The Evolution of the Polymerase Chain Reaction to Diagnose Childhood Infections.

Children's healthcare has evolved over the years, and the pediatric laboratory has contributed to the clinical understanding of childhood disease through the application of new technology and knowledge. This article highlights the evolution of PCR technology to aid in the diagnosis of pediatric infections, from the discovery of the PCR, through the subsequent years when the clinical need exceeded the capability of the technology, until the current day, when application of the PCR is becoming commonplace.

Risk factors for quantity not sufficient sweat collection in infants 3 months or younger.

OBJECTIVES: The purpose is to identify demographic characteristics associated with a quantity not sufficient (QNS) sweat collection in infants 3 months or younger. METHODS: History of premature birth, infant race and sex, gestational age at delivery, and weight of the infant were compared with QNS collection. RESULTS: Of 221 sweat collections from 197 infants, 25 were QNS. Infant weight less than 3 kg and history of prematurity were associated with QNS collection (P < .001). Thirteen (30.2%) of 43 infants weighing less than 3 kg had QNS collections compared with 12 (7.9%) of 151 infants 3 kg or more. Twelve (46.2%) premature infants had QNS collections compared with 13 (7.6%) term infants. Lower birth gestational age and corrected gestational age were associated with QNS collections. Six (86%) of seven infants who weighed less than 3 kg, had a history of prematurity, and were more than 54 days old at testing had a QNS result. Sex and race did not correlate with QNS collections. CONCLUSIONS: Weight less than 3 kg and history of prematurity are associated with an increased chance of QNS sweat collections.

Multiplex viral polymerase chain reaction testing using the FilmArray device compared with direct fluorescent antibody testing.

Advances in fluidics, electronics, and instrument design have enabled automation of complex molecular assays. The FilmArray Respiratory Panel (RP) is one such assay; it allows for rapid detection of multiple viral and bacterial respiratory targets via nested polymerase chain reaction (PCR), with all components contained within a single pouch. We performed a detailed comparison of workflow between the FilmArray RP and direct fluorescent antibody (DFA) staining. The FilmArray RP proved to be more efficient, with as few as 91 total touches needed and as little as 4 minutes 52 seconds hands-on time. This compares with as many as 502 total touches and 12 minutes 45 seconds hands-on time for DFA staining. FilmArray RP detected a greater number of organisms (20 viruses and bacteria vs 7 viruses for DFA staining) and required less training compared with DFA staining. The ease of use of the FilmArray RP makes this molecular assay amenable to operation 24 hours a day, 7 days a week in routine laboratory and point-of-care settings.

Monocytopenia as a diagnostic clue to pediatric B-lymphoblastic leukemia with rare circulating blasts.

Abstract Background: B-lymphoblastic leukemia/lymphoma (B-LL) is the most common childhood cancer. Occasionally, circulating blasts in the peripheral blood are rare ({less than or equal to}1%) and may be missed, even when flow cytometric immunophenotyping is performed, leading to a false negative report. Methods: The records from all patients with a new diagnosis of B-LL at our institution were reviewed from Jan 2009-Dec 2011. Of 130 cases with peripheral blood flow cytometry, 15 had a blast count of {less than or equal to}1%, with 14 having electronic files for gating monocytes. The percentage of monocytes by flow cytometry and absolute monocyte counts (AMCs) were compared with peripheral blood samples that were negative by flow cytometry, sent due to at least one lineage cytopenia (n=39). Results: The monocytes from the patients with leukemia averaged 0.8%, and were statistically lower than the negative controls, which averaged 7.1% (p<0.001). 11 of the 14 (79%) patients with leukemia had monocytes <1%, compared to only 3 (8%) of the negative controls. The AMCs were also significantly lower (p<0.001), with 93% of the leukemia group having an AMC of <100 cells/µL, compared to only 28% of the negative controls. Conclusions: In patients presenting with cytopenias, assessment of percentage monocytes may be an important diagnostic clue in determining the presence of occult leukemia. If flow cytometry is performed, acquisition of more than the standard 10,000 events is necessary to adequately assess for leukemia. If monocytes are <1% by flow cytometry in the setting of cytopenias, bone marrow examination is recommended, even with negative peripheral blood flow cytometry.

