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.

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.

Neisseria meningitidis (Nm), Haemophilus influenzae (Hi), and Streptococcus pneumoniae (Sp) 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 Nm ctrA, Hi hpd and Sp lytA, and serogroup-specific genes in the cap locus for Nm 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 B-synD, W-synG, and Y-synF showed 100% specificity for detecting Nm species, with high sensitivity [B-synD, 99%(75/76); W-synG, 97%(38/39); and Y-synF, 100%(66/66)]. The lower limit of detection (LLD) was 9, 43 and 10 copies/reaction for B-synD, W-synG, and Y-synF assays, respectively, a significant improvement compared to the previous singleplex assays. We developed three multiplex real-time PCR assays for detection of: (i) Nm ctrA, Hi hpd and Sp lytA (NHS); (ii) Nm serogroups A, W135 and X (AWX), and (iii) Nm serogroups B, C and Y (BCY). Each multiplex assay was 100% specific for detecting its target organisms or serogroups, and the LLD was similar to that for singleplex. Pairwise comparison of real-time PCR between multiplex and singleplex showed that cycle threshold values of the multiplex were similar to those for singleplex. There were no substantial differences in the sensitivity and specificity between these multiplex and singleplex real-time PCR assays.

An increase in invasive Haemophilus influenzae type b (Hib) cases occurred in Minnesota in 2008 after the recommended deferral of the 12-15 months Hib vaccine boosters during a US vaccine shortage. Five invasive Hib cases (one death) occurred in children; four had incomplete Hib vaccination (three refused/delayed); one was immunodeficient. Subsequently, we evaluated Hib carriage and vaccination. From 18 clinics near Hib cases, children (aged 4 weeks-60 months) were surveyed for pharyngeal Hib carriage. Records were compared for Hib, diphtheria-tetanus-acellular pertussis (DTaP), and pneumococcal (PCV-7) vaccination. Parents completed questionnaires on carriage risk factors and vaccination beliefs. In 1631 children (February-March 2009), no Hib carriage was detected; Hib vaccination was less likely to be completed than DTaP and PCV-7. Non-type b H. influenzae, detected in 245 (15%) children, was associated with: male sex, age 24-60 months, daycare attendance >15 h/week, a household smoker, and Asian/Pacific Islander race/ethnicity. In 2009, invasive Hib disease occurred in two children caused by the same strain that circulated in 2008. Hib remains a risk for vulnerable/unvaccinated children, although Hib carriage is not widespread in young children.

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.

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.