Tag: pediatrics

Case History

A 2 week old African American female presented to the pediatric emergency department (ED) with erythema, swelling and copious mucopurulent discharge from the right eye. One week earlier, her Mom noted similar symptoms in the left eye which spontaneously resolved. Mom denied fever, irritability, lethargy, rash, and respiratory or urinary symptoms. The baby was born at term through a spontaneous vaginal delivery with no complications. Mom received regular prenatal care and all screening tests were negative. The baby received erythromycin eye ointment at birth prior to initial discharge. Complete blood count showed a slight leukocytosis (WBC 15.7 TH/cm²) and cerebral spinal fluid (CSF) values were unremarkable. A complete sepsis work up was performed with blood, CSF, eye swabs and urine sent for bacterial cultures. Given the high suspicion for a sexually transmitted infection, an eye swab was also collected for Neisseria gonorrhoeae and Chlamydia trachomatis polymerase chain reaction (PCR). Herpes simplex virus PCR from the CSF was also performed. The patient was started on IV ampicillin, cefotaxime and oral erythromycin in the ED.

Lab results

The organism was positive for both catalase and oxidase and identified by matrix-assisted light desorption ionization- time of flight (MALDI-TOF) as Neisseria meningitidis. The health department also confirmed the identification. PCR of the eye swab was negative for Neisseria gonorrhoeae and Chlamydia trachomatis. Bacterial cultures from the blood, CSF and urine were all negative.

Discussion

Neisseria meningitidis is an encapsulated Gram negative diplococcus (Image 2) that is usually transmitted through large droplet secretions from the oropharynx from colonized individuals. It can cause invasive meningococcal disease, which can present as meningitis (high fever, stiff neck, and headache), acute sepsis or a combination of both. Waterhouse Friderichsen-syndrome can result in severe dissemination forms of the disease and is characterized by petechial hemorrhages, involvement of the adrenal glands, and disseminated intravascular coagulopathy (DIC). Rarely, N. meningitidis can cause acute bacterial conjunctivitis (1.5 % – 2.5% of cases). Local complications, including corneal ulcers or a more systemic disease, may occur as well.

N. meningitidis produces multiple virulence factors that help cause disease and evade human immune defense mechanisms. The polysaccharide capsule represents the major virulence factor and is also the basis of meningococcal serotyping. Twelve different capsular serotypes can be distinguished, with serotypes A, B, C, W, X, and Y accounting for most invasive disease worldwide. Other virulence factors include pili, which helps the bacteria attach to host surfaces, and IgA protease, an enzyme that cleaves IgA and allows the bacteria to escape the humoral portion of the immune system.

In the laboratory, N. meningitidis grows well on both blood and chocolate agars after 24 hours of incubation (Image 1) and it is positive for both catalase and oxidase. Traditionally, sugar fermentation was used to differentiate Neisseria species from one another. N. meningitidis ferments both glucose and maltose whereas N. gonorrhoeae is only capable of fermenting glucose. Currently, more rapid identification methods (MALDI-TOF, PCR and sequencing) are being increasingly used in most laboratories for a faster and more accurate identification of Neisseria species. The work up of suspected N. meningitidis isolates must be performed using BSL 2 standards, as aerosols created during mobilization from culture plates or performance of biochemical testing has been known to cause invasive disease in laboratory workers.

In general, N. meningitidis is susceptible to penicillin and cefotaxime, but susceptibility testing by disk or gradient diffusion is recommended. Both rifampin and ciprofloxacin can be used for chemoprophylaxis in close contacts of the patient and healthcare & laboratory workers. In addition, a number of meningococcal vaccines are available in the United States (US) and the Centers for Disease Control & Prevention (CDC) recommends vaccinating all adolescents and people at high risk for infection (college students, military recruits, those who had a splenectomy and patients with complement deficiencies). The most common vaccine is a quadrivalent polysaccharide-protein conjugate vaccine which covers serotypes A, C, W and Y. Recently in 2014, the Food and Drug Administration (FDA) approved Trumenba, a vaccine effective against serotype B, which a common serotype causing invasive disease in the US.

 

-Akram Shalaby, MD, is a first year anatomical and 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 the Microbiology and Serology Laboratories. Her interests include infectious disease histology, process and quality improvement and resident education.

