CBPH assay for the highest sensitive detection of SARS-COV-2 in the semen.

BACKGROUND: Great concerns have been raised on SARS-CoV-2 impact on men's andrological well-being, and many studies have attempted to determine whether SARS-CoV-2 is present in the semen and till now the data are unclear and somehow ambiguous. However, these studies used quantitative real-time (qRT) PCR, which is not sufficiently sensitive to detect nucleic acids in clinical samples with a low viral load. METHODS: The clinical performance of various nucleic acid detection methods (qRT-PCR, OSN-qRT-PCR, cd-PCR, and CBPH) was assessed for SARS-CoV-2 using 236 clinical samples from laboratory-confirmed COVID-19 cases. Then, the presence of SARS-CoV-2 in the semen of 12 recovering patients was investigated using qRT-PCR, OSN-qRT-PCR, cd-PCR, and CBPH in parallel using 24 paired semen, blood, throat swab, and urine samples. RESULTS: The sensitivity and specificity along with AUC of CBPH was markedly higher than the other 3methods. Although qRT-PCR, OSN-qRT-PCR and cdPCR detected no SARS-CoV-2 RNA in throat swab, blood, urine, and semen samples of the 12 patients, CBPH detected the presence of SARS-CoV-2 genome fragments in semen samples, but not in paired urine samples, of 3 of 12 patients. The existing SARS-CoV-2 genome fragments were metabolized over time. CONCLUSIONS: Both OSN-qRT-PCR and cdPCR had better performance than qRT-PCR, and CBPH had the highest diagnostic performance in detecting SARS-CoV-2, which contributed the most improvement to the determination of the critical value in gray area samples with low vrial load, which then provides a rational screening strategy for studying the clearance of coronavirus in the semen over time in patients recovering from COVID-19. Although the presence of SARS-CoV-2 fragments in the semen was demonstrated by CBPH, COVID-19 is unlikely to be sexually transmitted from male partners for at least 3 months after hospital discharge.

Interspecies Phylogenetic Analysis of Clonorchis sinensis in High-incidence Areas of Hunan Province, China.

OBJECTIVE: This study aims to investigate the infection of Clonorchis sinensis ( C. sinensis) in high-incidence areas of Hunan Province, China. The phylogenetic analysis of the C. sinensis species in the highly infected areas was carried out. METHOD: Infection of the definitive human host and intermediate fish host by C. sinensis was investigated, and the mitochondrial genes cox1 and Nad1were used as genetic markers for phylogenetic analysis. RESULTS: In 2016-2020, the average population infection rate of Hunan was 1.38%, while in Tongdao County the rate was up to 26.90%, and the highest fish infection rate was detected in Qiyang County (99.44% in the dorsal fin of crucian carp). High genetic sequence similarity was observed in the samples from Qiyang and Lengshuitan which exhibited high homology with those from Guangdong and Gansu, whereas the parasitic species from Tongdao was highly homologous with those located in high-latitude areas. Moreover, no significant difference was found in the gene sequence of the parasitic species in definitive hosts dogs and cats. CONCLUSION: The systematically study of C. sinensis infection in the high-incidence areas will contribute greatly to the prevention and effectively controlling the spread of Clonorchis sinensis in Hunan Province The endemic of C. sinensis infection in Hunan Province is the result of co-action of local and foreign parasite species.

Risks associated with cryopreserved semen in a human sperm bank during and after the COVID-19 pandemic.

RESEARCH QUESTION: What are the risks associated with cryopreserved semen collected during and after the coronavirus disease 2019 (COVID-19) pandemic wave in Wuhan, China? DESIGN: Retrospective cohort study involving young adult men who were qualified sperm donors at the Hunan Province Human Sperm Bank (China) during the pandemic wave (1 January 2020 to 30 January 2020) and after the wave and return to work (7 April 2020 to 30 May 30 2020). One hundred paired semen and blood specimens from 100 donors were included. One-step single-tube nested quantitative real-time polymerase chain reaction (OSN-qRT-PCR) was used to detect SARS-CoV-2. Moreover, to control the unacceptable risk of false-negative results, a second round of screening was performed with pooled RNA from negative semen samples using crystal digital PCR (cd-PCR). RESULTS: For individual blood and semen samples, the target genes, namely the nucleocapsid protein (N) and open reading frame (ORF-1ab) genes, tested negative in all of the 100 paired samples. Further, as per cd-PCR results, there were >20,000 droplets per well in the RNA for each combined sample and no positive droplets were present for either of the aforementioned target genes. A total of 100 paired semen and blood samples from these two groups tested negative for SARS-CoV-2. CONCLUSIONS: Cryopreserved semen at the Hunan Province Human Sperm Bank during and after the COVID-19 pandemic wave was free of SARS-CoV-2 and was judged safe for external use in the future.

