A TaqMan minor groove binder probe-based quantitative reverse transcription polymerase chain reaction for detection and quantification of chikungunya virus.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus with widespread distribution across the globe. Since 2016, CHIKV re-emerged in several countries including Indian subcontinent and Southeast Asia. A proper diagnostic tool for early diagnosis of CHIKV infection is crucial to facilitate patient management and control virus transmission at the earliest stage of outbreak. Therefore, a TaqMan minor groove binder (MGB) probe-based quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay was developed to detect and quantify the CHIKV. The primers and probe were designed based on a conserved genomic region of 730 global CHIKV sequences that is located between nsP1 and nsP2 genes. The nucleotide mismatches of primers and probe with 730 global CHIKV sequences and 13 alphaviruses were then analysed in silico. In this study, the last 5 nucleotides at 3' end of primers and 5' end of probe were considered to be the critical regions for priming. In silico analysis revealed that the critical regions of primers and probe were at least 99.6% matched with the 730 global CHIKV sequences. Besides, the primers and probe showed at least 5/20 (25.0%) and 4/17 (23.5%) nucleotide mismatches with 13 alphaviruses respectively. The amplification efficiency of qRT-PCR assay was 100.59% (95% CI= 93.06, 109.33) with a R2 score of 0.957. Its limit of detection (LOD) at 95% probability level was 16.6 CHIKV RNA copies (95% CI= 12.9, 28.9). The qRT-PCR assay was specific to CHIKV without cross-reacting with all dengue virus serotypes, Getah virus, Tembusu virus and Zika virus. The diagnostic results of qRT-PCR assay were perfectly agreed (k=1.000, p=0.003) with a commercial trioplex assay, with sensitivity of 100% (95% CI= 61, 100) and specificity of 100% (95% CI= 44, 100). Overall, the developed qRT-PCR assay is ideal for rapid, sensitive and specific detection as well as quantification of CHIKV.

Metformin as a potential disease-modifying drug in osteoarthritis: a systematic review of pre-clinical and human studies.

OBJECTIVE: Osteoarthritis causes significant pain and disability with no approved disease-modifying drugs. We systematically reviewed the evidence from both pre-clinical and human studies for the potential disease-modifying effect of metformin in osteoarthritis. METHODS: Ovid Medline, Embase and CINAHL were searched between inception and June 2021 using MeSH terms and key words to identify studies examining the association between metformin use and outcome measures related to osteoarthritis. Two reviewers performed the risk of bias assessment and 3 reviewers extracted data independently. Qualitative evidence synthesis was performed. This systematic review is registered on PROSPERO (CRD42021261052 and CRD42021261060). RESULTS: Fifteen (10 pre-clinical and 5 human) studies were included. Most studies (10 pre-clinical and 3 human) assessed the effect of metformin using knee osteoarthritis models. In pre-clinical studies, metformin was assessed for the effect on structural outcomes (n = 10); immunomodulation (n = 5); pain (n = 4); and molecular pathways of its effect in osteoarthritis (n = 7). For human studies, metformin was evaluated for the effect on structural progression (n = 3); pain (n = 1); and immunomodulation (n = 1). Overall, pre-clinical studies consistently showed metformin having a chondroprotective, immunomodulatory and analgesic effect in osteoarthritis, predominantly mediated by adenosine monophosphate-activated protein kinase activation. Evidence from human studies, although limited, was consistent with findings in pre-clinical studies. CONCLUSION: We found consistent evidence across pre-clinical and human studies to support a favourable effect of metformin on chondroprotection, immunomodulation and pain reduction in knee osteoarthritis. Further high-quality clinical trials are needed to confirm these findings as metformin could be a novel therapeutic drug for the treatment of osteoarthritis.