Detection of monkeypox virus using helicase dependent amplification and recombinase polymerase amplification combined with lateral flow test.

The monkeypox virus (MPXV) is a zoonotic DNA virus that belongs to the poxvirus family. Conventional laboratory methods for detecting MPXV are complex and expensive, making them unsuitable for detecting the virus in regions with limited resources. In this study, we using the Helicase dependent amplification (HDA) method and the Recombinase polymerase amplification (RPA) technique in combination with the lateral flow test (LFT), together with a self-designed qPCR technique for the detection of the MPXV specific conserved fragment F3L, to compare the sensitivity and specificity of the three assays. By analyzing the sensitivity detection results using Probit, it can be seen that the limit of detection (LOD) of the HDA-LFT detection target is 9.86 copies/µL (95% confidence interval, CI 7.52 copies/µL lower bound), the RPA-LFT detection target is 6.97 copies/µL (95% CI 3.90 copies/µL lower bound), and the qPCR detection target is 479.24 copies/mL (95% CI 273.81 copies/mL lower bound). The specificity test results showed that the specificity of the three methods mentioned above was higher than 90% in detecting pseudoviruses of the same genus of MPXV. The simple, highly sensitive, and specific MPXV assay developed in this study is anticipated to provide a solid foundation for future applications in the early screening, diagnosis, and evaluation of the efficacy of MPXV. This is the first time the HDA-LFT assay has been utilized to detect MPXV infection.

Distributive characteristics of the CYP2C9 and AGTR1 genetic polymorphisms in Han Chinese hypertensive patients: a retrospective study.

BACKGROUND: There is an individual variation in response to antihypertensive effect of the angiotensin II receptor antagonist. This study aimed to determine the allele and genotype frequencies of CYP2C9 and AGTR1 genetic polymorphisms and explore the potential role of these polymorphisms in guiding the selection of angiotensinIIreceptor antagonist in Han Chinese hypertensive patients. METHODS: Totally 2419 Han Chinese hypertensive patients and 126 normotensive controls were recruited in this study. Venous blood samples were collected from each patient, and the genetic polymorphisms of CYP2C9 and AGTR1 were assessed using a gene chip platform. The allele and genotype frequency of each gene and the combined genotypes in this study were analyzed respectively. RESULTS: The gene chip analysis identified an allelic frequency of 96.51% for CYP2C9*1 and 3.49% for CYP2C9*3 in the cohort of Han Chinese hypertensive patients. Statistical analysis showed that the frequency of wild-type homozygous for CYP2C9*1/*1 was 93.30%, while the frequency of heterozygous for *1/*3 or mutant homozygous for *3/*3 was 6.41% or 0.29%. Meanwhile, we detected allelic frequencies of 95.06% and 4.94% for the A and C allele of AGTR1, respectively. While the genotype frequency of wild-type homozygous for AA was 90.41%, the frequency of heterozygous for AC or mutant homozygous for CC was 9.30% or 0.29%. Notably, we observed that 84.66% (2048/2419) of the subjects exhibited a combined genotype of CYP2C9 and AGTR1 as *1/*1 + AA, while the combined genotypes *3/*3 + AC or *3/*3 + CC were not detected in hypertension patients. Besides, no significant association was found between normotensive controls and hypertensive patients, or among the three grades of hypertensive patients. CONCLUSIONS: These data revealed the polymorphisms characteristics of CYP2C9 and AGTR1 in Han Chinese hypertensive patients, providing valuable information for genotype-based antihypertension therapy in prospective clinical studies in the future.

Rapid point-of-care testing for SARS-CoV-2 virus nucleic acid detection by an isothermal and nonenzymatic Signal amplification system coupled with a lateral flow immunoassay strip.

