Comparative genome characterization of Leptospira interrogans from mild and severe leptospirosis patients

Leptospirosis is a zoonotic disease caused by spirochetes from the genus Leptospira. In Thailand, Leptospira interrogans is a major cause of leptospirosis. Leptospirosis patients present with a wide range of clinical manifestations from asymptomatic, mild infections to severe illness involving organ failure. For better understanding the difference between Leptospira isolates causing mild and severe leptospirosis, illumina sequencing was used to sequence genomic DNA in both serotypes. DNA of Leptospira isolated from two patients, one with mild and another with severe symptoms, were included in this study. The paired-end reads were removed adapters and trimmed with Q30 score using Trimmomatic. Trimmed reads were constructed to contigs and scaffolds using SPAdes. Cross-contamination of scaffolds was evaluated by ContEst16s. Prokka tool for bacterial annotation was used to annotate sequences from both Leptospira isolates. Predicted amino acid sequences from Prokka were searched in EggNOG and David gene ontology database to characterize gene ontology. In addition, Leptospira from mild and severe patients, that passed the criteria e-value < 10e-5 from blastP against virulence factor database, were used to analyze with Venn diagram. From this study, we found 13 and 12 genes that were unique in the isolates from mild and severe patients, respectively. The 12 genes in the severe isolate might be virulence factor genes that affect disease severity. However, these genes should be validated in further study.

Comparison of Four Human Papillomavirus Genotyping Methods: Next-generation Sequencing, INNO-LiPA, Electrochemical DNA Chip, and Nested-PCR

BACKGROUND: Human papillomavirus (HPV) infection causes cervical cancer, thus necessitating early detection by screening. Rapid and accurate HPV genotyping is crucial both for the assessment of patients with HPV infection and for surveillance studies. METHODS: Fifty-eight cervicovaginal samples were tested for HPV genotypes using four methods in parallel: nested-PCR followed by conventional sequencing, INNO-LiPA, electrochemical DNA chip, and next-generation sequencing (NGS). RESULTS: Seven HPV genotypes (16, 18, 31, 33, 45, 56, and 58) were identified by all four methods. Nineteen HPV genotypes were detected by NGS, but not by nested-PCR, INNO-LiPA, or electrochemical DNA chip. CONCLUSIONS: Although NGS is relatively expensive and complex, it may serve as a sensitive HPV genotyping method. Because of its highly sensitive detection of multiple HPV genotypes, NGS may serve as an alternative for diagnostic HPV genotyping in certain situations.

Multiplex real-time RT-PCR for detecting chikungunya virus and dengue virus

OBJECTIVE@#To develop diagnostic test for detection chikungunya virus (CHIKV and Dengue virus (DENV) infection.@*METHODS@#We have performed a rapid, accurate laboratory confirmative method to simultaneously detect, quantify and differentiate CHIKV and DENV infection by single-step multiplex real-time RT-PCR.@*RESULTS@#The assay's sensitivity was 97.65%, specificity was 92.59% and accuracy was 95.82% when compared to conventional RT-PCR. Additionally, there was no cross-reaction between CHIKV, DENV, Japanese encephalitis virus, hepatitis C, hepatitis A or hepatitis E virus.@*CONCLUSIONS@#This rapid and reliable assay provides a means for simultaneous early diagnosis of CHIKV and DENV in a single-step reaction.