Marilyn S. Hamilton

Clinical utility of polymerase chain reaction testing for enteroviral meningitis.

BACKGROUND During summer enteroviral meningitis is a common cause of febrile illness in children, who are typically hospitalized for 2 to 3 days if bacterial infection is suspected. It has been hypothesized that a sensitive polymerase chain reaction (PCR) assay could quickly confirm the diagnosis and subsequently decrease hospitalization costs. However, to have maximum impact results should be available within 24 h. This necessitates daily assays on small numbers of samples. METHODS We examined the clinical utility of a PCR assay during two summers, comparing length of stay and charges. Only during the second summer were results reported to clinicians. Case controls were patients with negative PCR assay results but uncomplicated, presumed viral infections. We determined the cost per case identified with and without pleocytosis as a screen for PCR testing. RESULTS During the first summer 25% (5/20) of patients with positive PCR assay results remained hospitalized for >2 days. During the second summer 10.2% (6 of 59) of children with positive enteroviral PCR assay results but 37.9% (25 of 66) of case controls remained hospitalized for >2 days. The mean length of hospitalization was significantly (P < 0.05) shorter for patients with positive PCR test results than for case controls. The material cost was approximately

Clinical Evaluation of the ZstatFlu-II Test: a Chemiluminescent Rapid Diagnostic Test for Influenza Virus

238 per case identified. CONCLUSIONS PCR testing has clinical utility for diagnosis of enteroviral meningitis. Although the demands for daily testing make the test expensive, it appears to be cost-effective with savings related to shorter hospital stays.

Pediatric Laboratory MedicineCurrent Challenges and Future Opportunities

ABSTRACT Exploiting the high sensitivity of the chemiluminescence phenomenon, an accurate and sensitive point-of-care test, called the ZstatFlu-II test (ZymeTx, Inc., Oklahoma City, Okla.), was developed to detect influenza virus infections. The ZstatFlu-II test takes 20 min and requires approximately 2 min of “hands-on” time for operational steps. The ZstatFlu-II test does not distinguish between infections with influenza virus types A and B. ZstatFlu-II test results are printed on Polaroid High-Speed Detector Film, allowing test results to be archived. A prototype version of the ZstatFlu-II test was evaluated during the 2000-to-2001 flu season with 300 nasal aspirate specimens from children at a pediatric hospital. Compared to culture, the ZstatFlu-II test had 88% sensitivity and 92% specificity. The Directigen test had a sensitivity of 75% and a specificity of 93%. The sensitivity of the ZstatFlu-II test was significantly higher than that of the Directigen test (P < 0.0574).

High Frequency of Competitive Inhibition in the Roche Cobas AMPLICOR Multiplex PCR for Chlamydia trachomatis and Neisseria gonorrhoeae

The practice of pediatric laboratory medicine involves unique challenges related to development, nutrition, growth, and diseases during different periods of infancy, childhood, and adolescence. This article discusses key aspects of pediatric laboratory medicine faced by clinical pathologists, clinical laboratory scientists, and clinicians, including point-of-care testing, preanalytic variables, analytic factors, age-specific reference intervals, esoteric laboratory tests, clinical impact, and future opportunities. Although challenging, pediatric laboratory testing offers many opportunities for improved patient care, clinical- and laboratory-based research, and education.

Plasma cell-rich acute cellular rejection of a transplanted kidney associated with antibody to the red cell Kidd antigen

