Semi-nested RT-PCR enables sensitive and high-throughput detection of SARS-CoV-2 based on melting analysis.

BACKGROUND: Asymptomatic transmission was found to be the Achilles' heel of the symptom-based screening strategy, necessitating the implementation of mass testing to efficiently contain the transmission of COVID-19 pandemic. However, the global shortage of molecular reagents and the low throughput of available realtime PCR facilities were major limiting factors. METHODS: A novel semi-nested and heptaplex (7-plex) RT-PCR assay with melting analysis for detection of SARS-CoV-2 RNA has been established for either individual testing or 96-sample pooled testing. The complex melting spectrum collected from the heptaplex RT-PCR amplicons was interpreted with the support of an artificial intelligence algorithm for the detection of SARS-CoV-2 RNA. The analytical and clinical performance of the semi-nested RT-PCR assay was evaluated using RNAs synthesized in-vitro and those isolated from nasopharyngeal samples. RESULTS: The LOD of the assay for individual testing was estimated to be 7.2 copies/reaction. Clinical performance evaluation indicated a sensitivity of 100% (95% CI: 97.83-100) and a specificity of 99.87% (95% CI: 99.55-99.98). More importantly, the assay supports a breakthrough sample pooling method, which makes possible parallel screening of up to 96 samples in one real-time PCR well without loss of sensitivity. As a result, up to 8,820 individual pre-amplified samples could be screened for SARS-CoV-2 within each 96-well plate of realtime PCR using the pooled testing procedure. CONCLUSION: The novel semi-nested RT-PCR assay provides a solution for highly multiplex (7-plex) detection of SARS-CoV-2 and enables 96-sample pooled detection for increase of testing capacity. .

Índices biométricos oculares en vietnamitas de 46 a 65 años de edad / Ocular biometric indices in Vietnamese 46 to 65 years of age

Introducción: La longitud axial ocular, la profundidad de la cámara anterior y el grosor corneal central, son tres índices biométricos oculares importantes. Estas medidas son útiles para mostrar los cambios en la población vietnamita con presbicia. Objetivos: Determinar los índices biométricos oculares, longitud axial ocular, profundidad de la cámara anterior y espesor corneal central, en población vietnamita y evaluar la correlación entre ellos y con la edad y el sexo. Métodos: Se realizó un estudio transversal en población vietnamita, con edad de 46 a 65 años. Se recogieron los datos de longitud axial ocular, profundidad de la cámara anterior y grosor corneal central. Se utilizaron la prueba t de Student y ANOVA para comparar las medias de los índices, agrupados por edad y sexo. La relación entre los índices biométricos oculares fue probada mediante la correlación de Pearson, con un nivel de significación de p < 0,05. Resultados: Se analizaron 390 ojos de 195 personas. La longitud media del eje ocular fue 23,13 ± 0,66 mm, la profundidad de la cámara anterior, 3,15 ± 0,36 mm, el grosor corneal central, 529,15 ± 30,57 µm. Los tres índices biométricos disminuyeron con la edad y fueron mayores en los hombres (p < 0,05). La longitud del eje ocular tuvo relación positiva con la profundidad de la cámara anterior (r = 0,411 y p < 0,001) y el espesor corneal central (r = 0,141 y p < 0,001). No hubo relación entre la profundidad de la cámara anterior y el grosor corneal central (r = 0,039 y p = 0,44). Conclusión: Los tres índices biométricos oculares disminuyeron con la edad y fueron mayores en los hombres. La longitud del eje ocular se relacionó con la profundidad de la cámara anterior y el grosor de la córnea central(AU)

Introduction: Ocular axial length, anterior chamber depth and central corneal thickness are three important ocular biometric indices. These measurements are useful to show changes in the Vietnamese population with presbyopia. Objectives: To determine the ocular biometric indices, ocular axial length, anterior chamber depth and central corneal thickness, in Vietnamese population and evaluate the correlation between these indices. Methods: A cross-sectional study was carried out in a Vietnamese population, aged 46 to 65 years. Data on ocular axial length, anterior chamber depth and central corneal thickness were collected. The Student's t test and ANOVA were used to compare the means of the indices, grouped by age and sex. The relationship between the ocular biometric indices was tested using Pearson's correlation, with a significance level of p <0.05. Results: 390 eyes of 195 people were analyzed. The mean length of the ocular axis was 23.13 ± 0.66 mm, the depth of the anterior chamber, 3.15 ± 0.36 mm, and the central corneal thickness, 529.15 ± 30.57 µm. The three biometric indices decreased with age and were higher in men (p <0.05). The length of the ocular axis had a positive relationship with the depth of the anterior chamber (r = 0.411 and p <0.001) and the central corneal thickness (r = 0.141 and p <0.001). There was no relationship between anterior chamber depth and central corneal thickness (r = 0.039 and p = 0.44). Conclusion: Three ocular biometric indices decreased with age and were higher in men. The length of the ocular axis was related to the depth of the anterior chamber and the thickness of the central cornea(AU)

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.

Expression of aryl hydrocarbon receptor, inflammatory cytokines, and incidence of rheumatoid arthritis in Vietnamese dioxin-exposed people.

Many Vietnamese citizens have been and continue to be inadvertently exposed to dioxins and dioxin-like compounds deposited in the country during the Vietnam War. Dioxins may be involved in the pathogenesis of inflammatory diseases in part via by affecting expression of aryl hydrocarbon receptor (Ahr) and inflammatory cytokines in animal models. As the role of the Ahr in dioxin-exposed people is not well defined, a study was conducted to examine gene expression levels of Ahr, inflammatory cytokines, and the incidence of diseases in dioxin-exposed citizens who had/still resided near a heavily dioxin-contaminated area in Vietnam. Whole blood from citizens at/around Da Nang airbase and control individuals living in unsprayed areas was collected. Serum levels of dioxins were analyzed by using a dioxins-responsive chemical-activated luciferase gene expression bioassay. Gene expression of Ahr, interleukin (IL)-1ß, TNFα, IL-6, and IL-22 in whole blood was examined by quantitative real-time PCR. The results showed levels of dioxins and expression of Ahr, IL-1ß, TNFα, and IL-6 were up-regulated while IL-22 expression was down-regulated in dioxin-exposed people. Various disease incidences in the study subjects was also examined. Interestingly, the incidence of rheumatoid arthritis (RA) in these individuals was increased compared to the estimated prevalence of this disease in the general Vietnamese population. Analyses also showed that expression levels of Ahr correlated to those of IL-6 and IL-22 in the dioxin-exposed people. Taken together, dioxins might be involved in an up-regulated expression of Ahr that might possibly relate to changes in level of inflammatory cytokines and, ultimately, in the incidence of select diseases in residents of Vietnam who had/continue to live near a dioxins-contaminated site.