Establishment of an in-house real-time RT-PCR assay for the detection of severe acute respiratory syndrome coronavirus 2 using the first World Health Organization international standard in a resource-limited country.

BACKGROUND: The COVID-19 pandemic caused by SARS-CoV-2 remains public health burdens and many unresolved issues worldwide. Molecular assays based on real-time RT-PCR are critical for the detection of SARS-CoV-2 in clinical specimens from patients suspected of COVID-19. OBJECTIVE: We aimed to establish and validate an in-house real-time RT-PCR for the detection of SARS-CoV-2. METHODOLOGY: Primers and probes sets in our in-house real-time RT-PCR assay were designed in conserved regions of the N and E target genes. Optimized multiplex real-time RT-PCR assay was validated using the first WHO International Standard (NIBSC code: 20/146) and evaluated clinical performance. RESULTS: The limit of detection validated using the first WHO International Standard was 159 IU/ml for both E and N target genes. The evaluation of clinical performance on 170 clinical samples showed a positive percent agreement of 100% and the negative percent agreement of 99.08% for both target genes. The Kappa value of 0.99 was an excellent agreement, the strong correlation of Ct values observed between two tests with r2  = 0.84 for the E gene and 0.87 for the N gene. Notably, we assessed on 60 paired saliva and nasopharyngeal samples. The overall agreement was 91.66%, and Kappa value of 0.74 showed a high agreement between two types of samples. When using nasopharyngeal swabs as the reference standard, positive percent agreement, and negative percent agreement were 91.83% and 90.90%, respectively. CONCLUSION: In the present study, we established and validated an in-house real-time RT-PCR for molecular detection of SARS-CoV-2 in a resource-limited country.

BK Polyomavirus Subtypes IVc-1 and Ib-1 in Vietnamese Renal Transplant Recipients.

We report here the nearly complete genome sequences of two human BK polyomavirus (BKV) strains recovered from two Vietnamese renal allograft recipients and belonging to subtypes IVc1 (strain VN_PBK185) and Ib1 (strain VN_PBK212). The genome sequences of VN_PBK185 and VN_PBK212 were highly similar (99.9% nucleotide identity) to the reference BKV strains VNM-1 and VNM-9, respectively.

Establishment of Recombinase Polymerase Amplification assay for rapid and sensitive detection of Orientia tsutsugamushi in Southeast Asia.

Scrub typhus, caused by Orientia tsutsugamushi, is a common fever in parts of Southern and Southeast Asia. As delayed diagnosis of scrub typhus leads to inappropriate treatment and high mortality rates, of up to 70%, sensitive and rapid detection of O. tsutsugamushi is required for timely and appropriate treatment. Molecular assays, such as PCR and real-time PCR, have been shown to be more sensitive than conventional immunoassay, however, they are only available in centralized laboratories. In contrast to PCR assays, Recombinase Polymerase Amplification (RPA) is conducted under a constant temperature ranging from 24°C to 45°C. Therefore, this technology is very promising for nucleic acid testing in the field, and in resource-limited areas. An RPA assay for the detection of O. tsutsugamushi based on the target gene encoding for the 47 kDa outer membrane protein has been reported, but the primer and probe sequences of this assay are suboptimal for detection of the majority of recently published sequences of O. tsutsugamushi isolates from Southeast Asia. We have established a real-time RPA assay with primer and probe sequences that are optimized for most Southeast Asia's isolates of O. tsutsugamushi. As a result, the new RPA assay showed better performance than the previous assay in detecting O. tsutsugamushi in clinical samples of scrub typhus cases found in Vietnam. The specificity of RPA assay was also evaluated using genomic DNA from microorganisms commonly encountered in the differential diagnosis of scrub typhus, and blood samples from healthy controls and O. tsutsugamushi negative confirmed cases.

Evaluation of the expression levels of BRAFV600E mRNA in primary tumors of thyroid cancer using an ultrasensitive mutation assay.

