Establishment and evaluation of MIRA-qPCR assay for the rapid and sensitively detection of Mycoplasma pneumoniae.

Aim: Mycoplasma pneumoniae (MP) is a common cause of respiratory infections, and its incidence has increased post-COVID-19 due to "immune debt." Real-time quantitative polymerase chain reaction (qPCR) is the standard for detecting MP, but it has a lengthy detection time. This study aimed to establish a highly sensitive rapid detection method for MP.Materials & methods: We developed an integrated assay combining multienzyme isothermal rapid amplification (MIRA) with qPCR, referred to as MIRA-qPCR, for the rapid detection of MP, delivering results within approximately 40 min.Results: The analytic sensitivity of the MIRA-qPCR assay was 10 copies per reaction, and it exhibited no cross-reactivity with other respiratory pathogens, ensuring high specificity. Clinical sample analysis demonstrated higher sensitivity for MIRA-qPCR compared to qPCR reported in the literature, and 100% concordance with commercial qPCR kit.Conclusion: The MIRA-qPCR method established in this study is a promising tool for the clinical detection of MP, offering significant advantages for the rapid diagnosis of MP infections.

Mycoplasma pneumoniae is a bacteria that can make us sick. It mainly affects the lungs and can cause a sickness called "walking pneumonia". This is because it can make you poorly, but not so badly that you are unable to walk around. This bacteria spreads when someone that is infected sneezes or coughs. It is important that M. pneumoniae can be diagnosed quickly. This article looks at a new, fast way to identify infection called MIRA-quantitative PCR.

Safety and immunogenicity of aerosolised Ad5-nCoV, intramuscular Ad5-nCoV, or inactivated COVID-19 vaccine CoronaVac given as the second booster following three doses of CoronaVac: a multicentre, open-label, phase 4, randomised trial.

BACKGROUND: Aerosolised Ad5-nCoV is the first approved mucosal respiratory COVID-19 vaccine to be used as a booster after the primary immunisation with COVID-19 vaccines. This study aimed to evaluate the safety and immunogenicity of aerosolised Ad5-nCoV, intramuscular Ad5-nCoV, or inactivated COVID-19 vaccine CoronaVac given as the second booster. METHODS: This is an open-label, parallel-controlled, phase 4 randomised trial enrolling healthy adult participants (≥18 years) who had completed a two-dose primary immunisation and a booster immunisation with inactivated COVID-19 vaccines (CoronaVac only) at least 6 months before, in Lianshui and Donghai counties, Jiangsu Province, China. We recruited eligible participants from previous trials in China (NCT04892459, NCT04952727, and NCT05043259) as cohort 1 (with the serum before and after the first booster dose available), and from eligible volunteers in Lianshui and Donghai counties, Jiangsu Province, as cohort 2. Participants were randomly assigned at a ratio of 1:1:1, using a web-based interactive response randomisation system, to receive the fourth dose (second booster) of aerosolised Ad5-nCoV (0·1 mL of 1·0 × 1011 viral particles per mL), intramuscular Ad5-nCoV (0·5 mL of 1·0 × 1011 viral particles per mL), or inactivated COVID-19 vaccine CoronaVac (0·5 mL), respectively. The co-primary outcomes were safety and immunogenicity of geometric mean titres (GMTs) of serum neutralising antibodies against prototype live SARS-CoV-2 virus 28 days after the vaccination, assessed on a per-protocol basis. Non-inferiority or superiority was achieved when the lower limit of the 95% CI of the GMT ratio (heterologous group vs homologous group) exceeded 0·67 or 1·0, respectively. This study was registered with ClinicalTrials.gov, NCT05303584 and is ongoing. FINDINGS: Between April 23 and May 23, 2022, from 367 volunteers screened for eligibility, 356 participants met eligibility criteria and received a dose of aerosolised Ad5-nCoV (n=117), intramuscular Ad5-nCoV (n=120), or CoronaVac (n=119). Within 28 days of booster vaccination, participants in the intramuscular Ad5-nCoV group reported a significantly higher frequency of adverse reactions than those in the aerosolised Ad5-nCoV and intramuscular CoronaVac groups (30% vs 9% and 14%, respectively; p<0·0001). No serious adverse events related to the vaccination were reported. The heterologous boosting with aerosolised Ad5-nCoV triggered a GMT of 672·4 (95% CI 539·7-837·7) and intramuscular Ad5-nCoV triggered a serum neutralising antibody GMT of 582·6 (505·0-672·2) 28 days after the booster dose, both of which were significantly higher than the GMT in the CoronaVac group (58·5 [48·0-71·4]; p<0·0001). INTERPRETATION: A heterologous fourth dose (second booster) with either aerosolised Ad5-nCoV or intramuscular Ad5-nCoV was safe and highly immunogenic in healthy adults who had been immunised with three doses of CoronaVac. FUNDING: National Natural Science Foundation of China, Jiangsu Provincial Science Fund for Distinguished Young Scholars, and Jiangsu Provincial Key Project of Science and Technology Plan.

