Development and Implementation of Multiplex TaqMan Array Cards for Specimen Testing at Child Health and Mortality Prevention Surveillance Site Laboratories.

Child Health and Mortality Prevention Surveillance (CHAMPS) laboratories are employing a variety of laboratory methods to identify infectious agents contributing to deaths of children <5 years old and stillbirths in sub-Saharan Africa and South Asia. In support of this long-term objective, our team developed TaqMan Array Cards (TACs) for testing postmortem specimens (blood, cerebrospinal fluid, lung tissue, respiratory tract swabs, and rectal swabs) for >100 real-time polymerase chain reaction (PCR) targets in total (30-45 per card depending on configuration). Multipathogen panels were configured by syndrome and customized to include pathogens of significance in young children within the regions where CHAMPS is conducted, including bacteria (57 targets covering 30 genera), viruses (48 targets covering 40 viruses), parasites (8 targets covering 8 organisms), and fungi (3 targets covering 3 organisms). The development and application of multiplex real-time PCR reactions to the TAC microfluidic platform increased the number of targets in each panel while maintaining assay efficiency and replicates for heightened sensitivity. These advances represent a substantial improvement in the utility of this technology for infectious disease diagnostics and surveillance. We optimized all aspects of the CHAMPS molecular laboratory testing workflow including nucleic acid extraction, quality assurance, and data management to ensure comprehensive molecular testing of specimens and high-quality data. Here we describe the development and implementation of multiplex TACs and associated laboratory protocols for specimen processing, testing, and data management at CHAMPS site laboratories.

Evaluation of new biomarker genes for differentiating Haemophilus influenzae from Haemophilus haemolyticus.

PCR detecting the protein D (hpd) and fuculose kinase (fucK) genes showed high sensitivity and specificity for identifying Haemophilus influenzae and differentiating it from H. haemolyticus. Phylogenetic analysis using the 16S rRNA gene demonstrated two distinct groups for H. influenzae and H. haemolyticus.

Clinical validation of multiplex real-time PCR assays for detection of bacterial meningitis pathogens.

Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae are important causes of meningitis and other infections, and rapid, sensitive, and specific laboratory assays are critical for effective public health interventions. Singleplex real-time PCR assays have been developed to detect N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA and serogroup-specific genes in the cap locus for N. meningitidis serogroups A, B, C, W135, X, and Y. However, the assay sensitivity for serogroups B, W135, and Y is low. We aimed to improve assay sensitivity and develop multiplex assays to reduce time and cost. New singleplex real-time PCR assays for serogroup B synD, W135 synG, and Y synF showed 100% specificity for detecting N. meningitidis species, with high sensitivity (serogroup B synD, 99% [75/76]; W135 synG, 97% [38/39]; and Y synF, 100% [66/66]). The lower limits of detection (LLD) were 9, 43, and 10 copies/reaction for serogroup B synD, W135 synG, and Y synF assays, respectively, a significant improvement compared to results for the previous singleplex assays. We developed three multiplex real-time PCR assays for detection of (i) N. meningitidis ctrA, H. influenzae hpd, and S. pneumoniae lytA (NHS assay); (ii) N. meningitidis serogroups A, W135, and X (AWX assay); and (iii) N. meningitidis serogroups B, C, and Y (BCY assay). Each multiplex assay was 100% specific for detecting its target organisms or serogroups, and the LLD was similar to that for the singleplex assay. Pairwise comparison of real-time PCR between multiplex and singleplex assays showed that cycle threshold values of the multiplex assay were similar to those for the singleplex assay. There were no substantial differences in sensitivity and specificity between these multiplex and singleplex real-time PCR assays.

sodC-based real-time PCR for detection of Neisseria meningitidis.

Real-time PCR (rt-PCR) is a widely used molecular method for detection of Neisseria meningitidis (Nm). Several rt-PCR assays for Nm target the capsule transport gene, ctrA. However, over 16% of meningococcal carriage isolates lack ctrA, rendering this target gene ineffective at identification of this sub-population of meningococcal isolates. The Cu-Zn superoxide dismutase gene, sodC, is found in Nm but not in other Neisseria species. To better identify Nm, regardless of capsule genotype or expression status, a sodC-based TaqMan rt-PCR assay was developed and validated. Standard curves revealed an average lower limit of detection of 73 genomes per reaction at cycle threshold (C(t)) value of 35, with 100% average reaction efficiency and an average R(2) of 0.9925. 99.7% (624/626) of Nm isolates tested were sodC-positive, with a range of average C(t) values from 13.0 to 29.5. The mean sodC C(t) value of these Nm isolates was 17.6±2.2 (±SD). Of the 626 Nm tested, 178 were nongroupable (NG) ctrA-negative Nm isolates, and 98.9% (176/178) of these were detected by sodC rt-PCR. The assay was 100% specific, with all 244 non-Nm isolates testing negative. Of 157 clinical specimens tested, sodC detected 25/157 Nm or 4 additional specimens compared to ctrA and 24 more than culture. Among 582 carriage specimens, sodC detected Nm in 1 more than ctrA and in 4 more than culture. This sodC rt-PCR assay is a highly sensitive and specific method for detection of Nm, especially in carriage studies where many meningococcal isolates lack capsule genes.