Case History

14 year old cystic fibrosis (CF) patient was admitted to the hospital for a CF exacerbation with a known rhinovirus infection. The patient reported congestion and cough with production of greenish sputum. The following was isolated from a sputum culture.

Laboratory Culture

 

Image A. MacConkey Agar plate with mucoid Pseudomonas aeruginosa colonies.

Image B. Kirby-Bauer Method of antimicrobial susceptibility.

Discussion

Cystic fibrosis patients have complex polymicrobial respiratory flora. Routine cultures may reveal different bacterial species that can contribute to the difficulty in treating and preventing infections in these patients. Staphylococcus aureus, Haemophilus influenza and Pseudomonas aeruginosa are main contributors to the infections found in CF patients.

In this case, we isolated a mucoid Pseudomonas aeruginosa (Image A). Pseudomonas aeruginosa is an oxidase-positive, gram negative rod-shaped bacterium that is commonly found in the environment. It is considered a severe and frequent pathogen in patients with cystic fibrosis. In chronic infections, it is thought that P. aeruginosa can undergo a “mucoid switch” where the bacterium can acquire mutations that lead to the mucoid phenotype. The phenotype is so impressive that excess polysaccharide will often drip onto the lid of the plate when stored upside down during incubation.

One of the key features of mucoid strains of P. aeruginosa is their ability to form biofilms. Biofilms consist of a matrix of polysaccharide, protein and DNA. This provides not only a protective barrier from antibiotics and the immune system, but also may contribute to the growth of other bacteria within the microenvironment.

With all the excess polysaccharide, it can be difficult to standardize the inoculum of mucoid isolates of P. aeruginosa which is an essential starting point for the microbroth dilution method of antimicrobial susceptibility testing. For this reason, susceptibility testing of mucoid isolates is often performed by Kirby-Bauer (KB) disk diffusion method. The KB method is a test of antimicrobial susceptibility that is based on the zone of inhibition surrounding disks that contain antimicrobial drugs (Image B). The strain of mucoid P. aeruginosa isolated in this case was found to be susceptible to aztreonam, ceftazidime, piperacillin/tazobactam and resistant to amikacin, cefepime, ciprofloxacin, gentamicin, levofloxacin, meropenem and tobramycin, by the Kirby Bauer method.

Case Follow Up

The patient was ultimately treated with IV piperacillin/tazobactam, as well as with chest physical therapy and hypertonic saline inhalation. They clinically improved and were ultimately discharged home after a 2 week hospital stay.

References

The mucoid switch in Pseudomonas aeruginosa represses quorum sensing systems and leads to complex changes to stationary phase virulence factor regulation. Ben Ryall, Marta Carrara, James EA Zlosnik, Volker Behrends, Xiaoyun Lee, Zhen Wong, Kathryn E. Lougheed, Huw D. Williams. PLOS ONE, May 2014, Vol. 9, Iss. 5, Pages 1-11.

Pseudomonas aeruginosa biofilms in cystic fibrosis. Niels Høiby, Oana Ciofu, and Thomas Bjarnsholt. Future Microbiology, November 2010, Vol. 5, No. 11, Pages 1663-1674.

Insights into Cystic Fibrosis Polymicrobial Consortia: The Role of Species Interactions in Biofilm Development, Phenotype, and Response to In-Use Antibiotics. Magalhaes AP, Lopes SP, Pereira MO. Frontiers in Microbiology, January 13, 2017, Vol. 7, Article 2146.

Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. Gary W. Procop, Deirdre L. Church, Geraldine S. Hall, William M. Janda, Elmer W. Koneman, Paul C. Schreckenberger; Gail L. Woods. Seventh Edition. 2017. Pages 343, 1110.

 

-Megan B. Wachsmann, MD, MSCS, is a 4th year Anatomic and Clinical Pathology Resident and Chief Resident at UT Southwestern Medical Center.

–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.