Safety and immunogenicity of heterologous boost immunisation with an orally administered aerosolised Ad5-nCoV after two-dose priming with an inactivated SARS-CoV-2 vaccine in Chinese adults: a randomised, open-label, single-centre trial.

BACKGROUND: Due to waning immunity and protection against infection with SARS-CoV-2, a third dose of a homologous or heterologous COVID-19 vaccine has been proposed by health agencies for individuals who were previously primed with two doses of an inactivated COVID-19 vaccine. METHODS: We did a randomised, open-label, controlled trial to evaluate the safety and immunogenicity of heterologous boost immunisation with an orally administered aerosolised adenovirus type-5 vector-based COVID-19 vaccine (Ad5-nCoV) in Chinese adults (≥18 years old) who had previously received two doses of an inactivated SARS-CoV-2 vaccine-Sinovac CoronaVac. Eligible participants were randomly assigned (1:1:1) to receive a heterologous booster vaccination with a low dose (1·0 × 1011 viral particles per mL; 0·1 mL; low dose group), or a high dose (1·0 × 1011 viral particles per mL; 0·2 mL; high dose group) aerosolised Ad5-nCoV, or a homologous intramuscular vaccination with CoronaVac (0·5 mL). Only laboratory staff were masked to group assignment. The primary endpoint for safety was the incidence of adverse reactions within 14 days after the booster dose. The primary endpoint for immunogenicity was the geometric mean titres (GMTs) of serum neutralising antibodies (NAbs) against live SARS-CoV-2 virus 14 days after the booster dose. This study was registered with ClinicalTrials.gov, NCT05043259. FINDINGS: Between Sept 14 and 16, 2021, 420 participants were enrolled: 140 (33%) participants per group. Adverse reactions were reported by 26 (19%) participants in the low dose group and 33 (24%) in the high dose group within 14 days after the booster vaccination, significantly less than the 54 (39%) participants in the CoronaVac group (p<0·0001). The low dose group had a serum NAb GMT of 744·4 (95% CI 520·1-1065·6) and the high dose group had a GMT of 714·1 (479·4-1063·7) 14 days after booster dose, significantly higher than the GMT in the CoronaVac group (78·5 [60·5-101·7]; p<0·0001). INTERPRETATION: We found that a heterologous booster vaccine with an orally administered aerosolised Ad5-nCoV is safe and highly immunogenic in adults who have previously received two doses of CoronaVac as the primary series vaccination. FUNDING: National Natural Science Foundation of China and Jiangsu Provincial Key Research and Development Program.

Outbreak of Vaccinia Virus Infection from Occupational Exposure, China, 2017.

Human infections with vaccinia virus (VACV), mostly from laboratory accidents or contact with infected animals, have occurred since smallpox was eradicated in 1980. No recent cases have been reported in China. We report on an outbreak of VACV from occupational exposure to rabbit skins inoculated with VACV.

Severe human infection with a novel avian-origin influenza A(H7N4) virus.

Human infections with influenza H7 subtypes, such as H7N9, have raised concerns worldwide. Here, we report a human infection with a novel influenza A(H7N4) virus. A 68 years-old woman with cardiovascular and cholecystic comorbidities developed rapidly progressed pneumonia with influenza-like-illness as initial symptom, recovered after 23 days-hospitalization including 8 days in ICU. Laboratory indicators for liver and blood coagulation dysfunction were observed. Oseltamivir phosphate, glucocorticoids and antibiotics were jointly implemented, with nasal catheterization of oxygen inhalation for this patient. We obtained the medical records and collected serial respiratory and blood specimens from her. We collected throat, cloacal and/or feces samples of poultry and wild birds from the patient's backyard, neighborhood, local live poultry markets (LPMs) and the nearest lake. All close contacts of the patient were followed up and sampled with throat swabs and sera. Influenza viruses and other respiratory pathogens were tested by real-time RT-PCR, viral culturing and/or sequencing for human respiratory and bird samples. Micro-neutralizing assay was performed for sera. A novel reassortant wild bird-origin H7N4 virus is identified from the patient and her backyard poultry (chickens and ducks) by sequencing, which is distinct from previously-reported avian H7N4 and H7N9 viruses. At least four folds increase of neutralizing antibodies to H7N4 was detected in her convalescent sera. No samples from close contacts, wild birds or other poultry were tested positive for H7N4 by real-time RT-PCR.

