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 (lipL32, flaB, gyrB, lfb1, secY and ligB) 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 ligB-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 (lipL32-PCR, 95.5%;flaB-PCR, 90.9%; gyrB-PCR, 77.3%; lfb1-PCR, 59.1%; secY-PCRs, 40.9% G1/G2-PCR, 36.4%; ligB-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.

Influenza vaccination among Malaysian healthcare workers: a survey of coverage and attitudes

Background: Healthcare workers (HCWs) are at increased risk of getting influenza than the general population, therefore putting patients at risk of nosocomial infection. Influenza vaccination coverage among HCWs is low despite the availability of a safe and effective vaccine. However, the reasons for such a poor uptake are not well reported in Malaysia. This study aimed at assessing the rate of influenza vaccination uptake, knowledge and attitude of healthcare workers regarding influenza, and employers’ policy on influenza vaccination. Methods: A cross-sectional questionnaire survey was conducted in three hospitals in the Klang Valley. Mann–Whitney test was used to assess possible differences in knowledge and attitude towards flu vaccination and the χ2 was used for categorical variables. Analyses were performed with SPSS 22.0. Results: A total of 690 questionnaires were distributed; 527 were returned (giving a response rate of 76.4%. The vaccine uptake was 51.4% with the majority (83.5%) of those believing they were vaccinated to protect themselves. Higher proportion of vaccinated HCWs (p <0.05) agreeing to the fact that influenza is a serious threat to their health, however, 10% were not sure of its safety. Eighty-three (15.7%) claimed their employers did not have a vaccination policy, while 43.3% were not sure if their employers have vaccination policy. Conclusion: This study has demonstrated more than half of the healthcare workers were vaccinated, with a significant proportion of the healthcare workers believed they were vaccinated to protect themselves, while most of those that were not vaccinated claimed they are worried about the safety of the vaccine. Most employers did not have a flu vaccination policy in place. Hence, the need for government to enforce such policy and make annual flu vaccination free and compulsory for all healthcare workers KEY WORDS:

Systemic antibody response to nano-size calcium phospate biocompatible adjuvant adsorbed HEV-71 killed vaccine

PURPOSE: Since 1980s, human enterovirus-71 virus (HEV-71) is one of the common infectious disease in Asian Pacific region since late 1970s without effective commercial antiviral or protective vaccine is unavailable yet. The work examines the role of vaccine adjuvant particle size and the route of administration on postvaccination antibody response towards HEV-71 vaccine adsorbed to calcium phosphate (CaP) adjuvant. MATERIALS AND METHODS: First, CaP nano-particles were compared to a commercial micro-size and vaccine alone. Secondly, intradermal reduced dosage was compared to the conventional intramuscular immunization. Killed HEV-71 vaccines adsorbed to CaP nano-size (73 nm) and commercial one of micro-size (1.7 microm) were administered through intradermal, intramuscular, rabbits received vaccine alone and unvaccinated animals. RESULTS: CaP nano-particles adsorbed HEV-71 vaccine displayed higher antibody than the micro-size or unadsorbed vaccine alone, through both parenteral immunization routes. Moreover, the intradermal route (0.5 microg/mL) of 0.1-mL volume per vaccine dose induced equal IgG antibody level to 1.0-mL intramuscular route (0.5 microg/mL). CONCLUSION: The intradermal vaccine adsorbed CaP nano-adjuvant showed safer and significant antibody response after one-tenth reduced dose quantity (0.5 microg/mL) of only 0.1-mL volume as the most suitable protective, cost effective and affordable formulation not only for HEV-71; but also for developing further effective vaccines toward other human pathogens.

Detection and characterization of viruses causing hand, foot and mouth disease from children in Seri Kembangan, Malaysia

Hand, foot and mouth disease (HFMD) is a common viral infection among infants and children. The major causative agents of HFMD are enterovirus 71 (EV71) and coxsackievirus A16 (CVA16). Recently, coxsackievirus A6 (CVA6) infections were reported in neighboring countries. Infected infants and children may present with fever, mouth/throat ulcers, rashes and vesicles on hands and feet. Moreover, EV71 infections might cause fatal neurological complications. Since 1997, EV71 caused fatalities in Sarawak and Peninsula Malaysia. The purpose of this study was to identify and classify the viruses which detected from the patients who presenting clinical signs and symptoms of HFMD in Seri Kembangan, Malaysia. From December 2012 until July 2013, a total of 28 specimens were collected from patients with clinical case definitions of HFMD. The HFMD viruses were detected by using semi-nested reverse transcription polymerase chain reaction (snRT-PCR). The positive snRTPCR products were sequenced and phylogenetic analyses of the viruses were performed. 12 of 28 specimens (42.9%) were positive in snRT-PCR, seven are CVA6 (58.3%), two CVA16 (16.7%) and three EV71 (25%). Based on phylogenetic analysis studies, EV71 strains were identified as sub-genotype B5; CVA16 strains classified into sub-genotype B2b and B2c; CVA6 strains closely related to strains in Taiwan and Japan. In this study, HFMD in Seri Kembangan were caused by different types of Enterovirus, which were EV71, CVA6 and CVA16.

Expression analysis of SIR2 and SAPs1-4 gene expression in Candida albicans treated with allicin compared tofluconazole

One of the main factors for virulence of fungus such as Candida albicans is the ability to change its morphology from yeast to hyphae. Allicin, one of the volatile sulfur-oil compounds from freshly crushed garlic, has a variety of antifungal activities. In this study, the effect of allicin on growth and hyphae production in C. albicans as compared to fluconazole, an antifungal drug was investigated using survival time in vitro and microscopic image at different time intervals. Additionally, the expression of selected genes involved in hyphae formation and development such as SIR2 and SAP1-4 was evaluated by semi-quantitative RTPCR and relative real time RT-PCR. Allicin was shown to down-regulate the expression of SIR2 (5.54 fold), similar to fluconazole (3.48 fold) at 2x MIC concentrations. Interestingly, allicin had no effect on SAPs1-4 expression, whereas fluconazole was able to suppress SAP4 expression. Our findings showed that allicin was effective in suppressing hyphae development of C. albicans to an extent that is sometimes equal or more than fluconazole. Moreover, allicin and fluconazole seemed to share a common anti-Candida mechanism through inhibition of SIR2 gene, while fluconazole appeared to also exert its fungistatic effect through another pathway that involved SAP4 suppression.

Laboratory confirmation of respiratory viruses: is it necessary?

Respiratory infections caused by viral agents are very common and have caused tremendous burden to many countries economically. In the past, laboratory confirmation of viral agents in respiratory infections was regarded as low priority and not cost-effective. With the changing trends in managing such patients, laboratory confirmation of selective agents is reported to be beneficial in providing appropriate health care to patients, especially in hospital settings. In the long run, it has been shown that laboratory confirmation of respiratory viruses can reduce cost and contribute significantly to the better management of the patients