Evaluation of Maxwell® 16 for automated DNA extraction from whole blood and formalin-fixed paraffin embedded (FFPE) tissue.

BACKGROUND: The aim of the study was to assess the performance of Promega, Maxwell® 16 for the extraction of genomic DNA from whole blood and FFPE tissue. METHODS: DNA was extracted from 10 whole blood and 10 FFPE specimens using six different commercial kits. RESULTS: For whole blood, the mean DNA concentration obtained by Maxwell® 16 was significantly greater than either easyMAG® (p<0.0001) or QIAamp® Blood DNA kit (p<0.001). For FFPE, the mean DNA concentration obtained by the AllPrep® FFPE specific DNA/RNA kit was significantly greater than either the Maxwell® 16 (p<0.0001) or the general AllPrep® DNA/RNA kit (p<0.0001). CONCLUSIONS: Comparative evaluation of the six DNA extraction kits indicated that the semi-automated Maxwell® 16 was superior for whole blood extraction while the manual AllPrep® FFPE DNA/RNA kit (Qiagen) performed better for FFPE DNA extraction in terms of quantity of DNA obtained. All six extraction methods (blood and FFPE) performed well in terms of purity. Although there were variances in the quantity of DNA obtained, there were no significant differences in the efficiency of these methods in yielding amplifiable DNA extracts, as demonstrated by ß-actin for whole blood specimens. In evaluation of FFPE DNA extraction methods, the Qiagen AllPrep® FFPE DNA/RNA Mini Kit was the best for applications requiring larger amplicons, but for smaller amplicons the Maxwell was most consistent.

Avascular villi, increased syncytial knots, and hypervascular villi are associated with pregnancies complicated by factor V Leiden mutation.

There is controversy about whether pathologic abnormalities are associated with pregnancies complicated by factor V Leiden (FVL) mutation. The purpose of this study was to evaluate 105 placentas delivered to mothers heterozygous for FVL mutation to determine if there are pathologic changes suggestive of hypoxia or thrombosis, which correlate with mutation status. We examined placentas obtained as part of a prospective study of 5188 pregnancies analyzed for the presence of FVL mutation in either the mother or the infant. One hundred five placentas from mothers heterozygous for the mutation were compared with 225 controls matched for maternal age, race, and geographic site. Of the 330 pregnancies, 50 infants were FVL mutation heterozygotes. Maternal FVL heterozygote status was associated with more frequent increased numbers of syncytial knots (13% vs 4%); the difference remained significant after controlling for hypertension, preeclampsia, small-for-gestational-age infants, and delivery prior to 35 weeks of gestation (odds ratio 3.6, 95% confidence interval 1.5-8.7, P  =  0.004). Maternal FVL heterozygotes had more hypervascular villi (10% vs 3%), with significance retained controlling for delivery route (odds ratio 3.4, 95% confidence ratio 1.2-9.4, P  =  0.018). Placentas from infants heterozygous for FVL mutation had more avascular villi than controls (odds ratio 2.9, 95% confidence interval 1.5-5.6, P  =  0.001). Fetal or maternal FVL heterozygosity was not associated with infarcts, small-for-gestational-age placentas, or fetal thrombotic vasculopathy. This analysis demonstrates that pathologic findings associated with placental hypoxia, specifically focal avascular villi, increased numbers of syncytial knots, and hypervascular villi, also correlate with FVL heterozygosity in infants or mothers.

Enterovirus is not present in placentas from cases of perinatal depression using polymerase chain reaction analysis.

