Category: microbiology

Case History

A 5 month old girl was brought to her pediatrician by her mother for redness of the medial canthus of her left eye. There was some associated thick discharge as well. The mother mentioned that her daughter had excessive tearing from the left eye since birth. The pediatrician diagnosed dacryocystitis, and prescribed a course of oral cefdinir. However, the swelling and redness continued to worsen, and the infant began to have low-grade fevers. She was sent to pediatric ophthalmology for a consult, and admitted to the hospital on IV clindamycin. The following day, she was taken to the operating room, where they opened the obstructed nasolacrimal duct and sent the contents for culture.

Laboratory Work-Up

Gram stain and smear: No neutrophils, no bacteria seen.

Routine bacterial culture showed growth of small, smooth and translucent colonies on the chocolate agar only. The colonies had a distinct wet-mouse odor.

Repeat review of the gram stain showed moderate neutrophils, with gram negative coccibacilli both extracellular and intracellular.

Discussion

The gram stain and colony morphology described above are consistent with Haemophilus influenzae. Identification of the organism was confirmed by MALDI-TOF.

Congenital nasolacrimal duct obstruction is a common problem, affecting up to 6% of newborns. The vast majority of cases will resolve without treatment by the time the child is 6 months old. However, complications can arise, include acute or chronic dacryocystitis. Acute dacryocystitis causes swelling, warmth, and erythema with or without purulent discharge. The organisms most commonly implicated are alpha-hemolytic streptococci, Staphylococcus epidermidis, and Staphylococcus aureus. In chronic dacryocystitis, there is purulent drainage from the eye but no other signs or symptoms. The most common organisms isolated in these cases are Streptococcus pneumonia, Haemophilus influenzae, Pseudomonas aeruginosa, and viridans group Streptococci.  Acute dacryocystitis usually requires systemic antibiotics, while chronic can be treated with topical antibiotics. In this case, the infant had clinical features of acute dacryocystitis, but infection with an organism more typically associated with chronic dacryocystitis.

H. influenzae is a gram negative coccobacillus, which grows only on chocolate agar due to its requirement for factors V and X. However, H. influenzae can grow on blood agar if it is growing around an organism that hemolyzes the red blood cells in the media, releasing factor V (i.e. Staphylococcus aureus), a phenomenon known as satelliting.

As illustrated by this case, reviewing gram smears inconsistent with the final culture is an important aspect of quality assurance in the microbiology laboratory. This allows the opportunity to provide continuous feedback to technologists on their technique, and lets us keep track of any trends or common mistakes that may be occurring. In this instance, on review of the gram smear it appears the original reader examined only the very thick portions of the smear, which were uninterpretable. However, by moving out to the edges, neutrophils and bacteria were clearly visible. Feedback was provided to the original reader. The provider was immediately called and notified that a corrected report would be issued; the patient was switched to oral Augmentin based on these results.

-Alison Krywanczyk, MD is a 3^rd 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 Assistant Professor at the University of Vermont.

Case History

A 29 year old G2P1 woman presented in labor at 39+2 weeks gestational age. Her pregnancy had been previously uncomplicated. Prenatal infectious disease testing showed that she was negative for HIV and Hepatitis C, but that she was positive for Group B Streptococcus. No test results were available for rubella, VZV, toxoplasmosis, or syphilis.

A term male infant was born shortly afterwards by spontaneous vaginal delivery; the mother received less than 4 hours of antibiotics.  The baby was noted to be covered in petechiae, and in a moderate amount of respiratory distress. A CBC showed thrombocytopenia to 23 K/cmm. The baby was transported emergently to the neonatal intensive care unit, where platelet transfusions were given. Blood cultures were drawn. The baby was started empirically on ampicillin/gentamycin, and the following day, once platelet counts were improved, a lumbar puncture was performed. The cell counts in the CSF were unremarkable. A cranial ultrasound showed scattered bilateral parenchymal calcifications, mineralized vasculature of the lenticulostriate arteries, and a subependymal cyst. Urine PCR testing was positive for CMV.

Laboratory Work-up

• Bacterial Culture and Smear, CSF: No neutrophils, no bacteria. No growth.
• CSF Viral PCR: Negative for HSV and VZV.
• Urine CMV PCR: Positive.
• The placenta was not sent for pathologic examination.