An outbreak of a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), began in December 2019. Accurate, rapid, convenient, and relatively inexpensive diagnostic methods for SARS-CoV-2 infection are important for public health and optimal clinical care. The current gold standard for diagnosing SARS-CoV-2 infection is reverse transcription-polymerase chain reaction (RT-PCR). However, RTPCR assays are designed for use in well-equipped laboratories with sophisticated laboratory infrastructure and highly trained technicians, and are unsuitable for use in under-equipped laboratories and in the field. In this study, we report the development of an accurate, rapid, and easy-to-implement isothermal and nonenzymatic signal amplification system (a catalytic hairpin assembly (CHA) reaction) coupled with a lateral flow immunoassay (LFIA) strip-based detection method that can detect SARSCoV-2 in oropharyngeal swab samples. Our method avoids RNA isolation, PCR amplification, and elaborate result analysis, which typically takes 6-8 h. The entire CHA-LFIA detection method, from nasopharyngeal sampling to obtaining test results, takes less than 90 min. Such methods are simple and require no expensive equipment, only a simple thermostatically controlled water bath and a fluorescence reader device. We validated our method using synthetic oligonucleotides and clinical samples from 15 patients with SARS-CoV-2 infection and 15 healthy individuals. Our detection method provides a fast, simple, and sensitive (with a limit of detection (LoD) of 2000 copies/mL) alternative to the SARS-CoV-2 RT-PCR assay, with 100 % positive and negative predictive agreements.

Comparison of Indirect Immunofluorescence and Passive Particle Agglutination for the Detection of Mycoplasma Pneumoniae Antibodies.

BACKGROUND: Mycoplasma pneumoniae (MP) is the most predominant pathogen causing pneumonia. The present study compares two serological assays, the indirect immunofluorescence assay (IFA) and the passive particle agglutination assay (PPA), in order to assist clinicians in selecting accurate diagnosis methods. METHODS: Sera from 127 patients suffering from mycoplasma pneumonia and 76 from the healthy group were analyzed simultaneously by PPA and IFA. Receiver operating characteristic analyses were performed to evaluate the detection value of PPA and IFA for mycoplasma pneumoniae. The kappa coefficient was analyzed to evaluate the agreement between the IFA and PPA assay. RESULTS: The AUC of PPA and IFA was more than 0.70, suggesting both assays were acceptable in clinical efficacy for detecting mycoplasma pneumoniae. When ± 1:40 antibody titers were interpreted as negative, PPA showed the highest specificity, Youden index, and AUC (86.84%, 65.58%, and 0.828, respectively), and the kappa coefficient between PPA and IFA was 0.360. CONCLUSIONS: IFA and PPA assays have advantages and disadvantages in the detection of MP antibodies. MP anti-bodies ± 1:40 antibody titers should be interpreted as negative to improve PPA detection abilities, and the consistency of the two methods was regular agreement. Clinicians should detect MP antibodies simultaneously with two methods or analyze paired samples with one method for diagnosing whether or not MP infection is present.

The aetiology of community associated pneumonia in children in Nanjing, China and aetiological patterns associated with age and season.

BACKGROUND: Viral and atypical bacterial pathogens play an important role in respiratory tract infection. Using the Pneumoslide IgM test, the presented study explored the aetiology of community-acquired pneumonia and investigated further whether there was an association between age or season and aetiological organisms. METHODS: Serum samples, taken between August 2011 and August 2013, from patients with CAP were tested with the Pneumoslide IgM kit. The Pneumoslide IgM technology can simultaneously diagnose 9 viral and atypical bacterial pathogens: Legionella pneumophila serogroup 1 (LP1), Mycoplasma pneumoniae (MP), Coxiella burnetii (COX), Chlamydophila pneumonia (CP), Adenovirus (ADV), Respiratory syncytial virus (RSV), Influenza A (INFA), Influenza B (INFB), Parainfluenza 1, 2 and 3 (PIVs). The data was analyzed by using Statistical Package for the Social Sciences for Windows (SPSS, version 11.0). RESULTS: Of a total of 1204 serum samples tested, 624 samples were positive. M. pneumoniae was the dominant pathogen, with INFB, PIVs, and RSV ranking second to fourth, respectively. The positive percentages of MP, INFB, PIVs and RSV were found to be associated with age, especially MP, INFB and PIVs. The positive percentages of MP, PIVs and RSV were also found to be associated with season. The positive percentage of MP in autumn was the highest. The positive percentages of LP1 in August and September, ADV in June and INFB in March were relatively higher than that in other months. CONCLUSIONS: The results show there were 4 main viral and atypical bacterial pathogens causing CAP in our study. Some pathogens were found to be associated with age and season. M. pneumoniae was the most predominant pathogen among these 9 pathogens. It is necessary to take preventative measures in order to prevent the spread of these pathogens in susceptible age groups during peak season.

Accessing HIV testing and treatment among men who have sex with men in China: a qualitative study.

