Microbiology Case Study: an 11 Month Old with a 3 Day History of Gastroenteritis and High Fever

Clinical History:

An 11 month old female with no significant past medical history was admitted with a fever of 104 degrees Fahrenheit, nausea and vomiting for 3 days (now resolved), watery diarrhea 4-5 times/day (resolved), and a new onset of acute pharyngitis/bilateral cervical adenitis. ER staff was concerned for a bacterial superinfection. She appeared sick with pale skin, but vital signs were stable, and labs were unremarkable except for an elevated CRP (15.7) and an absolute monocytosis (though no elevation in total WBCs). Exam showed a hyperemic pharynx without exudates, and no lymph nodes larger than 1 cm. A CT neck shows bilateral cervical adenitis, left greater than right, with some suggestion of necrotic nodes, as well as a likely left 3rd or 4th branchial cleft cyst. Blood cultures were drawn, and they turned up positive in a matter of hours, with the gram stain and plate morphology seen below:

Gram stain of positive blood culture broth showing Gram positive cocci in chains
Gram stain of positive blood culture broth showing Gram positive cocci in chains
Large zones of beta-hemolysis around colonies growing on 5% sheep blood agar
Large zones of beta-hemolysis around colonies growing on 5% sheep blood agar

Laboratory Identification:

Gram positive cocci in chains were seen, with small, glossy, gray-white, translucent colonies on blood agar having a wide zone of surrounding beta hemolysis. Catalase testing was negative, PYR testing was positive, and latex agglutination testing for Lancefield antigens was positive for Group A. MALDI-TOF confirmed the presumptive identification of Streptococcus pyogenes.

Discussion:

S. pyogenes (aka Group A Streptococcus [GAS]) is a ubiquitous gram positive cocci that causes a wide range of disease in humans. It is the leading cause of acute pharyngitis, particularly in children aged 5-15, although 15-25% of school aged children are asymptomatically colonized. S. pyogenes can also cause cellulitis, impetigo, necrotizing fasciitis, scarlet fever, toxic shock syndrome, otitis media, osteomyelitis, pneumonia, or even rarely meningitis/brain abscess. It additionally causes several serious post-infectious sequelae, particularly in untreated cases, including acute rheumatic fever with potential rheumatic heart disease, and poststreptococcal glomerulonephritis.

S. pyogenes has numerous virulence factors, most importantly the M protein, of which there are nearly 80 variants. M protein binds fibrinogen, inhibits complement binding, and prevents phagocytosis. As it is the major antigenic target of antibodies formed following infection, immunity is conferred only to the infecting strain, and none of the others, which complicates vaccine development efforts. Streptolysin O is responsible for the organism’s b-hemolysis on blood agar, and is also immunogenic, causing elevated Anti-Streptolysin O antibodies (ASO), which can be useful in diagnosing a recent infection, rheumatic fever, or poststreptococcal glomerulonephritis.

GAS pharyngitis is uncommon in children less than 3 years old, as is the bacteremia seen in the patient, which was presumably secondary to the severity of the throat infection. A later CT scan showed an enlarging abscess in the left lateral neck, corresponding to the earlier supposed branchial cleft cyst, despite IV clindamycin which was begun in the ER. Ceftriaxone was added, and later changed to Piperacillin/Tazobactam to complete a seven day course, though future blood cultures were negative. The additional antimicrobial coverage was due to the concern for other infectious microorganisms, as S. pyogenes is universally susceptible to penicillin.

Interestingly, this patient also developed a severe absolute neutropenia, with her ANC dropping from 7,100 at admission to 300 two days later, and then to 60 after two further days. The hematology/oncology service was consulted, and they determined that this likely represented a reaction to the infection rather than a more sinister bone marrow pathology. Several more days into therapy her ANC did begin to recover.

References:

  1. Henningham A, Barnett TC, Maamary PG, Walker MJ. 2012. Pathogenesis of group A streptococcal infections. Discovery medicine 13:329-342.
  2. Cunningham MW. 2000. Pathogenesis of group A streptococcal infections. Clinical microbiology reviews 13:470-511.
  3. Red Book 2015
  4. Journal of Clinical Microbiology, 10th editionCunningham MW. 2000. Pathogenesis of group A streptococcal infections. Clinical microbiology reviews 13:470-511.

-Paul Yell, M.D. is a 2nd year anatomic and clinical pathology resident at the University of Texas 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.

Pediatric Labs are Different

Why do I say pediatric labs are different? A blood test is a blood test, right? Of course it is, however there are a lot of aspects of operating a pediatric lab that really are different from the way operations run in a lab whose clientele is mainly adults.

Most people can think of the most obvious difference: age-related reference intervals. The concentration of various biomarkers in the body changes as an infant grows and develops into an adult. In some cases, there’s not a lot of change in the biomarker. A blood pH is pretty constant over the course of a person’s life, as are the electrolytes. The reference intervals for these may be broader in infancy, but in general they don’t change a lot. However some biomarkers change so drastically that normal levels in childhood would be considered pathological in an adult. Without reference intervals tied to age or developmental state, these tests are not able to be properly interpreted. Alkaline phosphatase during bone growth and steroid hormones during puberty are two good examples of this.