BACKGROUND: The BRAFV600E gene encodes for the mutant BRAFV600E protein, which triggers downstream oncogenic signaling in thyroid cancer. Since most currently available methods have focused on detecting BRAFV600E mutations in tumor DNA, there is limited information about the level of BRAFV600E mRNA in primary tumors of thyroid cancer, and the diagnostic relevance of these RNA mutations is not known. METHODS: Sixty-two patients with thyroid cancer and non-malignant thyroid disease were included in the study. Armed with an ultrasensitive technique for mRNA-based mutation analysis based on a two step RT-qPCR method, we analysed the expression levels of the mutated BRAFV600E mRNA in formalin-fixed paraffin-embedded samples of thyroid tissues. Sanger sequencing for detection of BRAFV600E DNA was performed in parallel for comparison and normalization of BRAFV600E mRNA expression levels. RESULTS: The mRNA-based mutation detection assay enables detection of the BRAFV600E mRNA transcripts in a 10,000-fold excess of wildtype BRAF counterparts. While BRAFV600E mutations could be detected by Sanger sequencing in 13 out of 32 malignant thyroid cancer FFPE tissue samples, the mRNA-based assay detected mutations in additionally 5 cases, improving the detection rate from 40.6 to 56.3%. Furthermore, we observed a surprisingly large, 3-log variability, in the expression level of the BRAFV600E mRNA in FFPE samples of thyroid cancer tissue. CONCLUSIONS: The expression levels of BRAFV600E mRNA was characterized in the primary tumors of thyroid cancer using an ultrasensitive mRNA-based mutation assay. Our data inspires further studies on the prognostic and diagnostic relevance of the BRAFV600E mRNA levels as a molecular biomarker for the diagnosis and monitoring of various genetic and malignant diseases.

T-817MA, a neurotrophic agent, ameliorates the deficits in adult neurogenesis and spatial memory in rats infused i.c.v. with amyloid-beta peptide.

BACKGROUND AND PURPOSE: Adult neurogenesis occurs throughout life in the subgranular zone and the dentate gyrus of the hippocampus. Deficient neurogenesis may be responsible for deficient hippocampal functions in neurodegenerative disorders such as Alzheimer's disease (AD). T-817MA [1-{3-[2-(1-Benzothiophen-5-yl)ethoxy] propyl}-3-azetidinol maleate] is a newly synthesized agent for AD treatment with neuroprotective effects against toxicity from amyloid-beta peptide (Abeta) and actions promoting neurite outgrowth in vitro. Furthermore, systemic administration of T-817MA ameliorated cognitive dysfunctions caused by neurodegeneration in a rat model of AD, induced by intracerebroventricular (i.c.v.) infusion of Abeta. The present study investigated quantitative relationships between spatial memory performance in Abeta-infused rats and hippocampal neurogenesis, and the effects of T-817MA on neuronal proliferation in vivo. EXPERIMENTAL APPROACH: Seven weeks after infusion of Abeta (peptide 1-40; 300 pmol.day(-1); i.c.v.), rats were tested in a place learning task in which they were required to alternately visit two reward places in an open field to obtain intracranial self-stimulation as rewards. KEY RESULTS: Rats given Abeta infusions for 10 weeks displayed spatial memory impairments and a decrease in neurogenesis compared with those infused with vehicle. Treatment of the Abeta-infused rats with T-817MA (8.4 mg.kg(-1).day(-1), p.o.) significantly increased hippocampal neurogenesis and ameliorated spatial learning impairments. Furthermore, spatial learning in the task was significantly correlated with neurogenesis. CONCLUSIONS AND IMPLICATIONS: These results suggest that defective hippocampal neurogenesis is a new target for AD treatment. The neurotrophic compound T-817MA increased hippocampal neurogenesis in an AD model and might be useful for treatment of AD patients.

Ameliorative effects of a neuroprotective agent, T-817MA, on place learning deficits induced by continuous infusion of amyloid-beta peptide (1-40) in rats.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by cognitive decline due to neuronal loss and neural network dysfunction. It has been postulated that progressive neuronal loss in AD is consequence of the neurotoxic properties of the amyloid-beta peptide (Abeta). In the present study, we investigated the effect of T-817MA (1-{3-[2-(1-benzothiophen-5-yl)ethoxy] propyl}-3-azetidinol maleate), a newly synthesized neurotrophic compound, on place learning deficits in rats with hippocampal damages. To induce granule cell loss in the dentate gyrus (DG) of the hippocampus, Abeta (1-40) was continuously infused (300 pmol/day) into the cerebral ventricle using a mini-osmotic pump for 5 weeks. Three weeks after the Abeta infusion, the rats were tested in a place learning task, which required them to alternatively visit two diametrically opposed areas in an open field to obtain intracranial self-stimulation reward. The results indicated that the Abeta-infused rats without treatment of T-817MA displayed learning impairment in the task; their performance level was significantly inferior to that of the vehicle rats. Treatment of T-817MA (8.4 mg/kg/day, p.o.) significantly improved the task performance of the Abeta-infused rats. Furthermore, T-817MA prevented granule cell loss due to Abeta-infusion, which was correlated to task performance of the rats. However, other cognitive enhancer, an acetylcholinesterase inhibitor, had no such effects. The results demonstrated that T-817MA ameliorated learning deficits induced by Abeta infusion, which might be attributed to neuroprotection in the hippocampus.