Barriers to HIV testing and HIV care and treatment pose significant challenges to HIV prevention among men who have sex with men (MSM) in China. We carried out a qualitative study to identify barriers and facilitators to HIV testing and treatment among Chinese MSM. In 2012, seven focus group (FG) discussions were conducted with 49 MSM participants in Nanjing, China. Purposive sampling was used to recruit a diverse group of MSM participants. Semi-structured interviews were conducted to collect FG data. Major barriers to testing included gay- and HIV-related stigma and discrimination, relationship type and partner characteristics, low perception of risk or threat, HIV is incurable or equals death, concerns of confidentiality, unaware that testing is offered for free, and name-based testing. Key facilitators of testing included engaging in high-risk sex, sense of responsibility for partner, collectivism, testing as a part of standard/routine medical care, MSM-friendly medical personnel, increased acceptance of gay/bisexual men by the general public, legal recognition and protection of homosexuals, and home self-testing. Barriers to treatment included negative coping, nondisclosure to families, misconceptions of domestically produced antiretroviral drugs (ARVs) and the benefits of treatment, and costs associated with long-term treatment. Facilitators of treatment included sense of hopefulness that a cure would be found, the cultural value of longevity, peer social support and professional psychological counseling, affordable and specialized treatment and care, and reduced HIV-related stigma and discrimination. Finally, for both testing and treatment, more educational and promotional activities within MSM communities and among the general public are needed.

Two-Stage Mixed-Dye-Based Isothermal Amplification with Ribonuclease-Cleavable Enhanced Probes for Dual-Visualization Detection of SARS-CoV-2 Variants of Interest.

Rapid and visual detection of SARS-CoV-2 variants is vital for timely assessment of variant transmission in resource-limited settings. Here, a closed-tube, two-stage, mixed-dye-based isothermal amplification method with ribonuclease-cleavable enhanced probes (REP), termed REP-TMAP, for dual-visualization detection of SARS-CoV-2 variants including JN.1, BA.2, BA.4/5, and Delta is introduced. The first stage of REP-TMAP is reverse transcription recombinase polymerase amplification and the second stage is dual-visualization detection synergistically mediated by the REP and the mixed dyes of cresol red and hydroxy naphthol blue. In REP-TMAP reaction, the color change under ambient light indicates SARS-CoV-2 infection, while the fluorescence change under blue light excitation specifies variant type. On detecting transcribed RNA of SARS-CoV-2 spike gene, this assay is rapid (within 40 min), highly sensitive (10-200 copies per reaction), and highly specific (identification of single-base mutations). Furthermore, the assay has been clinically validated to accurately detect JN.1, BA.2, and BA.4/5 variants from 102 human oropharyngeal swabs. The proposed assay therefore holds great potentials to provide a rapid, dual-visualization, sensitive, specific, point-of-care detection of SARS-CoV-2 variants and beyond.

Ferritin and procalcitonin serve as discriminative inflammatory biomarkers and can predict the prognosis of severe fever with thrombocytopenia syndrome in its early stages.

Introduction: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with high mortality. The pathophysiology of SFTS remains unclear. Hence, the identification of inflammatory biomarkers for SFTS is crucial for the timely management and prevention of disease severity. Methods: A total of 256 patients with SFTS were divided into a survivor group and a non-survivor group. Classical inflammatory biomarkers such as ferritin, procalcitonin (PCT), C-reactive protein (CRP), and white blood cells were investigated for their association with viral load and the clinical significance for predicting the mortality of patients with SFTS. Results: Serum ferritin and PCT showed a positive association with viral load. Ferritin and PCT levels in non-survivors were significantly higher than those in survivors at 7-9 days from symptom onset. The area under the receiver operating characteristic curve (AUC) values of ferritin and PCT for predicting the fatal outcome of SFTS were 0.9057 and 0.8058, respectively. However, the CRP levels and WBC counts exhibited a weak association with viral load. The AUC value of CRP for predicting mortality was more than 0.7 at 13-15 days from symptom onset. Discussion: Ferritin and PCT levels, especially ferritin, could be potential inflammatory biomarkers for predicting the prognosis of patients with SFTS in its early stages.

CYP2D6 and ADRB1 genetic polymorphisms and the selection of antihypertensive beta-receptor blockers for hypertensive patients.

