RESUMO
Molecular analysis of rabies virus can provide accurate diagnosis and information on its genetic diversity. The transportation of rabies brain samples from remote areas to a central laboratory is challenging owing to biohazard risks and decomposability. We investigated the utility of used lateral flow devices (LFDs) for subsequent molecular analysis and assessed the necessary storage temperatures. Using RNA extracted from used LFD strips, we performed conventional reverse transcription-PCR (RT-PCR) using an LN34 primer set to amplify short fragments (165 bp) for rabies virus detection and the P1-304 primer set to amplify long fragments of the entire N gene amplicon (1,506 bp) for phylogenetic analysis. Among 71 used LFDs stored in a refrigerator and 64 used LFDs stored at room temperature, the LN34 assay showed high sensitivities (96.2% and 100%, respectively) for the diagnosis of rabies, regardless of the storage temperature. A significant reduction in the sensitivity of rabies diagnosis was observed when using the P1-304 primer set for used LFDs stored at room temperature compared to those stored at refrigeration temperature (20.9% versus 100%; P < 0.05). Subsequent sequencing and phylogenetic analysis were successfully performed using the amplicons generated by the P1-304 RT-PCR assays. Used LFDs are thus promising resources for rabies virus RNA detection and sequence analysis. Virus detection via RT-PCR, amplifying a short fragment, was possible regardless of the storage temperature of the used LFDs. However, refrigerated storage is recommended for RT-PCR amplification of long fragments for phylogenetic analysis.
Assuntos
Vírus da Raiva , Raiva , Humanos , Vírus da Raiva/genética , Raiva/diagnóstico , Filogenia , RNA Viral/genética , RNA Viral/análise , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Expansion of the use of lateral flow devices (LFD) for animal rabies diagnosis can help mitigate the widespread underreporting of rabies. However, this has been hindered by the limited number and small sample size of previous studies. To overcome this limitation, we conducted a multicenter study with a larger sample size to assess the diagnostic accuracy of the ADTEC LFD for postmortem rabies diagnosis in animals. Thirteen governmental animal diagnostic laboratories in the Philippines were involved in this study, and 791 animals suspected of having rabies were tested using both the direct fluorescence antibody test (DFAT) and ADTEC LFD between August 2021 and October 2022. The LFD demonstrated a sensitivity of 96.3% [95% confidence interval (CI): 94.1%-97.9%] and a specificity of 99.7% (95% CI: 98.4%-100%). Notably, false-negative results were more likely to occur in laboratories with lower annual processing volumes of rabies samples in the previous years (adjusted odds ratio 4.97, 95% CI: 1.49-16.53). In this multicenter study, the high sensitivity and specificity of the LFD for the diagnosis of animal rabies, compared to that of the DFAT, was demonstrated, yet concerns regarding false-negative results remain. In areas with limited experience in processing rabies samples, it is essential to provide comprehensive training and careful attention during implementation.
Assuntos
Doenças do Cão , Vírus da Raiva , Raiva , Animais , Cães , Raiva/diagnóstico , Raiva/veterinária , Filipinas , Laboratórios , Doenças do Cão/diagnóstico , Sensibilidade e EspecificidadeRESUMO
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease and a possible cause of Crohn's disease in humans. A total of 70 blood and fecal samples were collected from water buffaloes in selected municipalities of Nueva Ecija for ELISA and qPCR assay. Results revealed presence of antibodies of MAP in 3 serum samples for ELISA. The qPCR assay was carried out using standard curve method targeting the MAP specific insertion element IS900. Results revealed that 10 of the samples were positive for MAP DNA in qPCR. ELISA was able to detect antibodies for MAP showing 2.48% infection rate among the 70 buffaloes tested using blood serum samples. On the other hand, qPCR was able to detect MAP using IS900 showed 14.28% infection rate among buffaloes tested using fecal samples. Nucleotide sequence of isolated MAP showed high homology (99-100%) among the reported MAP isolates in the GenBank.