The term multiplex PCR refers to simultaneous amplification of more than one target in a single PCR. This method has some advantages but presents the possibility of competition between multiple targets for a finite number of reagents, which may invalidate the assay. The Roche Cobas AMPLICOR multiplex PCR for Chlamydia trachomatis and Neisseria gonorrhoeae, a U.S. Food and Drug Administration-approved assay, can detect both C. trachomatis and N. gonorrhoeae from a single specimen. PCR amplification of C. trachomatis and N. gonorrhoeae proceeds in one tube with a shared enzyme and shared nucleotides but independent biotinylated primers. An optional internal control (IC) permits detection of amplification inhibition. The IC DNA has primer binding sequences identical to those of the C. trachomatis target. Detection is accomplished by using oligonucleotide probes that are unique for C. trachomatis, N. gonorrhoeae, and IC, respectively, with colorimetric quantification by spectophotometer. A negative assay result is valid if the IC optical density (OD) is ≥0.2, indicating successful amplification (Method Manual, Cobas Amplicor, Roche Diagnostics, 12/1999, Revision 3.0). We performed 580 multiplex PCRs on endocervical and urethral swab specimens, and 58 assays yielded positive results for C. trachomatis alone (OD ≥ 2.0), 13 assays yielded positive results for N. gonorrhoeae alone (OD ≥ 3.5), and 4 assays yielded positive results for both. Of the 58 assays positive for C. trachomatis, 13 (22.4%) had IC OD values that were <0.2 (mean OD, 0.059). The results of these 13 assays were correctly interpreted as C. trachomatis positive (mean C. trachomatis OD, 3.187). Because of the failure of the IC to amplify, the N. gonorrhoeae OD values for these 13 were invalid. The limiting reagent could be the primer, shared by C. trachomatis and IC. In the absence of IC amplification, it was not clear that there were a sufficient number of reagents, aside from the primer, to amplify N. gonorrhoeae. Of these 13 assays, 11 had OD values interpreted as negative for N. gonorrhoeae (mean N. gonorrhoeae OD, 0.047). Five of these specimens from the 11 assays were cultured and yielded negative results. Two additional specimens that turned out to be N. gonorrhoeae culture positive had equivocal N. gonorrhoeae OD values of 2.013 and 3.492, respectively, resolvable as positive by duplicate repeat testing. A Roche Molecular Systems study suggested that competitive inhibition occurs when the relative concentration of one target is extremely high and that the competition is for reagents other than the primer. This Roche paper discusses the optional use of the IC for increased sensitivity as well as retesting of existing specimens to eliminate nonspecific, labile polymerase inhibition, which we saw in five specimens not discussed here (1). However, we had a very high rate of competitive inhibition not correctable by repetition. Experimental dilution of the specimens did result in IC amplification, but also, in one case, converted a positive C. trachomatis assay result to negative. The Molecular Pathology Checklist of the College of American Pathologists states that “the laboratory should be able to distinguish a true negative result from a false negative result due to PCR failure.” ICs accomplish this. The competitive inhibition described could be resolved by repeat amplification with only the N. gonorrhoeae primer. For the two equivocal specimens, positive for C. trachomatis, the initial OD value cutoff for N. gonorrhoeae positivity could be lower than the stated 3.5; perhaps 2.0 could be used as the cutoff OD value for duplicate repeat testing of specimens that produce equivocal results. Competitive inhibition is not fully addressed in the Method Manual. Undetected false negatives, due to failure to include the so-called optional IC, are misleading.

Teaching pediatric laboratory medicine to pathology residents

Abstract:  Plasma cell‐rich acute cellular rejection of a transplanted kidney is described in association with the identification of serum antibody to the red cell Kidd antigen, Jk‐b, which is also found in the kidney. This antibody was formed without a history of a recent blood transfusion or exposure to intravenous immunoglobulin. The role this antibody could have in the rejection of the transplant is discussed.

Prophylactic versus reactive transfusion of thawed plasma in patients undergoing surgical repair of craniosynostosis: a randomized clinical trial

CONTEXT Laboratory data are essential to the medical care of fetuses, infants, children, and adolescents. However, the performance and interpretation of laboratory tests on specimens from these patients, which may constitute a significant component of the workload in general hospitals and integrated health care systems as well as specialized perinatal or pediatric centers, present unique challenges to the clinical pathologist and the laboratory. Therefore, pathology residents should receive training in pediatric laboratory medicine. OBJECTIVE Childrens Health Improvement through Laboratory Diagnostics, a group of pathologists and laboratory scientists with interest and expertise in pediatric laboratory medicine, convened a task force to develop a list of curriculum topics, key resources, and training experiences in pediatric laboratory medicine for trainees in anatomic and clinical pathology or straight clinical pathology residency programs and in pediatric pathology fellowship programs. DATA SOURCES Based on the experiences of 11 training programs, we have compiled a comprehensive list of pediatric topics in the areas of clinical chemistry, endocrinology, hematology, urinalysis, coagulation medicine, transfusion medicine, immunology, microbiology and virology, biochemical genetics, cytogenetics and molecular diagnostics, point of care testing, and laboratory management. This report also includes recommendations for training experiences and a list of key texts and other resources in pediatric laboratory medicine. CONCLUSIONS Clinical pathologists should be trained to meet the laboratory medicine needs of pediatric patients and to assist the clinicians caring for these patients with the selection and interpretation of laboratory studies. This review helps program directors tailor their curricula to more effectively provide this training.

QuantiFERON-TB Gold In-Tube Testing for Tuberculosis in Healthcare Professionals

Surgical repair of craniosynostosis in young children is associated with copious bleeding and often coagulopathy. Typically, a reactive transfusion strategy is used to treat coagulopathy whereby fresh frozen plasma (FFP) is given only after clinical manifestation of clotting abnormality. This prospective, randomized clinical trial was designed to test the hypothesis that prophylactic FFP during craniofacial surgery reduces blood loss and blood transfusion requirements compared to a reactive FFP transfusion strategy.

