RESUMEN

This study aimed to design and evaluate a universal primer for Polymerase Chain Reaction (PCR)- based detection of mucormycosis-causing fungi by targeting their Internal Transcribed Spacer (ITS) sequences. In-silico analysis was conducted to assess primer suitability. Using Clustal Omega and Primer-BLAST, ITS sequences of 32 fungi species causing mucormycosis were aligned and subjected to primer design. Generated primers were sorted and in silico PCR simulations were performed to identify primers capable of amplifying all fungal species. Instead of identifying one pair of universal primer, in silico PCR analysis identified a panel of 14 primer pairs designed from full-length ITS sequences, and a panel of 12 primer pairs designed from conserved regions, that could detect 31 species. The study recommends a panel of 12 primers for multiplex-PCR to detect mucormycosis-causing fungi instead of a long list of PCR analyses for each fungus in diagnosing mucormycosis.

Asunto(s)

Cartilla de ADN , ADN de Hongos , Mucormicosis , Mucormicosis/diagnóstico , Mucormicosis/microbiología , Humanos , Cartilla de ADN/genética , ADN de Hongos/genética , Simulación por Computador , ADN Espaciador Ribosómico/genética , Reacción en Cadena de la Polimerasa/métodos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos

RESUMEN

This dataset includes data from febrile patients recruited for a large hospital-based study in Sri Lanka from 2016 to 2019. The variables include primary socio-demographic data, exposure data, clinical data, biochemical and investigation data. Some of these data are available as serial data from admission to discharge daily. Microscopic agglutination test, quantitative PCR of whole blood, urine and serum and culture isolation was performed to diagnose the patients with leptospirosis.

RESUMEN

BACKGROUND: Leptospirosis has globally significant human mortality and morbidity, yet estimating the clinical and public health burden of leptospirosis is challenging because timely diagnosis remains limited. The goal of the present study was to evaluate leptospirosis undercounting by current standard methods in both clinical and epidemiological study settings. METHODOLOGY/PRINCIPAL FINDINGS: A prospective hospital-based study was conducted in multiple hospitals in Sri Lanka from 2016 to 2019. Culture, whole blood, and urine samples were collected from clinically suspected leptospirosis cases and patients with undifferentiated fever. Analysis of biological samples from 1,734 subjects confirmed 591 (34.1%) cases as leptospirosis and 297 (17.1%) were classified as "probable" leptospirosis cases. Whole blood quantitative PCR (qPCR) did identify the most cases (322/540(60%)) but missed 40%. Cases missed by each method include; urine qPCR, 70% (153/220); acute sample microscopic agglutination test (MAT), 80% (409/510); paired serum sample MAT, 58% (98/170); and surveillance clinical case definition, 53% (265/496). qPCR of negative culture samples after six months of observation was of diagnostic value retrospectively with but missed 58% of positives (109/353). CONCLUSION: Leptospirosis disease burden estimates should consider the limitations of standard diagnostic tests. qPCR of multiple sample types should be used as a leading standard test for diagnosing acute leptospirosis.

Asunto(s)

Leptospira , Leptospirosis , Pruebas de Aglutinación/métodos , Humanos , Leptospira/genética , Leptospirosis/diagnóstico , Leptospirosis/epidemiología , Estudios Prospectivos , Estudios Retrospectivos

RESUMEN

Leptospirosis is an important cause of acute undifferentiated fever and complex multisystem febrile diseases in the tropics and subtropics. Understanding the evolution of Leptospira especially as related to the clinical pathogenesis of leptospirosis is facilitated by systematic comparative genomic analysis of human-infecting isolates. Here, we announce the complete genome sequences of three Leptospira strains that were isolated from blood of humans with undifferentiated fever in Sri Lanka.