with a history of pre-B cell acute lymphocytic leukemia with a failed bone marrow transplant was transferred to a regional children’s hospital for leukodepletion and participation in an experimental clinical trial. At that time, his CBC was significant for 10% polymorphonuclear cells and 50% blasts. He was subsequently transferred to the ICU in respiratory failure and developed papulonecrotic lesions on his face, trunk, and bilateral legs. Prior to this, he was pancytopenic with no blasts present with cell counts of 100 WBC, hemoglobin 8.3 and 37,000 platelets. His Fungitell assay, which detects (1-3)-β-d-glucan, was positive.

Routine blood culture, fungal culture from the endotracheal tube, and fungal culture from the skin lesion biopsy specimens all had fungal elements on KOH stain. Young growth of a whitish, fluffy mold was present on all cultures within two days. Histopathology on the punch biopsy of a skin lesion on the thigh showed septate hyphae within the dermis, epidermis, and invading the vasculature that was particularly apparent with GMS stain (Figure 1a and 1b). Within a few days, the fungal cultures showed septate hyphae with microconidia using lactophenol cotton blue tape preparation, and shortly thereafter the mold developed into macroconidia with multiple septations taking on canoe-like forms (Figure 2). The white, cotton-like colonies developed a pink tinge (Figure 3). These characteristics allowed for the identification of the growth as Fusarium sp.

Septate hyphae on GMS stained section of the skin punch biopsy.
Septate hyphae on GMS stained section of the skin punch biopsy.
Septate hyphae on GMS stained section of the skin punch biopsy.
Septate hyphae on GMS stained section of the skin punch biopsy.
Microscopic identification of Fusarium by lactophenol cotton blue stain.
Microscopic identification of Fusarium by lactophenol cotton blue stain.
Colony of Fusarium growing on inhibitory mold agar (IMA).
Colony of Fusarium growing on inhibitory mold agar (IMA).

Fusarium is an opportunistic hyaline mold with infection most commonly seen in immunocompromised hosts. It can cause keratitis through contamination of contact lenses, penetration due to trauma, or use of immunosuppressive steroid ophthalmic solution. It is increasingly becoming the cause of disseminated infection in neutropenic hosts with a broader spectrum of disease, which includes: skin lesions, fungemia, rhinocerebral involvement and pneumonia. In these cases, without an immune system to fight the infection, mortality is high. Inhalation of airborne conidia, ingestion from water sources or access through mucosal membranes are all potential points of entry.

The colony growth on plated fungal media is rapid, usually maturing within four days. On microscopic examination, Fusarium hyphae are septate, approximately 3-6 microns wide with acute angle branching. Microconidia are small, oval-shaped, and no larger than 4 x 8 microns in size. These can look like Acremonium sp. Macroconidia are canoe- or sickle-shaped with the largest dimension being about 80 microns in length, exhibiting 3-5 septatations.

 

Jodi Music, MD, is an AP/CP resident at UT Southwestern Medical Center.

-Erin McElvania TeKippe, Ph.D., D(ABMM), is the Director of Clinical Microbiology at Children’s Medical Center in Dallas Texas and an Assistant Professor of Pathology and Pediatrics at University of Texas Southwestern Medical Center.