One-step, multiplex, dual-function oligonucleotide of loop-mediated isothermal amplification assay for the detection of pathogenic Burkholderia pseudomallei.

This study highlights the development of a multiplex real-time loop-mediated isothermal amplification assay. The developed assay employed a dual-function oligonucleotide (DFO) which simultaneously monitors the emitted amplification signals and accelerates the amplification process. The DFO was a modification of loop primer (LP); the 5'-end and 3'-end of the LP was tagged with fluorophore and quencher, respectively. The DFO was quenched in its unbound state and fluoresces only when it anneals to the specific target during the amplification process. With the same working mechanism as LP, DFO allowed the detection of target genes in less than 1 h in a real time monitoring system. We demonstrated this detection platform with Burkholderia pseudomallei, the causative agent of melioidosis. An internal amplification control (IAC) was incorporated in the assay to rule out false negative result and to demonstrate that the assay was successfully developed in a multiplex system. The assay was 100% specific when it was evaluated against 96 B. pseudomallei clinical isolates and 48 other bacteria species. The detection limit (sensitivity) of the developed assay was 1 fg/µl of B. pseudomallei genomic DNA and 18.2 CFU/ml at the bacterial cell level. In spiked blood samples, the assay's detection limit was 14 CFU/ml. The assay's diagnostic evaluation showed 100% diagnostic sensitivity, diagnostic specificity, positive predictive value, and negative predictive value. An integrated multiplex LAMP and real-time monitoring system was successfully developed, simplifying the workflow for the rapid and specific nucleic acid diagnostic test.

Development of monoclonal antibodies against recombinant LipL21 protein of pathogenic Leptospira through phage display technology.

Leptospirosis is a potentially fatal zoonosis that is caused by spirochete Leptospira. The signs and symptoms of leptospirosis are usually varied, allowing it to be mistaken for other causes of acute febrile syndromes. Thus, early diagnosis and identification of a specific agent in clinical samples is crucial for effective treatment. This study was aimed to develop specific monoclonal antibodies against LipL21 antigen for future use in leptospirosis rapid and accurate immunoassay. A recombinant LipL21 (rLipL21) antigen was optimized for expression and evaluated for immunogenicity. Then, a naïve phage antibody library was utilized to identify single chain fragment variable (scFv) clones against the rLipL21 antigen. A total of 47 clones were analysed through monoclonal phage ELISA. However, after taking into consideration the background OD405 values, only 4 clones were sent for sequencing to determine human germline sequences. The sequence analysis showed that all 4 clones are identical. The in silico analysis of scFv-lip-1 complex indicated that the charged residues of scFv CDRs are responsible for the recognition with rLipL21 epitopes. The generated monoclonal antibody against rLipL21 will be evaluated as a detection reagent for the diagnosis of human leptospirosis in a future study.

Evaluation of beak and feather disease virus, avian polyomavirus and avian papillomavirus of captives psittacine birds in Seri Kembangan, Selangor, Malaysia

Aims@#Psittacine birds such as parrots, macaws, cockatoos, lovebirds and parakeets, are widely reared as household pets or at aviary due to their attractive features. However, the status of virus-causing diseases of psittacine species in Malaysia is fairly under-documented. Therefore, this study was aimed to detect the presence of three common avian viruses that infect psittacine birds, i.e. beak and feather disease virus (BFDV), avian polyomavirus and avian papillomavirus. @*Methodology and results@#Faecal samples from twelve asymptomatic captive psittacine birds of different species were collected from an undisclosed animal garden in Serdang, Selangor, Malaysia. Briefly, the sample was homogenised and resuspended with SM buffer with the ratio 1:1 (weight of sample/g: volume of SM buffer/mL) before centrifugation at 1,000 × g for 20 min. The supernatant was collected and filtered before subjected to genomic DNA extraction using a commercialised kit. Polymerase chain reaction (PCR) technique was used to screen the V1, VP1 and L1 genes of beak and feather disease virus (BFDV), avian polyomavirus and avian papillomavirus, respectively. Findings revealed that the samples were negative for BFDV and avian polyomavirus. However, positive results of 1.5 kbp PCR amplicon were detected for avian papillomavirus in four out of the 12 samples (33.33%), which was from the white-crested cockatoo, African grey parrot, yellow-collared macaw and Senegal parrot. Sequence analysis of the L1 gene from the Senegal parrot Poicephalus senegalus revealed 93% identity to a reference Psittacus erithacus timneh avian papillomavirus.@*Conclusion, significance and impact of study@#This study added to the limited prevalence data of three important avian viruses which infect captive psittacines in Seri Kembangan, Selangor, Malaysia. Avian papillomavirus, but not BFDV and avian polyomavirus, was detected in the collected captive psittacine birds. Therefore, a routine screening can be performed to monitor the health status of birds despite their asymptomatic manifestation, in order to prevent possible virus transmission.

Molecular Characterization of Leptospira spp. in Environmental Samples from North-Eastern Malaysia Revealed a Pathogenic Strain, Leptospira alstonii.

The presence of pathogenic Leptospira spp. in the environment poses threats to human health. The aim of this study was to detect and characterize Leptospira spp. from environmental samples. A total of 144 samples comprised of 72 soil and 72 water samples were collected from markets and recreational areas in a north-eastern state in Malaysia. Samples were cultured on Ellinghausen and McCullough modified by Johnson and Harris media. Leptospires were positive in 22.9% (n = 33) of the isolates. Based on partial sequences of 16S rRNA, a pathogenic leptospire, Leptospira alstonii (n = 1/33), was identified in 3% of the isolates followed by intermediate leptospire (L. wolffii, n = 1/33, and L. licerasiae, n = 7/33) and nonpathogenic leptospire, L. meyeri (n = 22/33) in 24.2% and 66.7%, respectively. This study demonstrates the presence of a clinically significant pathogenic L. alstonii in the environments which could pose health risks to the occupants and visitors.