Prospective case-control analysis of the aetiologies of acute undifferentiated fever in Vietnam

Le-Viet, Nhiem; Le, Viet-Nho; Chung, Hai; Phan, Duc-Tuan; Phan, Quang-Duong; Cao, Thanh-Van; Abat, Cédric; Raoult, Didier; Parola, Philippe

The DNA products isolated from the whole blood specimens were tested using quantitative real-time PCR (qPCR) with genus-specific primers and probes targeting specific sequences of O. tsutsugamushi, Rickettsia spp., R. felis, R. typhi, Anaplasma spp., Bartonella spp., Borrelia spp., and C. burnetii (Table S4) . These DNA products were also tested by qPCR for Leptospira spp., Salmonella spp., Salmonella enterica serovar Typhi/Paratyphi, Shigella spp., S. aureus, S. pneumoniae, Streptococcus pyogenes, Tropheryma whipplei and Burkholderia pseudomallei. The DNA products isolated from the eschar swab specimens were tested for O. tsutsugamushi, Rickettsia spp., R. felis and R. typhi, whereas the DNA products isolated from urine specimens were tested for Leptospira spp. using specific qPCR systems. The DNA/RNA products obtained from the plasma samples were tested by real-time reverse-transcription PCR (qRT-PCR) for dengue viruses ( Salmonella spp.; and Pneumocystis jirovecii. PCR assays were performed with a CFX Connect TM Real-Time PCR Detection System (Bio-Rad, USA). Negative control and positive controls for the corresponding pathogens were included in each run. The PCR procedures were performed according to the manufacturer's instructions. Quantitative PCR and quantitative reverse-transcription PCR assays were considered positive if the cycle threshold (Ct-value) was <35. All the AUFs and Controls were tested in the same panel of these pathogens.