Additional case of acute cellular kidney rejection associated with the presence of antibodies to the red blood cell Kidd antigen.

OBJECTIVE To assess the performance of the QuantiFERON-TB Gold in-tube (QFT-GIT) assay for tuberculosis (TB) screening using a convenience sample from among a population of healthcare provider (HCP) employees of a hospital. METHODS For the individuals in our cohort, we reviewed occupational health records, including TB risk factors, and the results of QFT-GIT testing. We considered a QFT-GIT result of greater than 0.35 IU/mL to be positive; when we obtained a positive result from a specimen from a particular individual, we repeated testing on a fresh specimen from that individual. RESULTS Of the 758 HCP employees whose specimens we screened, 439 had negative QFT-GIT results with negative TB risk factors and 268 had a negative QFT-GIT result but had positive TB risk factors. QFT-GIT results were positive in 47 subjects. Of the positive participants, 12 had a mean TB antigen value (antigen minus nil stimulated concentrations [Ag-Nil]) of 0.61 on initial testing and had a negative result on repeat testing, 22 had a TB Ag-Nil of 1.19 on initial testing and had a positive result on repeat testing (P = .01). CONCLUSIONS The QFT-GIT assay is useful for screening HCPs. However, false-positive results occur, particularly in a borderline zone of less than 1 IU/ml. Re-evaluation by repeat testing of fresh specimens from the same individual should be considered in subjects whose specimens test within the low-level positive cutoff.

Pediatric Laboratory Medicine

Earlier we reported a case of plasma cell-rich acute cellular rejection of a transplanted kidney, which was associated with a serum antibody to the red blood cell (RBC) Kidd antigen, Jk-b, without a history of recent transfusion. We presented data to suggest that the plasma cells in the kidney made antibodies to the Jk-b antigen, which is also present in the kidney and may, in turn, have contributed to the rejection process (1). Since that time, we have identified a second patient with a milder but similar clinical course and antibodies to Jk-a, the polymorphic allele Kidd antigen. The patient was a 19-yr-old female who had received a kidney transplant from her father (three of six HLA match) seven yr earlier after developing end-stage renal disease secondary to focal segmental glomerulosclerosis. Her transplant status had been stable with a serum creatinine of 1.2–1.5 mg/dL with her immunosuppression consisting of alternate day prednisone, mycophenolate mofetile, and cyclosporine. She had a history of nephrolithiasis in the transplanted kidney and had recently experienced several urinary tract infections. A rise in her serum creatinine to 2.5 mg/dL and a recent history of medication non-adherence, as reflected by undetectable cyclosporine levels, prompted the performance of a kidney biopsy. The needle biopsy of the renal allograft revealed chronic allograft nephropathy with acute cellular rejection. The interstitium was infiltrated by lymphocytes and plasma cells that occupied 60% of its area. The plasma cells comprised less than 5% of the infiltrating mononuclear inflammatory cells. The tubules were infiltrated by lymphocytes that ranged from four to 10 lymphocytes per tubular cross-section. The blood vessels were free of inflammation. There were no viral inclusions or cytopathic viral changes. Immunohistochemical studies for CMV, EBV, and BK virus were negative. An immunofluorescence study for C4d using indirect immunofluorescence technique on frozen tissue with a monoclonal antibody (AbD Serotec, dilution 1:100) revealed no labeling of interstitial capillaries. The acute cellular rejection was graded as IA by the Banff criteria. Polymerase chain reaction (PCR) on blood was negative for BK and CMV virus, and there were only 500 copies/mL of EBV virus. PCR on urine revealed only 10 900 copies/mL of BK virus. The patient was treated with high dose steroids for three days and a repeat biopsy in one wk showed decreased lymphocytic infiltrates in the interstitium with only rare plasma cells. There was resolution of the acute cellular rejection with disappearance of lymphocytes from the tubular epithelium. The patient was discharged in stable condition and has remained so with a serum creatinine of 1.6–2.0 mg/dL. Although the patient had received two units of RBCs in association with her transplant seven yr earlier, her antibody screen for antibodies to red cells had been negative six months following the transplant and she had received no additional transfusions or treatment with intravenous immunoglobulin. Nevertheless, at the time of the first biopsy, the patient was noted to have low titer IgG serum antibodies to the RBC Kidd antigen, Jka. The patient s phenotype is Jk-a negative, Jk-b positive. The kidney donor was her father and his RBC phenotype is Jk-a positive, Jk-b positive. An eluate of the kidney biopsy contained anti-Jk-a antibodies. We previously discussed the possible functional relationship between kidney rejection and antibodies to the RBC Kidd antigen (1). The Pediatr Transplantation 2008: 12: 918–919 2008 Wiley Periodicals, Inc.