Comparative analysis of current diagnostic PCR assays in detecting pathogenic Leptospira isolates from environmental samples

Objective:To compare the efficiency of routine diagnostic PCR assays in detecting pathogenic Leptospira isolated from water and soils.Methods:Seven routine assays targeting six genes(lip L32,fla B,gyr B,lfb1,sec Y and lig B)were evaluated and compared on the cultures of two groups of pathogenic Leptospira from different sources.One group included 19 described reference strains recovered from infected human or animals,and another group included 22 environmental isolates from recreational and residential sites in Malaysia.The latter have been confirmed for presence of pathogenic Leptospira DNA.PCR positivity or detection sensitivity of each assay was determined and compared between the two groups.Results:Validation on reference strains showed 100.0%PCR sensitivity for all assays except lig B-PCR(95.0%)that failed to amplify Leptospira interrogans serovar Pomona.In marked contrast,there was a notable decline in sensitivity in the environmental isolates(lip L32-PCR,95.5%;fla B-PCR,90.9%;gyr B-PCR,77.3%;lfb1-PCR,59.1%;sec Y-PCRs,40.9%G1/G2-PCR,36.4%;lig B-PCR,13.6%),implying a large genetic distance between the two groups,as well as nucleotide polymorphism among environmental isolates.Conclusions:High proportion of false-negative PCR results suggests a need of prudent selection of primers in detecting environmental pathogenic Leptospira.These findings offer valuable insights on the extensive biodiversity of genus Leptospira and its impact on the efficacy and development of molecular detection tool.

An update on Gardneralla vaginalis associated bacterial vaginosis in Malaysia

Objective: To update the status of Gardnerella vaginalis(G. vaginalis) as a causative agent of bacterial vaginosis(BV) in Malaysia and to define its epidemiology, metronidazole resistance and virulence properties.Methods: It is a single-centre(Gynaecology clinic at the Hospital Kuala Lumpur,Malaysia) prospective study with laboratory-based microbiological follow up and analyses. Vaginal swabs collected from the patients suspected for BV were subjected to clinical BV diagnosis, isolation and identification of G. vaginalis, metronidazole susceptibility testing, vaginolysin and sialidase gene PCR, Piot's biotyping and amplified ribosomal DNA restriction analysis genotyping.Results: Among the 207 patients suspected for BV, G. vaginalis was isolated from 47 subjects. G. vaginalis coexisted with Trichomonas vaginalis and Candida albicans in 26 samples. Three G. vaginalis isolates were resistant to metronidazole. Biotyping revealed 1 and 7 as the common types. Amplified ribosomal DNA restriction analysis genotype II was found to be more common(n = 22; 46%) than I(n = 12; 25.53%) and III(n = 13;27.6%). All genotype I and III isolates carried the sialidase gene, while 91.6% and 84.6%contained the vaginolysin gene. Genotype I was significantly associated with postgynaecological surgical complications and abortions(P = 0.002).Conclusions: The existence of pathogenic G. vaginalis clones in Malaysia including drug resistant strains should not be taken lightly and needs to be monitored as these may bring more complications especially among women of child bearing age and pregnant women.