Cranial ultrasound demonstrating scattered parenchymal calcifications.

 

Discussion

Cytomegalovirus is one of the classic “TORCH” infections. TORCH is an acronym for a group of pathogens that can cause in-utero or intrapartum infections:

•                 T= Toxoplasmosis
•                 O= other (syphilis, VZV, parvovirus)
•                 R= rubella
•                 C= CMV
•                 H= HSV

Although these infections share several common signs and symptoms, there are clinically suggestive findings that can help target testing. The combination of thrombocytopenia and intracranial calcifications in this infant raised strong suspicion for congenital CMV. CMV is a member of the herpesvirus family.  It is a double-stranded DNA virus with both a viral capsid and envelope. While most babies born with congenital CMV are asymptomatic (~90%), congenital CMV infection is the main etiology of non-hereditary sensorineural hearing loss. This occurs in up to 50% of symptomatic infants and in 10-15% of asymptomatic infants. Symptomatic infants may be small for gestational age, and can be afflicted by thrombocytopenia, petechiae, intracranial calcifications, chorioretinitis, hepatosplenomegaly, microcephaly, and jaundice. While toxoplasmosis can also cause intracranial calcifications, it does not typically cause thrombocytopenia. Congenital HSV can cause thrombocytopenia, but is not associated with intracranial calcifications.

CMV infection during pregnancy is most often acquired by contact with young children. CMV has the ability to remain latent in the host, and become reactivated at a later time, so pregnancies can be affected by either primary infection or by reactivation of the virus. The risk of vertical transmission is much higher with primary CMV infection (32%) than with recurrent infection (1.4%). Although the rate of vertical transmission increases if the infection occurs later in pregnancy, infections acquired in early pregnancy are more likely to cause symptomatic disease. Treatment for the baby is generally supportive, with antivirals generally used only in symptomatic disease (their utility in asymptomatic infection is debated).

An audiology screen and ophthalmologic exam were both normal in the infant presented here. Oral valgancyclovir was started in addition to other supportive measures.

 

-Alison Krywanczyk, MD is a 3^rd 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 Assistant Professor at the University of Vermont.

Case History

After experiencing increasingly frequent asthma attacks and multiple episodes of pneumonia within the last two years, a 60 year-old woman with a longstanding history of allergic asthma presents to a pulmonologist complaining of increased shortness of breath and cough. The patient reports a history of abnormal lung infiltrates for which she was previously treated with a three month course of azithromycin. A repeat chest CT shows diffusely scattered, nodular, ground-glass opacities which have increased in number since her last CT two year prior. A bronchoalveolar lavage is performed and specimen is sent for bacterial, fungal, and AFB cultures as well as a respiratory virus PCR panel.

 

Laboratory Identification

The bacterial and fungal cultures do not grow any pathogens and the respiratory virus panel is negative. The AFB culture, however, grows beaded, Gram-Positive bacilli which are Auramine/Rhodamine negative and Modified Kinyoun positive. The organism grows well on 7H11, Chocolate, and Buffered Charcoal Yeast Extract (BCYE) agars forming irregular, chalky, white-pink colonies.

The organism is confirmed as Nocardia nova by molecular methods.

Discussion

Nocardia nova is a ubiquitous soil bacteria and one of several Nocardia species known to cause disease in humans. When contracted through traumatic inoculation, Nocardia may cause cutaneous diseases such as a mycetoma, superficial abscesses, or cellulitis. More commonly, however, Nocardia is contracted via inhalation and presents as a chronic, slowly progressive pulmonary infection with cough, shortness of breath, and fever. Complicated pulmonary infections may result in pleural effusions, empyema, pericarditis, chest wall abscesses, or dissemination to the brain and other deep organs. Due to low virulence, Nocardia primarily affects only the immunocompromised but those with pre-existing pulmonary disease are also susceptible to infection.

Nocardia is identified in the laboratory as an aerobic filamentous, beaded, Gram-Positive bacilli demonstrating right-angled branching. Nocardia is also weakly acid-fast and is usually identified by a Modified Kinyoun stain. While Nocardia grows within 3-5 days on blood and chocolate agar, it is often isolated on mycobacterial media or BCYE plates where it forms chalky white to faintly pigmented colonies. Accurate identification and speciation of Nocardia currently requires the use of molecular methods (primarily 16S ribosomal RNA gene sequencing). While many infections are successfully treated with a sulfonamide for 6 months to 1 year, the CDC recommends performing speciation and anti-microbial susceptibility testing on every clinical isolate due to species specific susceptibility profiles and multi-drug resistant strains. Nocardia farcinica, for example, is resistant to many antibiotics including 3rd generation cephalosporins.