Enteroviruses have been implicated as a cause of low Apgar scores in conjunction with perinatal seizures and respiratory insufficiency. Using in-situ reverse transcriptase polymerase chain reaction (in-situ PCR), Nuovo et al detected enterovirus in up to 86% of placentas from perinates exhibiting these symptoms. In-situ PCR has been the only method employed to assess for the presence ofenterovirus in this specific patient population. The purpose of our study was to use PCR amplification of enterovirus from extracted RNA to confirm these observations. RNA was extracted from 26 placentas of infants with low Apgar scores, perinatal seizures, and respiratory insufficiency. Each extraction was positive for beta-actin RNA, which confirmed that the integrity of RNA was maintained in the sample. Enterovirus RNA was not detected in any of the cases. Our results indicate that enterovirus is not present in placentas from neonates with the combination of low Apgar scores, respiratory insufficiency, and seizures, as previously reported.

Comparison of automated nucleic acid extraction methods with manual extraction.

Automated nucleic acid extractors can improve workflow and decrease variability in the clinical laboratory. We evaluated Qiagen EZ1 (Valencia, CA) and bioMérieux (Durham, NC) easyMAG extractors compared with Qiagen manual extraction using targets and matrices commonly available in the clinical laboratory. Pooled samples were spiked with various organisms, serially diluted, and extracted in duplicate. The organisms/matrices were Bordetella pertussis/bronchoalveolar lavage, herpes simplex virus II/cerebrospinal fluid, coxsackievirus A9/cerebrospinal fluid, BK virus/plasma, and Mycoplasma pneumoniae/endotracheal tube samples. Extracts were amplified in duplicate using real-time PCR assays, and amplification of the target at a cycle threshold of 35 using the manual method was used for comparison. Amplification efficiency of nucleic acids extracted by automated methods was similar to that by the manual method except for a loss of efficiency for M. pneumoniae in endotracheal tube samples. The EZ1 viral kit 2.0 gave better results for coxsackievirus A9 than the EZ1 viral kit version 1.0. At the lowest limit of detection (past a cycle threshold of 35), the easyMAG was more likely to produce amplifiable nucleic acid than were either the EZ1 or manual extraction. Operational complexity, defined as the number of manipulations required to obtain an extracted sample, was the lowest for the easyMAG. The easyMAG was the most expensive of the methods, followed by the EZ1 kit and manual extraction.

Characterization of inflammation in syphilitic villitis and in villitis of unknown etiology.

Chronic villitis is a histologic diagnosis that may be either associated with infection, or termed villitis of undetermined etiology (VUE). The lymphocytic infiltrate in VUE has been reported to consist of maternal lymphocytes, but the origin of the lymphocytic infiltrate in infectious villitis has not been identified. The purpose of our study was to compare the maternal vs. fetal origin of the infiltrating lymphocytes in VUE and syphilitic villitis, and to expand the immunophenotypic data provided by previous studies. Paraffin-embedded placentas from four males with VUE and two males with syphilitic villitis were subjected to fluorescence in situ hybridization (FISH) for the X and Y chromosomes. Serial sections were stained with antibodies to CD3, CD4, CD8, CD68, HLA-DR, and CD20. Quantitation of the relative number of cells marking with each antibody was done for four villi in each slide. CD3 lymphocytes predominated in both VUE and syphilitic villitis, with slightly more CD8 cells compared to CD4 cells. CD68 and HLA-DR positive cells were as frequent as CD3 cells, and B-lymphocytes were rare. Maternal cells were the predominant intravillous population in both VUE and syphilitic villitis, and neutrophils in syphilitic villitis were also maternal. These data indicate that the immune response in both syphilitic villitis and VUE is similar, raising the possibility of a similar immunopathogenetic pathway.

Adenovirus ascending cholangiohepatitis.

Three children, two with liver transplants and one with acquired human immunodeficiency virus (HIV) infection, presented with hepatitis accompanied by elevated gamma glutamyl transpeptidase. Biopsies revealed cholangiohepatitis caused by adenovirus infection. There was a progressive loss of interlobular bile ducts in two of the patients. In one patient, infection of the biliary tree was marked by a necrotizing cholangitis, with adenoviral inclusions noted in the biliary epithelium. In each patient, there was evidence of adenovirus gastrointestinal infection. This is the first report of adenoviral infection of the biliary tree in humans. It is hypothesized that adenovirus cholangiohepatitis occurs as a result of ascending infection from the gastrointestinal tract to the biliary tree.