Detection of bacterial pathogens in Mongolia meningitis surveillance with a new real-time PCR assay to detect Haemophilus influenzae.

Since the implementation of Haemophilus influenzae (Hi) serotype b vaccine, other serotypes and non-typeable strains have taken on greater importance as a cause of Hi diseases. A rapid and accurate method is needed to detect all Hi regardless of the encapsulation status. We developed 2 real-time PCR (rt-PCR) assays to detect specific regions of the protein D gene (hpd). Both hpd assays are very specific and sensitive for detection of Hi. Of the 63 non-Hi isolates representing 21 bacterial species, none was detected by the hpd #1 assay, and only one of 2 H. aphrophilus isolates was detected by the hpd #3 assay. The hpd #1 and #3 assays detected 97% (229/237) and 99% (234/237) of Hi isolates, respectively, and were superior for detection of both typeable and non-typeable Hi isolates, as compared to previously developed rt-PCR targeting ompP2 or bexA. The diagnostic sensitivity and specificity of these rt-PCR assays were assessed on cerebrospinal fluid specimens collected as part of meningitis surveillance in Ulaanbaatar, Mongolia. The etiology (Neisseria meningitidis, Hi, and Streptococcus pneumoniae) of 111 suspected meningitis cases was determined by conventional methods (culture and latex agglutination), previously developed rt-PCR assays, and the new hpd assays. The rt-PCR assays were more sensitive for detection of meningitis pathogens than other classical methods and improved detection from 50% (56/111) to 75% (83/111). The hpd #3 assay identified a non-b Hi that was missed by the bexA assay and other methods. A sensitive rt-PCR assay to detect both typeable and non-typeable Hi is a useful tool for improving Hi disease surveillance especially after Hib vaccine introduction.

Epidemiologic investigation and targeted vaccination initiative in response to an outbreak of meningococcal disease among illicit drug users in Brooklyn, New York.

BACKGROUND: An outbreak of serogroup C meningococcal disease that involved illicit drug users and their contacts occurred in Brooklyn, New York, during 2005 and 2006. METHODS: The objectives of this study were to identify the population at risk for meningococcal disease, describe efforts to interrupt disease transmission, and assess the impact of a vaccine initiative. Descriptive and molecular epidemiological analysis was used to define the extent of the outbreak and the common risk factors among outbreak-related cases. A vaccine initiative that used community-based service providers was targeted to illicit drug users and their close contacts. The vaccine initiative was assessed through cessation of outbreak-related cases and the reduction in carriage rate. RESULTS: The investigation identified 23 outbreak-related cases of serogroup C meningococcal disease; 17 isolates were indistinguishable and 4 isolates were closely related according to pulsed-field gel electrophoresis. Two additional culture-negative cases had epidemiological links to laboratory-confirmed cases. The median age of patients with outbreak-related cases was 41 years, and 19 (83%) of 23 patients reported an association with illicit drug use. There were 7 outbreak-related deaths. Vaccination was administered to 2763 persons at 29 community locations, including methadone treatment centers, syringe-exchange programs, and soup kitchens. Three additional cases of meningococcal disease due to strains with the same pulsed-field gel electrophoresis pattern were identified after the vaccination initiative. CONCLUSIONS: Community-based outbreaks of meningococcal disease are difficult to control, and the decision to vaccinate is not straightforward. Current national guidelines for implementing a vaccination campaign are not strict criteria and cannot be expected to accommodate the myriad of factors that occur in community-based invasive meningococcal disease outbreaks, such as the inability to enumerate the population at risk.

Emergence of ciprofloxacin-resistant Neisseria meningitidis in North America.

We report on three cases of meningococcal disease caused by ciprofloxacin-resistant Neisseria meningitidis, one in North Dakota and two in Minnesota. The cases were caused by the same serogroup B strain. To assess local carriage of resistant N. meningitidis, we conducted a pharyngeal-carriage survey and isolated the resistant strain from one asymptomatic carrier. Sequencing of the gene encoding subunit A of DNA gyrase (gyrA) revealed a mutation associated with fluoroquinolone resistance and suggests that the resistance was acquired by means of horizontal gene transfer with the commensal N. lactamica. In susceptibility testing of invasive N. meningitidis isolates from the Active Bacterial Core surveillance system between January 2007 and January 2008, an additional ciprofloxacin-resistant isolate was found, in this case from California. Ciprofloxacin-resistant N. meningitidis has emerged in North America.