A 10 year old girl presented to the ED with several month history of worsening CNS symptoms, including gait instability, left-sided weakness, nausea, vomiting, several witnessed seizure events and loss of consciousness. MRI revealed right frontal mass measuring 7.0 cm in greatest dimension with midline shift. The patient underwent a right-sided craniotomy with tumor resection and was closely followed in the PICU. On post-op day 3, the patient appeared pale and lethargic with a temperature of 39.9 C. Blood cultures were collected and the patient empirically started on ceftriaxone and vancomycin for meningitis coverage. Due to worsening fever and altered mental status, a lumbar puncture was performed with the following results:

• Opening pressure: 10.5 cm
• Appearance: slightly cloudy
• Cells: 91% PMN
• Gram negative rods were identified on gram smear

 

Photo credit: University Medical Center of Rotterdam, Dept. of Microbiology and Infectious Disease

Discussion

Pseudomonas stutzeri is an aerobic gram negative bacilli (GNB) with a single polar flagellum that is both catalase and oxidase positive. Several defining features help distinguish P. stutzeri from other Pseudomonas species, including its nutritional versatility (utilizes starch, maltose, and ethylene glycol), its inability to produce fluorescent pigment and its “coral-like” appearance on growth medium.  There are no known virulence factors for P. stutzeri. Hospital distribution rates of P. stutzeri are approximately 1-2% of Pseudomonas spp. Nosocomial infection via medical devices or solutions is the most common route of infection. P. stutzeri isolates are sensitive to beta lactam antibiotics—meropenem is commonly used.

 

-Christina Litsakos is a Pathology Student Fellow at 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 14 year-old-boy with Cystic Fibrosis had a respiratory culture collected at his routine clinic visit. It grew abundant mixed respiratory flora, and rare Gram-negative coccobacilli. This organism grew as non-lactose fermenting colonies on MacConkey agar (Figure 1) in approximately 36 hours and was oxidase and catalase positive. The isolate was identified by MALDI-TOF MS with a score of 2.39, which is acceptable for species-level identification.

Discussion

Our isolate was identified as Bordetella bronchiseptica. Bordetella spp. are small Gram-negative rods that often appear as coccobacilli. Like other Bordetella spp., our isolate was catalase positive. Oxidase results vary across the genus, but B. bronchiseptica is oxidase positive. Some Bordetella spp. including B. pertussis and B. parapertussis, are very sensitive to metabolites and toxic substances found in many types of microbiological media. For the best chance of recovering these fastidious Bordetella spp. in culture, specialized agar such as Regan-Lowe, Bordet-Gengou, or Stainer-Scholte medium along with extended incubation periods are used. Due to the difficulty of culturing B. pertussis and B. parapertussis, these days culture is only performed at large reference laboratories or public health facilities. Testing by PCR is the current clinical practice and has greatly improved the sensitivity of detection for these fastidious organisms. In contrast, other Bordetella spp. including B. bronchiseptica are routinely recovered in culture using standard laboratory methods.

The most clinically relevant Bordetella spp. in humans are B. pertussis, the infectious agent of whooping cough, and B. parapertussis, which causes a similar illness to B. pertussis but generally symptoms are less severe. B. bronchiseptica causes respiratory infections in many animals including cats and dogs, and is the infectious agent of kennel cough.  B. bronchiseptica can cause respiratory infection in humans who acquire the bacterium primarily from their infected pets. Human infection is rare, and most likely to occur in immunocompromised patients such as those with poorly controlled HIV or Cystic Fibrosis.

B. bronchiseptica produces a β-lactamase making the organism resistant to penicillin and many cephalosporins. Most strains are resistant to trimethoprim-sulfamethoxazole as well. In contrast, strains of B. bronchiseptica are generally susceptible to β-lactam/ β-lactamase inhibitor combinations, quinolones, aminoglycosides, and tetracycline.

Our patient was not having an exacerbation at the time of specimen collection, so he continues to do well. We expect to find B. bronchiseptica in his future sputum specimens, but the pathogenicity of B. bronchiseptica in such a low amount compared to respiratory flora is unclear.

–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

The patient was in his otherwise healthy state of being until 3 days prior when he developed non-bloody diarrhea. On the morning of presentation the stool had become bloody. The patient was afebrile, had some reduced intake of food, but drinking fine. Most notably, he periodically stops walking and bends over as if he is in pain. This happened 3-4 times the previous day and these episodes tended to last for about one minute, after which the boy would continue to play. He does not attend daycare and his immunizations are up to date.

