Diagnosis of Human Leptospirosis in a Clinical Setting: Real-Time PCR High Resolution Melting Analysis for Detection of Leptospira at the Onset of Disease:

Currently, direct detection of Leptospira can be done in clinical laboratories by conventional and by real-time PCR (qRT-PCR). We tested a biobank of paired samples of serum and urine from the same patient (202 patients) presenting at the hospital in an area endemic for leptospirosis using qRT-PCR followed by high resolution melting (HRM) analysis. The results were compared with those obtained by conventional nested PCR and with the serologic gold standard microscopic agglutination test (MAT). Differences were resolved by sequencing. qRT-PCR-HRM was positive for 46 of the 202 patients (22.7%, accuracy 100%) which is consistent with known prevalence of leptospirosis in the Azores. MAT results were positive for 3 of the 46 patients (6.5%). Analysis of paired samples allowed us to identify the illness point at which patients presented at the hospital: onset, dissemination or excretion. The melting curve analysis of Leptospira species revealed that 60.9% (28/46) of patients were infected with L. interrogans and 39.1% (18/46) were infected with L. borgpetersenii, both endemic to the Azores. We validated the use of qRT-PCR-HRM for diagnosis of leptospirosis and for identification of the Leptospira species at the earliest onset of infection in a clinical setting, in less than 2 hours.publishersversionpublishe

Human leptospirosis: seroreactivity and genetic susceptibility in the population of São Miguel Island (Azores, Portugal).

BackgroundLeptospirosis is a worldwide zoonotic and recognized neglected infectious disease. It has been observed that only a proportion of individuals exposed to pathogenic species of Leptospira become infected and develop clinically evident disease. Moreover, little information is available in subsequent reinfections. In the present study, we determine if a first infection with leptospirosis protects against subsequent reinfection, and investigate which of the host genetic factors are involved in the susceptibility and resistance to leptospirosis.Methodology and findingsWe conducted, in 2011, a retrospective hospital-based case-control study in the São Miguel Island population (Azores archipelago). In order to determine the seropositivity against pathogenic Leptospira after the first episode of leptospirosis, we performed a serological evaluation in 97 unrelated participants diagnosed with leptospirosis between 1992 and 2011. The results revealed that 46.4% of the 97 participants have circulating anti-Leptospira antibodies, and from these participants 35.6% maintained the seroprevalence for the same serogroup. Moreover, three of them were reinfected with unrelated Leptospira serovars. The genetic study was carried out by adding a control group composed of 470 unrelated healthy blood donors, also from São Miguel Island. Twenty five SNPs among twelve innate immune genes - IL1α, IL1β, IL6, IL10, IL12RB1, TLR2, TLR4, TLR9, CD14, CISH, LTA and TNF - were genotyped, as well as HLA class I (-A and -B) genes. Association analysis indicates that genotypes -511GG (OR=1.6, 95%CI 1.01-2.56, p=0.04) in IL1β, +1196CG (OR=2.0, 95%CI 1.26-3.27, p=0.003) in IL12RB1, -292TA (OR=1.8, 95% CI 1.06-2.1, p=0.03) and +3415CG (OR=1.8, 95% CI 1.08-3.08, p=0.02), both in CISH confer susceptibility to pathogenic Leptospira.ConclusionThe present study suggests some degree of long-term protection against leptospires with an attenuation of symptoms in case of reinfection. Moreover, our data supports the genetic influence of IL1β, IL12RB1 and CISH genes and the susceptibility to leptospirosis infection

MAT serological data obtained in 2011, after <i>n</i> years of first confirmed diagnosis.

<p>MAT serological data obtained in 2011, after <i>n</i> years of first confirmed diagnosis.</p

Significative genotype frequencies and susceptibility to leptospirosis.

<p>Bold refers to the significant association.</p><p>Significative genotype frequencies and susceptibility to leptospirosis.</p

Serological evaluation: comparison of Microscopic Agglutination Test (MAT) positive results (retrospective and in 2011).

<p>ND: not determined (borderline reactivity). A unique sample was collected from these individuals; the observed titles were below the cut-off (1∶160), value taken by the reference laboratory (IHMT) to endemic areas.</p><p>NA: not applicable.</p><p>Serological evaluation: comparison of Microscopic Agglutination Test (MAT) positive results (retrospective and in 2011).</p

Demographic, clinical and laboratory characterization of the 97 leptospirosis participants.

a<p>ALT: Alanine transaminase/ASP: Aspartate transaminase.</p>b<p>ALP: Alkaline phosphatase/GGT: Gamma glutamyltransferase.</p>c<p>Kreatinine Kinase.</p><p>Demographic, clinical and laboratory characterization of the 97 leptospirosis participants.</p