Using a field quantitative real-time PCR test to rapidly identify highly viremic rift valley fever cases

Abstract

Approximately 8% of Rift Valley fever (RVF) cases develop severe disease, leading to hemorrhage, hepatitis, and/or encephalitis and resulting in up to 50% of deaths. A major obstacle in the management of RVF and other viral hemorrhagic fever cases in outbreaks that occur in rural settings is the inability to rapidly identify such cases, with poor prognosis early enough to allow for more-aggressive therapies. During an RVF outbreak in Kenya in 2006 to 2007, we evaluated whether quantitative real-time reverse transcription-PCR (qRT-PCR) could be used in the field to rapidly identify viremic RVF cases with risk of death. In 52 of 430 RVF cases analyzed by qRT-PCR and virus culture, 18 died (case fatality rate [CFR] = 34.6%). Levels of viremia in fatal cases were significantly higher than those in nonfatal cases (mean of 10(5.2) versus 10(2.9) per ml; P < 0.005). A negative correlation between the levels of infectious virus particles and the qRT-PCR crossover threshold (C(T)) values allowed the use of qRT-PCR to assess prognosis. The CFR was 50.0% among cases with C(T) values of <27.0 (corresponding to 2.1 x 10(4) viral RNA particles/ml of serum) and 4.5% among cases with C(T) values of >or=27.0. This cutoff yielded 93.8% sensitivity and a 95.5% negative predictive value; the specificity and positive predictive value were 58% and 50%, respectively. This study shows a correlation between high viremia and fatality and indicates that qRT-PCR testing can identify nearly all fatal RVF cases.

FIG. 1.

Scatter plots to demonstrate the distribution patterns of acute RVF cases (n = 90), with their C_T values obtained from rapid qRT-PCR testing. (A) The distribution pattern of all cases (n = 90) against the cutoff C_T value of 27.0, which was indicative of prognosis. (B) The distribution among the subset of these cases (n = 51) that did not have detectable RVF antibodies (IgM or IgG). (C) The distribution among cases (n = 39) that were positive for RVF IgM (blue), IgG (purple), or both IgM and IgG (yellow).

FIG. 2.

(A) Correlation of C_T values with viral RNA copies among fatal (n = 18) and nonfatal (n = 36) cases of RVF. The RVF RNA concentration in serum was calculated using a standard curve developed using a qRT-PCR as described in Materials and Methods. The ≥95% chance limit of detection as determined by probit regression analysis by Drosten et al. (7) was 2,835 RNA copies/ml (95% confidence interval, 2,143 to 4,525) of serum. The C_T values were determined from a one-step real-time RT-PCR, with fluorescence read at the combined annealing-extension step at 57°C. Using a cutoff C_T value of 27.0, cases registering C_T values of ≤27.0 were associated with a CFR of ≥50.0, whereas those with C_T values of >27.0 had a CFR of 4.5%, a sensitivity of 93.8%, and a negative predictive value of 95.5%. (B) Correlation of levels of infectious RVF virus in serum (viremia), with C_T values among fatal (n = 18) and nonfatal (n = 36) cases of RVF. Viremia was determined by inoculating patient sera in Vero cells and expressed as TCID₅₀/ml of serum. The limit of detection was 100 infectious RVF virus particles per ml of serum. The mean infectious virus levels were fourfold higher in fatal cases than those in nonfatal cases of RVF. No infectious virus was detected in 20 of the 36 nonfatal cases, all of which had C_T values of >27.0, whereas infectious virus titers ranging from 10^1.3 to 10^7.8 TCID₅₀ were detected in all the fatal cases.