Stool culture was sent and a predominant organism was an oxidase negative, lactose-fermenting, Gram-negative rod (Figure 1). The organism was non-sorbitol fermenting based on growth on Sorbitol-MacConkey agar, and grew as mauve colonies on E. coli 0157 screening agar. MUG testing was negative.

Discussion

The isolate was E. coli O157. Isolates of E. coli O157 commonly produce shiga toxins (sxt1 and sxt2) which are responsible for diarrhea, hemorrhagic colitis, and most famously hemolytic-uremic syndrome (HUS). Typical illness starts with non-bloody diarrhea which becomes bloody after 2-3 days due to onset of hemorrhagic colitis. Often severe abdominal pain and low grade fever are present as well. HUS is a serious complication of E. coli O157 infection which results in acute renal dysfunction. HUS most often occurs in children < 5 years of age, of which 15% of those with laboratory confirmation of E. coli O157 developing this complication, compared to 6% in the general population. It is possible for other E. coli to produce shiga toxins, with 1% of HUS is caused by non-E. coli O157 infection.

It is recommended that all patients with suspected HUS should have stool cultured on selective and differential media for detection of E. coli O157 and direct shiga toxin detection should be performed to identify non-E. coli O157 isolates that are producing toxin. E. coli 0157 isolates look exactly the same as non-E. coli O157 normal fecal flora on 5% sheep blood, chocolate, and MacConkey agars. All E. coli ferment lactose on MacConkey agar (Figure 1A). E. coli O157 can be differentiated from other E. coli strains by growth on Sorbitol-MacConkey (SMAC) agar; E. coli O157 is a non-sorbitol fermenter while most other E. coli will ferment sorbitol (Figure 1B). Chromagenic agar for E. coli O157 is another option to screen stool specimens for E. coli O157. E. coli O157 grow mauve colored colonies on this particular agar (BBL CHROMagar O157 , Becton Dickinson) (Figure 1C). A summary of this data can be found in Table 1.

Growth of organisms suspicious for E. coli O157 on any media requires confirmation prior to reporting. Biochemical confirmation tests include E. coli O157 antiserum or latex agglutination and 4-methylumbelliferyl-beta-D-glucuronide (MUG) testing. For latex agglutination or antisera testing, it is essential to test the isolate of interest with the E. coli O157-specific reagent as well as a non-specific control to exclude non-specific binding. Unlike most E. coli strains, E. coli-O157 does not express beta-glucuronidase and is therefore MUG test negative (Table 1).

Table 1. Characteristics of E. coli O157 in comparison to other E. coli strains

Test	Non-E. coli O157	E. coli O157
Appearance on MacConkey agar	Lactose fermenter	Lactose fermenter
Appearance on Sorbitol-MacConky agar	Sorbitol fermenter	Non-sorbitol fermenter
MUG testing	Positive	Negative

For direct detection of shiga toxin, there are several commercially available immunoassays available for detection of shiga toxin protein. New on the market are multiplex gastrointestinal panels that can be used for molecular based detection of shiga toxin genes sxt-1 and sxt-2 among a host of other agents of gastrointestional disease.

E. coli O157 is spread via fecal oral route. It can be acquired directly from person to person or indirectly through food and water sources contaminated with fecal matter from infected humans and animals. Classic scenarios are undercooked ground beef, leafy greens, unpasteurized milk and juice, petting zoos, and contaminated drinking water. The incubation period prior to symptoms is 3-4 days (range 1-8 days).

Treatment for E. coli O157 is largely supportive consisting of fluids to prevent dehydration. The role of antibiotics is controversial with some studies suggesting antibiotics increase the risk of developing HUS while others found no association between the their use and increased HUS.

Following our patient’s stool culture result for E. coli O157, he was recalled to the Emergency Department for evaluation. He was still having diarrhea and vomiting, but it was reduced compared to the previous day. The patient was given fluids and sent home without antibiotic treatment and via phone conversation with his mother, his symptoms resolved a few days later.

-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.