Development of real-time PCR assays for the quantitative detection of Epstein-Barr virus and cytomegalovirus, comparison of TaqMan probes, and molecular beacons.

Human Epstein-Barr virus (EBV) and cytomegalovirus (CMV) can cause serious complications in immunocompromised patients. Rapid diagnosis of EBV and CMV infection is critical in the management of the disease so that anti-viral therapy can be started early. Here we describe the development of real-time PCR assays using TaqMan probes and molecular beacons and compare the performance of both assays with a well-established, validated, gel-based PCR method for the quantification of EBV and CMV in patients' samples. The TaqMan and molecular beacon assays were linear between 10 to 10(7) viral genomes/reaction. Both assays generated calibration curves with strong correlation and low intra-assay and interassay variation. Results of EBV and CMV viral load determination inpatient samples obtained by the gel-based and real-time PCR were very similar. The real-time PCR assays showed increases in viral load before clinical measures of viral disease and decreases in viral load during anti-viral therapy in two of six pediatric patients. The data indicate that these TaqMan and molecular beacon approaches are accurate, rapid, and reliable assays for the diagnosis and monitoring of EBV and CMV infections in patients.

Absence of the G1528C (E474Q) mutation in the alpha-subunit of the mitochondrial trifunctional protein in women with acute fatty liver of pregnancy.

Acute fatty liver of pregnancy (AFLP) is a rare and dreaded complication of pregnancy, almost exclusively seen in the third trimester. The histopathologic features of AFLP closely resemble those seen in metabolic disorders characterized by deficiency of fatty acid oxidative enzymes. Several reports have established a strong association between AFLP in the mother and fetal deficiency of the enzyme long-chain L-3-hydroxyacyl-CoA dehydrogenase (LCHAD). However, these studies have an inevitable selection bias resulting from ascertainment through an affected infant, rather than an unselected population of patients with AFLP. We retrospectively examined a series of 10 women with pregnancies complicated by AFLP to determine the prevalence of the common LCHAD mutation (G1528C) in this population. The existing LCHAD primers, which produce a 640-bp amplicon (IJlst L, Ruiter JP, Hoovers JM, Jakobs ME, Wanders RJ: J Clin Invest 98:1028-1033, 1996), were modified to make them amenable to analysis of fragmented DNA obtained from microdissected formalin-fixed material. None of the patients were found to harbor the common G1528C mutation. It is likely that AFLP arising in the context of fetal LCHAD deficiency represents only one of the possible etiologies for this uncommon disorder, and the metabolic basis of AFLP is more heterogeneous than previously believed.

Umbilical vein interleukin-6 levels correlate with the severity of placental inflammation and gestational age.

Interleukin-6 (IL6) and suppurating placental inflammation are markers of neonatal sepsis. The purpose of this study was to define a relationship between IL6 and acute chorioamnionitis and funisitis of the placenta, and to compare IL6 levels in term and preterm neonates. Umbilical venous IL6 was measured in 137 term and 110 preterm neonates. Acute chorioamnionitis was graded as none, mild, moderate, severe, and necrotizing. Funisitis was graded as none, 1 vessel, 2 vessels, 3 vessels, or necrotizing. A 2-way analysis of variance with interaction was used to compare the IL6 levels. There was a stepwise progression of IL6 levels with increasing severity of acute chorioamnionitis and funisitis. Term neonates showed an IL6 elevation with mild acute chorioamnionitis and single-vessel vasculitis, which increased progressively until the inflammation became severe. In contrast, IL6 levels in preterm neonates did not increase significantly until severe acute chorioamnionitis or 3-vessel vasculitis was seen. Statistically significant differences in IL6 levels were seen in term versus preterm infants when the acute chorioamnionitis was mild or moderate or when the funisitis involved either 1 or 2 vessels (P < 0.05). The difference may be related to the relative immaturity of the preterm immune system, as has been demonstrated in vivo and in vitro. However, differences in management could be confounding factors. In conclusion, umbilical venous IL6 levels correlate with the severity of acute placental inflammation, with greater IL6 elevations in term infants compared to preterm infants until the inflammation becomes severe.