 

-Elaine Amoresano, MD, is a 2^nd 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 Assistant Professor at the University of Vermont.

 

Case presentation

A 12-year-old female is seen in gastroenterology clinic following 2 weeks of abdominal pain. She is an otherwise healthy child with no significant past medical history. Her abdominal pain was diffuse, but it has worsened in the past 5 days and is now localized to the left upper quadrant and is sharp in nature. The pain was severe enough to prevent her from attending school last week. She was evaluated for appendicitis, which was ruled out. The patient was admitted for further management including an upper and lower endoscopy. During the endoscopy procedure, small, mobile worms were visualized in the ascending colon. Two worms were collected and removed for identification (Figure 1).

Figure 1. (A) Small, threadlike worms measuring 5-10 mm. Note pointed posterior tail. (B) Haematoxylin and eosin (H&E) stained cross section of the worm.

Figure 2. Cross section of the worms shows (A) anterior with cephalic inflations of the cuticle (arrows) and (B) a long pointed tail.

Figure 3. Internal eggs are 50-60 µm x 20-30 µm in size. They are elongated, flattened on one side, and have a thick colorless shell.

 

Discussion

The worms were identified as Enterobius vermicularis or pinworm. E. vermicularis is a nematode or round worm. Adult worms are 2.5 mm x 0.1 mm (males) and 8-13mm x 0.3-0.5mm (females). Both male and female worms have cephalic inflations of the cuticle at their anterior end (Figure 2A, arrows). Males have a wide blunt posterior tail while females have a long, pointed tail (Figure 1, 2B). Our worms were females as internal eggs were found in both worms. The eggs of E. vermicularis are 50-60 µm x 20-30 µm in size. They are elongated, flattened on one side, and have a thick colorless shell (Figure 3).

E. vermicularis infection is very common in preschool and school aged children as well as families and caregivers of infected children. Transmission occurs through the fecal-oral route. Embryonated eggs are ingested and travel to the small intestine. Adult worms reside in the colon. Gravid females migrate to the anus and deposit eggs onto the perianal area during the night. A single female can deposit as many as 10,000 fertilized eggs. Larvae within the eggs develop and become infective as quickly as 4-6 hours after they are deposited. The entire life cycle from ingestion until eggs are laid by a gravid female in the perianal area is 1-2 months. Perianal scratching and autoinfection are common as well as infection from contaminated fomites such as bedding, clothes, and shared toys.

The most common method of E. vermicularis detection is the Tape Prep method. Briefly, transparent (unfrosted) tape is used to touch the perianal region, after which the tape is placed on a glass slide for microscopic examination. The best time to detect E. vermicularis is 2-3 hours after the patient has gone to sleep due to the nocturnal cycle of the gravid females. Because E. vermicularis does not enter the stool stream, ova and parasite examination often fails to detect the parasite and is not recommended.

Discovery of E. vermicularis in our patient was an unexpected finding, as our patient had no perianal itching. Asymptomatic detection of E. vermicularis has been described in the past, so this finding is not unique. The patient was given a dose of albendazole and will receive another in two weeks, as the drug has reduced effectiveness at killing the eggs or larval stages of development. Her abdominal pain was attributed to overuse of nonsteroidal anti-inflammatory drugs (NSAIDS) and she is being monitored by gastroenterology outpatient clinic.

 

References

1. Ash and Orihel’s Atlas of Human Parasitology, 5^th
2. Red Book 2015 Report of the Committee on Infectious Diseases, 30^th

 

I would like to thank the staff of the Children’s Medical Center Histology Laboratory for sharing my enthusiasm for parasites as well as sectioning, staining, and taking images of the worms for educational purposes.

 

-Erin McElvania TeKippe, PhD, 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.