A novel and highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus.

The sensitivity of qRT-PCR assay is not adequate for the detection of the samples with lower viral load, particularly in the cerebrospinal fluid (CSF) of patients. Here, we present the development of a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of human enterovirus (HEV). The clinical performance of both RTN RT-PCR and qRT-PCR was also tested and compared using 140 CSF and fecal specimens. The sensitivities of RTN RT-PCR assay for EV71, Coxsackievirus A (CVA)16, CVA6 and CVA10 achieved 10-8 dilution with a corresponding Ct value of 38.20, 36.45, 36.75, and 36.45, respectively, which is equal to traditional two-step nested RT-PCR assay and approximately 2-10-fold lower than that of qRT-PCR assay. The specificity of RTN RT-PCR assay was extensively analyzed insilico and subsequently verified using the reference isolates and clinical samples. Sixteen qRT-PCR-negative samples were detected by RTN RT-PCR and a variety of enterovirus serotypes was identified by sequencing of inner PCR products. We conclude RTN RT-PCR is more sensitive than qRT-PCR for the detection of HEV in clinical samples.

An Improved Barcoded Oligonucleotide Primers-based Next-generation Sequencing Approach for Direct Identification of Viral Pathogens in Clinical Specimens.

OBJECTIVE: To provide a feasible and cost-effective next-generation sequencing (NGS) method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples. METHODS: Viruses from cell culture materials or clinical specimens were identified following an improved NGS procedure: reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide (random hexamer or non-ribosomal hexanucleotide) primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline. RESULTS: NGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease (HFMD) and identified other potential viruses or differentiated misdiagnosis events. CONCLUSION: The improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice.

Correlation analysis of EV71 detection and case severity in hand, foot, and mouth disease in the Hunan Province of China.

An increase in the incidence of hand, foot and mouth disease (HFMD) cases has been observed in the Hunan province of mainland China since 2009 with a particularly higher level of severe cases in 2010-2012. Intestinal viruses of the picornaviridae family are responsible for the human syndrome associated with HFMD with enterovirus 71 (EV71) and Coxsackievirus A16 (Cox A16) being the most common causative strains. HFMD cases associated with EV71 are generally more severe with an increased association of morbidity and mortality. In this study, the etiology surveillance data of HFMD cases in Hunan province from March 2010 to October 2012 were analyzed to determine if there is a statistically relevant linear correlation exists between the detection rate of EV71 in mild cases and the proportion of severe cases among all HFMD patients. As the cases progressed from mild to severe to fatal, the likelihood of EV71 detection increased (25.78%, 52.20% and 84.18%, respectively). For all cases in the timeframe evaluated in this study, the presence of virus was detected in 63.21% of cases; among cases showing positivity for virus, EV71 infection accounted for 50.14%. These results provide evidence to support the observed higher morbidity and mortality associated with this outbreak and emphasizes the importance of early detection in order to implement necessary prevention measures to mitigate disease progression.

[Risk factors of death cases of hand-foot-and-mouth disease in Hunan province].