A 6 year-old girl with a history of posterior fossa ependymoma presented with a one month history of fever, headaches, vomiting and more recently, neck stiffness. Additional history includes remote tumor resection followed by radiation and chemotherapy resulting in remission, with a residual ventriculoperitoneal shunt (VPS). Her parents reported she was in good health until approximately 1 month prior to presentation and is up to date on her immunizations. She was previously seen by her primary care physician for her symptoms and treated her with amoxicillin for suspected strep throat. Upon admission, she received supportive therapy for her symptoms after she was found to have tumor recurrence on imaging. The patient was scheduled for resection approximately two weeks after discharge and on post-operative day two she developed fever, vomiting and neck stiffness again. At this time, blood cultures were drawn and a lumbar puncture (LP) was performed. Cerebrospinal fluid (CSF) from both the LP and VPS submitted for fluid analysis (Table 1) and culture.

Table 1: Cerebrospinal Fluid Analysis

Spinal Fluid	LP	VPS
Appearance	Clear	Clear
Nucleated cells	1075 cells/μL	628 cells/μL
RBC	150 cells/μL	35 cells/μL
Polys	94%	87%
Lymphs	2%	6%
Mono/Macrophage	4%	7%
Glucose	68 mg/dL	13 mg/dL
Protein	69 mg/dL	164 mg/dL

 

Culture results:

The CSF Gram stain showed rare, paired, Gram-negative diplococci, which could raise suspicion for Neisseria meningitidis, however the typical flattened sides of adjacent bacteria were not observed. Rather, the morphology was more consistent with Gram-negative coccobaccilli, which is better demonstrated on Gram stain of the blood culture (Figure 1). Culture of both the CSF and blood specimens grew fairly large, smooth, round, opaque grey-yellow colonies on CAP, however showed no growth on BAP (Figure 2), suggesting a fastidious organism requiring growth factors. The colonies were both catalase and oxidase positive. The organism was identified as Haemophilus influenzae by MALDI-TOF MS (matrix-assisted laser desorption/ionizations time-of-flight mass spectrometry). This H. influenzae isolate was non-typeable by slide agglutination serotyping performed at the state public health laboratory.

Discussion:

H. influenzae are small, pleomorphic, gram-negative rods or coccobacilli that are non-motile. They are facultative anaerobes that grow best between 35-37°C with 5% CO2. H. influenzae is a fastidious species, requiring hemin (X factor) and nicotinamide-adenine-dinucleotide (NAD/V factor) for growth, which are both available in chocolate agar, but not blood agar. On chocolate agar, the colonies are non-hemolytic, typically large, smooth, round and convex with an opaque, colorless or grey hue. Encapsulated strains, including H. influenzae serotype b (Hib), appear mucoid and are typically small, grey colonies on CAP. Isolates are catalase and oxidase positive. H. influenzae displays the “satellite phenomenon” when grown near Staphylococcus aureus. This occurs when colonies of S. aureus lyse nearby red blood cells releasing hemin and NAD in the media. The presence of extracellular hemin and NAD allow colonies of H. influenzae to grow in the immediate vicinity of S. aureus.

H. influenzae is widely distributed in humans, colonizing the nose and throat and is spread from person-to-person via direct contact or respiratory droplets. Severe infections, including pneumonia, bacteremia and meningitis, affect predominantly infants and children. The American Academy of Pediatrics recommends routine vaccination with the Hib conjugate vaccine for infants aged 2 through 6 months (2 or 3 doses, depending on vaccine product) followed by a booster dose at age 12 through 15 months. Hib is the only serotype preventable by vaccine. Prior to routine vaccination in the US, approximately 20,000 children under the age of 5 were infected with H. influenzae and 3-6% died each year.

References:

1. Ledeboer N, Doern G. 2015. Haemophilus, p 667-684. In Jorgensen J, Pfaller M, Carroll K, Funke G, Landry M, Richter S, Warnock D (ed), Manual of Clinical Microbiology, 11th Edition. ASM Press, Washington, DC.
2. http://www.cdc.gov/meningitis/lab-manual/chpt09-id-characterization-hi.html
3. http://www.cdc.gov/hi-disease/index.html

 

-Petra Rahaman, MD is a 4th year Anatomic and Clinical Pathology 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.