Development of a TaqMan Array Card for Acute-Febrile-Illness Outbreak Investigation and Surveillance of Emerging Pathogens, Including Ebola Virus

Abstract

Acute febrile illness (AFI) is associated with substantial morbidity and mortality worldwide, yet an etiologic agent is often not identified. Convalescent-phase serology is impractical, blood culture is slow, and many pathogens are fastidious or impossible to cultivate. We developed a real-time PCR-based TaqMan array card (TAC) that can test six to eight samples within 2.5 h from sample to results and can simultaneously detect 26 AFI-associated organisms, including 15 viruses (chikungunya, Crimean-Congo hemorrhagic fever [CCHF] virus, dengue, Ebola virus, Bundibugyo virus, Sudan virus, hantaviruses [Hantaan and Seoul], hepatitis E, Marburg, Nipah virus, o'nyong-nyong virus, Rift Valley fever virus, West Nile virus, and yellow fever virus), 8 bacteria (Bartonella spp., Brucella spp., Coxiella burnetii, Leptospira spp., Rickettsia spp., Salmonella enterica and Salmonella enterica serovar Typhi, and Yersinia pestis), and 3 protozoa (Leishmania spp., Plasmodium spp., and Trypanosoma brucei). Two extrinsic controls (phocine herpesvirus 1 and bacteriophage MS2) were included to ensure extraction and amplification efficiency. Analytical validation was performed on spiked specimens for linearity, intra-assay precision, interassay precision, limit of detection, and specificity. The performance of the card on clinical specimens was evaluated with 1,050 blood samples by comparison to the individual real-time PCR assays, and the TAC exhibited an overall 88% (278/315; 95% confidence interval [CI], 84% to 92%) sensitivity and a 99% (5,261/5,326, 98% to 99%) specificity. This TaqMan array card can be used in field settings as a rapid screen for outbreak investigation or for the surveillance of pathogens, including Ebola virus.

FIG 1

Configuration of the TaqMan array card for detection of the agents causing acute febrile illness.

FIG 2

Paired comparison of C_qs from IRTP and TAC reactions. In general, the C_qs exhibited a linear relationship (R² = 0.632; P < 0.001). The results from different targets were pooled and compared with the Wilcoxon signed-rank test for the samples where IRTP and TAC yielded positive results. The difference was within 2 units (IRTP C_q – TAC C_q = −1.8 ± 4.1; P < 0.001). The data points at the top and right of the graph represented IRTP-positive/TAC-negative and IRTP-negative/TAC-positive results, respectively. There was no significant difference in the number of samples that were IRTP positive/TAC negative or IRTP negative/TAC positive (chi-square test; P = 0.205).

FIG 3

Optimal TAC C_q cutoffs using amplicon sequencing as the reference. The dashed line shows the cutoffs based on ROC analysis. On the x axis, symbols indicate that a sample was confirmed by sequencing (+) or not (−). TAC specificity and sensitivity versus sequencing at these cutoffs are shown in the box.