The Utility of Blood Culture Fluid for the Molecular Diagnosis of Leptospira: A Prospective Evaluation

Abstract

Leptospirosis is an important zoonosis worldwide, with infections occurring after exposure to contaminated water. Despite being a global problem, laboratory diagnosis remains difficult with culture results taking up to 3 months, serology being retrospective by nature, and polymerase chain reaction showing limited sensitivity. Leptospira have been shown to survive and multiply in blood culture media, and we hypothesized that extracting DNA from incubated blood culture fluid (BCF), followed by quantitative real-time polymerase chain reaction (qPCR) could improve the accuracy and speed of leptospira diagnosis. We assessed this retrospectively, using preincubated BCF of Leptospira spp. positive (N= 109) and negative (N= 63) febrile patients in Vientiane, Lao PDR. The final method showed promising sensitivities of 66% (95% confidence interval [CI]: 55-76) and 59% (95% CI: 49-68) compared with direct or direct and indirect testing combined, as the respective reference standards (specificities > 95%). Despite these promising diagnostic parameters, a subsequent prospective evaluation in a Lao hospital population (N= 352) showed that the sensitivity was very low (∼30%) compared with qPCR on venous blood samples. The disappointingly low sensitivity does suggest that venous blood samples are preferable for the clinical microbiology laboratory, although BCF might be an alternative if leptospirosis is only suspected postadmission after antibiotics have been used.

Figure 1.

Sensitivity and specificity characteristics plotted for different threshold values. Real-time quantitative polymerase chain reaction (qPCR) assays were analyzed using three different fluorescent thresholds (0.05, 0.075, 0.1), which were set manually. Receiver operator characteristics (ROC) analysis was used to determine sensitivity and specificity values at different Cq-values. Using (A) culture and (B) both culture and qPCR (blood) as gold standards for the ROC analysis.