Evaluation of different serological assays for early diagnosis of leptospirosis in Martinique (French West Indies).

Leptospirosis is a potentially life-threatening but curable zoonosis whose prognosis depends on accurate and timely diagnosis. Because of its non-specific clinical presentation, laboratory testing is essential to confirm the diagnosis. Here, we aimed to assess the performance of two enzyme-linked immunosorbent assays (ELISAs) (ELISA Serion and ELISA-Hb Pasteur) and one immunodot (GenBio) using quantitative PCR (qPCR) as gold standard, instead of the traditional microscopic agglutination test, for the diagnosis of acute leptospirosis in an endemic area.Between January 2011 and December 2012, a total of 122 patients were diagnosed with leptospirosis, as confirmed by qPCR at the University Hospital of Martinique. Among them, 103 had at least one serum sample available for analysis. Performance of each serological assay was evaluated according to days' post onset of symptoms (DPO) and local species diversity (which included L. santarosai, L. interrogans, L. kirschneri, L. borgpetersenii, L. noguchii, and L. kmetyi). Several thresholds were tested to optimize accuracy. When considering the manufacturer's threshold, the sensitivity of ELISA Serion, ELISA-Hb Pasteur and GenBio immunodot was 75%, 67% and 64%, while specificity was 92%, 98% and 100%, respectively. Moreover, the threshold optimization allowed a significant improvement in specificity for the ELISA Serion from 92% to 99% (p<0.05). During the first 5 DPO, sensitivities were 35%, 30% and 42% for ELISA Serion, ELISA-Hb Pasteur and GenBio immunodot, respectively. However, between 6─10 DPO, these sensitivities dramatically increased to reach 86%, 76% and 67%, respectively. Performances of the three assays were not affected by the species studied.All these serological assays showed the potential for diagnosing leptospirosis after (but not before) 6 days' post onset of symptoms. In a high prevalence setting, where highest specificities are needed, threshold optimizing should be performed for this purpose

Factors Associated with Severe Leptospirosis, Martinique, 2010–2013

To identify factors associated with disease severity, we examined 102 patients with quantitative PCR–confirmed leptospirosis in Martinique during 2010–2013. Associated factors were hypotension, chest auscultation abnormalities, icterus, oligo/anuria, thrombocytopenia, prothrombin time <68%, high levels of leptospiremia, and infection with L. interrogans serovar Icterohaemorrhagiae/Copenhageni

Sensivities and 95% confidence intervals of the 3 serological assays according to genomospecies.

<p>Sensivities and 95% confidence intervals of the 3 serological assays according to genomospecies.</p

Predictive values according to estimated prevalence before (top) and after (bottom) threshold optimization.

<p>Predictive values according to estimated prevalence before (top) and after (bottom) threshold optimization.</p

Serovar Diversity of Pathogenic <em>Leptospira</em> Circulating in the French West Indies

<div><p>Background</p><p>Leptospirosis is one of the most important neglected tropical bacterial diseases in Latin America and the Caribbean. However, very little is known about the circulating etiological agents of leptospirosis in this region. In this study, we describe the serological and molecular features of leptospires isolated from 104 leptospirosis patients in Guadeloupe (n = 85) and Martinique (n = 19) and six rats captured in Guadeloupe, between 2004 and 2012.</p> <p>Methods and Findings</p><p>Strains were studied by serogrouping, PFGE, MLVA, and sequencing 16SrRNA and <i>secY</i>. DNA extracts from blood samples collected from 36 patients in Martinique were also used for molecular typing of leptospires via PCR. Phylogenetic analyses revealed thirteen different genotypes clustered into five main clades that corresponded to the species: <i>L. interrogans</i>, <i>L. kirschneri</i>, <i>L. borgpetersenii</i>, <i>L. noguchi</i>, and <i>L. santarosai</i>. We also identified <i>L. kmetyi</i> in at least two patients with acute leptospirosis. This is the first time, to our knowledge, that this species has been identified in humans. The most prevalent genotypes were associated with <i>L. interrogans</i> serovars Icterohaemorrhagiae and Copenhageni, <i>L. kirschneri</i> serovar Bogvere, and <i>L. borgpetersenii</i> serovar Arborea. We were unable to identify nine strains at the serovar level and comparison of genotyping results to the MLST database revealed new <i>secY</i> alleles.</p> <p>Conclusions</p><p>The overall serovar distribution in the French West Indies was unique compared to the neighboring islands. Typing of leptospiral isolates also suggested the existence of previously undescribed serovars.</p> </div

Phylogenetic relationships of leptospirosis isolates based on <i>secY</i> sequences.

<p>The tree was drawn using the UPGMA (unweighted pair group method with arithmetic average) algorithm. The species and genotype are indicated. Circle sizes correspond to the numbers of strains of each genotype. Isolates from Martinique are highlighted by a red background.</p

Identification of Leptospira DNA from acute-blood samples.

<p>NA: non applicable.</p><p>Ictero: Icterohaemorragiae.</p>a<p>: Partial sequencing of the 5′ variable region of the 16S rRNA gene of 201102111 and 201103362 showed 99% (277/279 nucleotides) with the reference strain of <i>L. kmetyi</i>.</p>b<p>: Partial sequencing of the 5′ variable region of the 16S rRNA gene of 201102109 showed identities to both <i>L. kmetyi</i> (273/279 nucleotides) and <i>L. kirschneri</i> (272/279 nucleotides).</p>c<p>: size in bp of the PCR products for VNTR4, VNTR7, and VNTR10.</p>d<p>: serovars Icterohaemorragiae and Copenhageni cannot be distinguished by molecular methods.</p

Representative PFGE patterns of NotI-digested genomic DNA from isolates from Martinique and Guadeloupe.

<p>The genotype is indicated for each clinical isolate, and reference strains for serovars Panama, Icterohaemorragiae, Tbaquite, Bogvere, Beye, Szwajizak, Trinidad, Gorgas, Caribe, Ballum, Castellonis, Arborea, Sulzeae, Navet, Atchafalaya, Rama, Darien, Chagres, Bravo, Nicaragua, Bajan, Peruviana, and Atlantae. The molecular weight size marker is bacteriophage lambda DNA concatemers of 50 kb.</p

Phylogenetic tree of leptospiral 16S rRNA gene sequences of reference strains (<i>L. kirschneri</i> serovar Grippotyphosa strain MoskvaV, <i>L. interrogans</i> serovar Icterohaemorrhagiae strain Verdun, <i>L. santarosai</i> serovar Shermani strain 1342K, <i>L. borgpetersenii</i> serovar Ballum strain Mus27, and <i>L. noguchi</i> serovar Panama strain CZ214K) and a set of clinical isolates from Guadeloupe (G) and Martinique (M).

<p>The tree was drawn using the UPGMA (unweighted pair group method with arithmetic average) algorithm.</p