Comparison of real-time PCR assays for detection of pathogenic Leptospira spp. in blood and identification of variations in target sequences

Abstract

Leptospirosis is considered an underdiagnosed disease. Although several PCR-based methods are currently in use, there is little information on their comparability. In this study, four quantitative real-time PCR (qPCR) assays (SYBR green and TaqMan chemistries) targeting the secY, lfb1, and lipL32 genes were evaluated as diagnostic assays. In our hands, these assays can detect between 10(2) and 10(3) bacteria/ml of pure culture, whole-blood, plasma, and serum samples. In three independent experiments, we found a slightly higher sensitivity of the PCR assays in plasma than in whole blood and serum. We also evaluated the specificity of the PCR assays on reference Leptospira strains, including newly described Leptospira species, and clinical isolates. No amplification was detected for DNA obtained from saprophytic or intermediate Leptospira species. However, among the pathogens, we identified sequence polymorphisms in target genes that result in primer and probe mismatches and affect qPCR assay performance. In conclusion, most of these assays are sensitive and specific tools for routine diagnosis of leptospirosis. However, it is important to continually evaluate and, if necessary, modify the primers and/or probes used to ensure effective detection of the circulating Leptospira isolates.

Fig. 1.

Nucleotide sequence polymorphisms of secY and lipL32. Alignment of secY (A) and lipL32 (B) nucleotide sequences of L. interrogans serovar Copenhageni strain Fiocruz L1-130 (FioL1-130), L. kmetyi strain Bejo-Iso 9 (L. kmetyi), L. borgpetersenii strain 200801773 (Lborg1773), and L. borgpetersenii strain 200801929 (Lborg1929). Primer and probe sequences are represented with a black background (L. interrogans strain Fiocruz L1-130 serves as the template DNA for the published sequences of primers and probe). Nucleotides with a white background within the black background indicate mismatched positions.

Fig. 2.

Schematic representation of study design.

Fig. 3.

Box plot of the mean C_T for DNA extracted from whole blood, plasma, serum, and PBW spiked with 10³ L. interrogans/ml. Bacteremia was assessed using qPCR assay with the TaqMan probe targeting lipL32. A one-way ANOVA (Dunnett's multiple comparison test; *, P < 0.05) was used to compare the mean C_T of DNA extracted from whole blood (WB), plasma (PL), serum (SE), and PBW using the Qiagen (Q) and Maxwell (M) DNA purification kits. Each box represents the interquartile spread between the first and third quartiles (25th and 75th percentiles). The line inside the box is the median, and the lines extending from the box represent minimum and maximum values.