OBJECTIVE: To study risk factors of death cases of hand foot and mouth diseases (HFMD) in Hunan province, so as to provide scientific evidence for further prevention and control. METHODS: The 105 death cases of HFMD between January and October, 2010 in Hunan Province were selected as case group; and the 210 survival cases of serious HFMD, which were matched by gender and resident places with a ratio at 2:1 in the same period in Hunan were selected as control group. The basic information, hospitalized experience and previous medical history had been surveyed and the relevant risk factors were analyzed by single factor and multi-factor logistic regression. RESULTS: In case group, 79.05% (83/105) of the cases lived in rural area and 9.52% (10/105) of the cases lived in urban-rural midst area. In control group, 87.62% (184/210) of the cases lived in rural area and 11.43% (24/210) of the cases lived in urban-rural midst area. In case group, 59.05% (62/105) of the patients first visited rural (private) clinics and 20.00% (21/105) first visited community hospitals in villages and towns; while in control group, 43.81% (92/210) and 13.33% (28/210) chose rural (private) clinics and community hospitals in villages and towns as the first choice respectively.22.86% (24/105) of the case group and 39.05% (82/210) of the control group were diagnosed as HFMD in their first visit to hospital.27.62% (29/105) of the case group and 7.14% (15/210) in control group were provided pyrazolone in the treatment. For glucocorticoid, 80.95% (85/105) and 5.71% (6/105) of the case group were given as treatment by rural (private) clinics and community hospitals in villages and towns separately; while the proportions in the control group were 41.43% (87/210) and 0.48% (1/210) respectively. For antibiotics, 35.24% (37/105) and 23.81% (25/105) of the case group were prescribed by rural (private) clinics and community hospitals in villages and towns separately; while the percentages in the control group were 15.71% (33/210) and 7.14% (15/210). 3.81% (4/105) of the case group and 11.90% (25/210) of the control group were vaccinated in one month before the onset. The results of single-factor logistic regression indicated that living in rural areas (OR = 0.075, 95%CI: 0.016 - 0.343) and in rural-urban midst areas (OR = 0.069, 95%CI: 0.013 - 0.368), diagnosis of HFMD in the first visit to hospital (OR = 0.463, 95%CI: 0.271 - 0.788) and vaccination one month before the onset (OR = 0.293, 95%CI: 0.099 - 0.866) were four protective factors; while rural (private) clinics as the first choice (OR = 4.717, 95%CI: 1.891 - 11.767), community hospital in villages and towns as the first choice (OR = 5.250, 95%CI: 1.883 - 14.641), medication of pyrazolone (OR = 4.961, 95%CI: 2.520 - 9.766), medication of glucocorticoid in rural (private) clinics (OR = 6.009, 95%CI: 3.435 - 10.510) and in community hospital in villages and towns (OR = 12.667, 95%CI: 1.505 - 106.638), medication of antibiotics in rural (private) clinics (OR = 2.918, 95%CI: 1.690 - 5.040) and in community hospital in villages and towns (OR = 4.062, 95%CI: 2.036 - 8.108) were seven risk factors. The results of multi-factors logistic regression showed that medication of pyrazolone (OR = 2.311, 95%CI: 1.062 - 5.030), medication of glucocorticoid in rural (private) clinics (OR = 5.480, 95%CI: 3.039 - 9.880), medication of antibiotics in rural (private) clinics (OR = 2.430, 95%CI: 1.301 - 4.538) and medication of antibiotics in community hospitals in villages and towns (OR = 3.344, 95%CI: 1.477 - 7.569) were the risk factors of death of HFMD. CONCLUSION: The risk factors of HFMD deaths include the medication of pyrazolone, glucocorticoid and antibiotics by rural (private) clinics and medical institutions in villages and towns. The department concerned should revise the technical manual to standardize the medication of the above drugs.

[Immuno-screening of Schistosoma japonicum cercariae cDNA library by the sera of anti-soluble cercariae 66 to approximately 68 kD antigens].

OBJECTIVE: To obtain the coding genes related to Schistosoma japonicum (Sj) cercariae 66 to approximately 68 kD antigens,and to provide antigens for diagnosis and vaccine of schistosomiasis. METHODS: Sj cercariae cDNA library was screened using the monospecific anti-sera of rabbit against soluble cercariae 66 to approximately 68 kD antigens as probes.The inserted cDNA fragments of the positive clones were amplified with PCR and identified by agarose gel electrophoresis. Four strong positive clones were further sequenced and analyzed through the internet NCBI/BLAST software. RESULTS: Twenty-one positive clones were obtained, 10 of which revealed a single band (0.5 to approximately 3.0 kb).The 4 strong positive clones showed high identity to SJCHGC05187,SJCHGC05173,SJCHGC06989, and SJCHGC01894 at the nucleotide level. CONCLUSION: Four coding genes related with Sj antigens are obtained.