BACKGROUND: Genetic factors contribute to the variability in individual response to antihypertensive medications. We sought to investigate the frequencies of allele and genotype for CYP2D6 and ADRB1 genetic polymorphisms and explore their potential impact in influencing the selection of antihypertensive beta-receptor blockers. METHODS: The study population was selected from the Han Chinese patients in Zhongda Hospital, which contained 2419 Han Chinese hypertensive individuals and 151 normotensive controls. Each of the above participants underwent venous blood sampling. Then, the gene chip platform was adopted to evaluate the CYP2D6 and ADRB1 genetic polymorphisms. The allele as well as genotype frequencies for each gene, along with the combined genotypes, were subjected to analysis. RESULTS: The frequency of *1/*1 wild-type homozygous for CYP2D6 was 9.71%, while the frequency of *1/*10 heterozygous or *10/*10 mutant homozygous was 59.16% or 31.13%, respectively, as established by gene chip analysis. Similarly, we observed that the genotype frequencies of GG wild-type homozygous for ADRB1 was 10.29%, while that of GC heterozygous, or CC mutant homozygous was 44.98%, or 44.73%, respectively. Notably, combined genotypes *1/*10 + CC (25.88%) and *1/*10 + CG (27.78%) had the highest frequencies. Importantly, no substantial differences in the distributions of CYP2D6 and ADRB1 polymorphism were noted between hypertensive patients and normotensive controls, or among all different grades of hypertension. CONCLUSION: These findings provide insights into the CYP2D6 and ADRB1 polymorphisms in hypertensive patients from Han Chinese, which show significant differences compared to other geographic groups of Han Chinese hypertensive patients. These results offer valuable information for future prospective clinical studies on the antihypertensive effects of beta-receptor blockers in Han Chinese hypertensive patients.

Human leukocyte antigen (HLA)-Cw0303, HLA-Cw04, and HLA-Cw07 polymorphisms are associated with susceptibility of rheumatoid arthritis in Chinese Han patients from Southern China.

OBJECTIVES: This study aimed to investigate the association between human leukocyte antigen Cw (HLA-Cw) polymorphisms and rheumatoid arthritis (RA) in Chinese Han patients in the Jiangsu area (Southern China). MATERIALS AND METHODS: Polymerase chain reaction-sequence specific primers were used to detect HLA-Cw01-08 of 201 RA patients and 211 healthy individuals from Zhongda Hospital (China). The allele frequency distribution of HLA-Cw and genotypic differences between the two groups were analyzed. RESULTS: The frequency of HLA-Cw0303 in patients with RA was significantly higher than that in controls, while the frequency of HLA-Cw04 was lower than that in controls (P<0.05). The gene frequency of HLA-Cw07 in anti-cyclic citrullinated peptide (anti-CCP)-negative patients was higher than that in controls (P=0.044). The frequency of HLA-Cw04 was decreased in the short duration subgroup and increased in the long duration subgroup (P<0.05). Compared to controls, the frequency of HLA-Cw0303 in patients with RA and morning stiffness was increased (P=0.004), while the frequency of HLA-Cw04 was decreased ( 0.005). CONCLUSION: These results suggest that HLA-Cw0303 is a susceptibility gene for RA in Chinese Han patients in the Jiangsu area of southern China. The HLA-Cw04 gene may be a protective factor against RA, while HLA-Cw07 might play a protective role in the production of anti-CCP in the long-term course in patients with RA.

IFN-λ inhibits HIV-1 integration and post-transcriptional events in vitro, but there is only limited in vivo repression of viral production.

The lambda interferons (IL-28a, 28b, and IL-29) inhibit the replication of many viruses, but their role in the inhibition of HIV-1 infection remains unclear. During this study, we monitored IL-29 production in HIV-1 infected individuals and analyzed the in vitro and in vivo inhibition of HIV-1 production. Prior treatment with IL-28a or IL-29 induced an antiviral state in cultured primary T-cells, which suppressed HIV-1 integration and post-transcriptional events. The antiviral factors MxA, OAS, and PKR were up-regulated. In HIV-1 infected patients, IL-29 level was increased along with the depletion of CD4⁺ T-cells in peripheral blood, while the elevated IL-29 did not show a significantly negative correlation with viral load. Further analysis of HIV-1 infected individuals showed that IL-29 was positively correlated with IFN-ß and anti-inflammatory cytokine IL-10, and was negatively correlated with IFN-γ, which might suggest that IFN-λ participates in modulating antiviral immune responses during HIV-1 infection in vivo. Together, although IFN-λ impeded HIV-1 infection of T-cells in vitro, IFN-λ showed only limited in vivo repression of viral production. The modulation of IFN-λ on inflammatory factors might be worthy for further concentrating on for better understanding the host immune response during HIV-1 infection.