 

 

 

 

Case History

A 37 year old Indian male presents to the emergency department with complaints of a cyclic fever (102-103°F), chills, fatigue and a severe headache. He denies nausea, vomiting or diarrheal symptoms. His travel history is significant for a recent 2 month vacation to India and he is concerned he has malaria, given his symptoms. He reports no sick contacts or suspicious ingestions during his trip and his other family members are well. On physical exam, he is ill appearing, with dry mucous membranes but has no aversion to light or neck pain. Blood work revealed a normal white blood cell count and elevated liver enzymes (ALT 383 U/L, AST 282 U/L). Blood and CSF were collected and sent to the microbiology laboratory for Gram stain and culture. Additional tests for influenza, viral hepatitis and malaria were performed.

Laboratory Identification

Figure 1. Gram stain from a positive blood culture illustrating large Gram negative rods (100x, oil immersion).

Figure 2. Small, lactose negative colonies growing on MacConkey agar after 24 hours incubation in CO₂ at 35°C.

Figure 3. Green colonies growing on Hektoen enteric agar after 24 hours incubation in O₂ at 35°C.

Within a day of collection, multiple blood cultures were positive for a large Gram negative rods (Figure 1). The organism grew after 24 hours incubation in CO₂ at 35°C on blood, chocolate and MacConkey agars and was lactose negative (Figure 2). The identification by MALDI-TOF was Salmonella group. The isolate was then sent to the public health department for additional testing via molecular typing methods. The diagnosis of Salmonella serotype Typhi was confirmed. Testing for influenza, viral hepatitis and malaria were all negative.

Discussion

Salmonella serotype Typhi is a motile, Gram negative rod that is a member of Enterobacteriaceae family.  Typhoid fever is the cause of serious bloodstream infections in developing countries and the great majority of cases in the United States are identified after recent travel. Clinically, it presents with high fever and headaches, in the absence of gastrointestinal manifestations and causes a more severe illness than other Salmonella serotypes. The infecting organism, of which humans are the only known reservoir, is transmitted by food or drink contaminated with feces or person to person contact and has a low infectious dose. Healthy carriers that are able to asymptomatically shed the bacteria have been documented.

Salmonella serotype Typhi is more commonly isolated from blood rather than fecal specimens and grows well on a variety of media including blood, chocolate and Hektoen enteric agar (Figure 3). The characteristic reaction on a triple sugar iron (TSI) slate is alkaline/acid (K/A, only glucose fermented) with a small moustache of H₂S production at the site of inoculation and no gas production. In addition, a positive lysine decarboxylase reaction helps to distinguish Typhi from non-typhoidal Salmonella subspecies I members. MALDI-TOF mass spectrometry is successfully able to identify the isolate as Salmonella spp., but additional testing must be performed to determine the particular serotypes.

Traditionally, serotyping of the O (somatic), H (flagellar) and Vi (capsular) antigens and applying results to the Kauffmann-White scheme is useful in confirming the diagnosis of Salmonella and defining the serotype name. In the case of Salmonella serotype Typhi, the somatic antigen groups as D1 and the Vi antigen is present. The Vi antigen (heat labile) sometimes masks the identity of the O antigen (heat stable). In these cases, heating the bacterial suspension in boiling water for 15 minutes and repeating the O antigen serotyping yields the correct O antigen. Given the expansion in molecular testing, methods based on identifying the genes responsible for the serotype are gaining favor.

Due to the high mortality rate in untreated cases of typhoid fever, treatment with antibiotics is necessary and given the increasing levels of resistance reported, particularly to ciprofloxacin, susceptibility testing should be performed in all cases of Salmonella serotype Typhi. It is recommended that ampicillin, a fluoroquinolone, trimethoprim-sulfamethoxazole and a 3^rd generation cephalosporin be reported for all typhoid isolates based on the most current M100S-26 CLSI guidelines. Also, in the case of S. Typhi, reporting susceptibilities to azithromycin is encouraged (MIC ≤16 µg/mL S, ≥32 µg/mL R, interpretative criteria based on MIC distribution data).

In the case of our patient, he was started on ceftriaxone and continued to receive this IV antibiotic for seven days after blood cultures became negative. Susceptibility testing showed intermediate results for ciprofloxacin by disk diffusion and azithromycin was found to be susceptible with an MIC of ≤16 µg/mL. The patient has an uncomplicated hospital course and made a complete recovery.

 

-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. Currently, she oversees testing performed in both the Chemistry and Microbiology Laboratories.  Her interests include infectious disease histology, process and quality improvement and resident education.