The things that people tend not to think about that cause a pediatric lab to be different are predominately all centered on issues with sample volume. Especially in infancy, children have limited blood volume for testing, and this fact affects nearly every operation in a pediatric laboratory.

Front end processing of samples is affected, as often more than 60% of the tubes arriving in the lab are microtubes, which hold 1 mL or less. These tubes may not be able to hold a barcode label, nor fit on an instrument robotic system. The sample must be aliquotted into tubes that will fit a system, or hand fed into an instrument, and often hand-programmed into the instrument. More manual steps results in more opportunities for human error. In addition, the amount of sample used by any given instrument for testing must be considered. That sample volume includes the actual volume necessary for analysis, plus the volume necessary to run HIL (hemolysis, icterus, and lipemia) indices and the instrument dead volume (the volume below which an instrument cannot pipet the sample). Instruments with large dead volumes will not be found in pediatric labs.

Small sample volumes also affect the ability of the lab to add-on tests to samples already in the lab, or rerun samples later to check results. There may be insufficient volume to run more tests or rerun tests, or the sample may have evaporated and be unacceptable for running additional analyses. A study done using a 5 mL serum sample and a 0.1 mL serum sample and allowing both to sit open to the air for 4 hours showed that the 5 mL serum sample had less than 10% difference between original test values and those run 4 hours later. The 0.1 mL sample had more than 50% difference in its values. In addition, with just enough sample to run a test one time, if a dilution needs to be made, it may not be possible.

Lastly, there are a few test menu differences in a pediatric laboratory. Tests commonly found in pediatric labs, but essentially never in predominately adult labs include things like testing for inborn errors of metabolism and sweat chloride testing for Cystic Fibrosis. Conversely, common tests in an adult lab that are not performed in a pediatric lab include serum protein electrophoresis, PSA and other tumor markers. Some tests are performed in both labs but for different indications. For example, AFP is used as a tumor marker for hepatocellular carcinoma in pediatrics instead of as a maternal prenatal screening tool like in an adult lab.

These are some of the ways in which pediatric lab medicine differs from adult lab medicine, and offers unique challenges in day to day operations.

 

-Patti Jones PhD, DABCC, FACB, is the Clinical Director of the Chemistry and Metabolic Disease Laboratories at Children’s Medical Center in Dallas, TX and a Professor of Pathology at University of Texas Southwestern Medical Center in Dallas.

Pediatric Reference Intervals

All laboratory professionals are aware of the importance of reference intervals (RI). Without appropriate reference intervals, a test result is just a number. A numeric result for any given analyte cannot be used to diagnose or monitor treatment of disease unless there is an accompanying reference interval indicating what amount of that analyte should normally be present. In pediatrics, that’s even more important because as an infant develops through childhood and into adulthood, his or her biochemistry changes, adapts and develops with him or her. Using an adult RI to interpret a test result from an infant or child is likely to result in misinterpretation of the test, including misdiagnosis of disease states. A good example would be using adult alkaline phosphatase RI to interpret the results of a teenage boy’s test during accelerated bone growth in puberty.  His result will look pathological if interpreted using adult RI. Pediatric reference intervals (PRI) are age-related and often also gender-related intervals that must be used to interpret testing in the pediatric population.

Establishing reference intervals is problematic at the best of times because of the need to use 120 healthy individuals to establish them. In the pediatric population, especially in infancy, obtaining 120 healthy infants at each necessary time interval can be a daunting task. There are references available that present methods which allow the establishment of RI with smaller sample numbers (1,2). Also the CLSI document (2) allows the “validation” and “transfer” of current or literature RI, rather than complete “establishment” of RI in some cases.  Validating a current reference interval can be done with as few as 20 samples in a correlation study. “Transferring” a reference interval also involves using a correlation study and bias evaluation to adapt or adjust a current RI for use with a new assay. Transferring can also be performed with 20 – 60 patient samples.

These techniques especially come in handy with the hardest PRI to establish, the hormones during puberty. During this time, the RI are not really related to the child’s age, but related to the child’s phase of development, or Tanner Stage. To establish a PRI for these hormones, the healthy child donating the blood sample must also have his/her Tanner Stage determined, usually by an Endocrinologist.

Another consideration when dealing with PRI is that although all pediatric institutions use PRI, not all PRI are the same, even when the same instrument is used. An informal poll of 9 pediatric institutions using the same instrument resulted in 9 different PRI for common analytes such as electrolytes. There is a need to harmonize PRI, as we harmonize test results, in order to allow non-pediatric institutions a set of PRI that they can use also.

  1. Horn PS, Pesce AJ. Reference Intervals: a User’s Guide. AACC Press, Washington DC, 2005
  2. Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory: Approved Guideline – Third ed. CLSI C28-A3

 

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-Patti Jones PhD, DABCC, FACB, is the Clinical Director of the Chemistry and Metabolic Disease Laboratories at Children’s Medical Center in Dallas, TX and a Professor of Pathology at University of Texas Southwestern Medical Center in Dallas.