Probable person to person transmission of novel avian influenza A (H7N9) virus in Eastern China, 2013: epidemiological investigation.

OBJECTIVE: To determine whether the novel avian influenza H7N9 virus can transmit from person to person and its efficiency. DESIGN: Epidemiological investigations conducted after a family cluster of two patients with avian H7N9 in March 2013. SETTING: Wuxi, Eastern China. PARTICIPANTS: Two patients, their close contacts, and relevant environments. Samples from the patients and environments were collected and tested by real time reverse transcriptase-polymerase chain reaction (rRT-PCR), viral culture, and haemagglutination inhibition assay. Any contacts who became ill had samples tested for avian H7N9 by rRT-PCR. Paired serum samples were obtained from contacts for serological testing by haemagglutination inhibition assays. MAIN OUTCOMES MEASURES: Clinical data, history of exposure before the onset of illnesses, and results of laboratory testing of pathogens and further analysis of sequences and phylogenetic tree to isolated strains. RESULTS: The index patient became ill five to six days after his last exposure to poultry. The second patient, his daughter aged 32, who provided unprotected bedside care in the hospital, had no known exposure to poultry. She developed symptoms six days after her last contact with her father. Two strains were isolated successfully from the two patients. Genome sequence and analyses of phylogenetic trees showed that both viruses were almost genetically identical. Forty three close contacts of both patients were identified. One had mild illness but had negative results for avian H7N9 by rRT-PCR. All 43 close contacts tested negative for haemagglutination inhibition antibodies specific for avian H7N9. CONCLUSIONS: The infection of the daughter probably resulted from contact with her father (the index patient) during unprotected exposure, suggesting that in this cluster the virus was able to transmit from person to person. The transmissibility was limited and non-sustainable.

A family cluster of infections by a newly recognized bunyavirus in eastern China, 2007: further evidence of person-to-person transmission.

BACKGROUND: Seven persons in one family living in eastern China developed fever and thrombocytopenia during May 2007, but the initial investigation failed to identify an infectious etiology. In December 2009, a novel bunyavirus (designated severe fever with thrombocytopenia syndrome bunyavirus [SFTSV]) was identified as the cause of illness in patients with similar clinical manifestations in China. We reexamined this family cluster for SFTSV infection. METHODS: We analyzed epidemiological and clinical data for the index patient and 6 secondary patients. We tested stored blood specimens from the 6 secondary patients using real time reverse transcription polymerase chain reaction (RT-PCR), viral culture, genetic sequencing, micro-neutralization assay (MNA), and indirect immunofluorescence assay (IFA). RESULTS: An 80-year-old woman with fever, leucopenia, and thrombocytopenia died on 27 April 2007. Between 3 and 7 May 2007, another 6 patients from her family were admitted to a local county hospital with fever and other similar symptoms. Serum specimens collected in 2007 from these 6 patients were positive for SFTS viral RNA through RT-PCR and for antibody to SFTSV through MNA and IFA. SFTSV was isolated from 1 preserved serum specimen. The only shared characteristic between secondary patients was personal contact with the index patient; none reported exposure to suspected animals or vectors. CONCLUSIONS: Clinical and laboratory evidence confirmed that the patients of fever and thrombocytopenia occurring in a family cluster in eastern China in 2007 were caused by a newly recognized bunyavirus, SFTSV. Epidemiological investigation strongly suggests that infection of secondary patients was transmitted to family members by personal contact.

2009 pandemic characteristics and controlling experiences of influenza H1N1 virus 1 year after the inception in Hangzhou, China.

This paper described the epidemiology and controlling experiences of influenza H1N1 in Hangzhou in the past 1 year. A total of 2,078 cases confirmed by real-time quantitative PCR till March 31, 2010, were analyzed by SPSS 12.0 software. During the early pandemic stage, a patient must be tested for H1N1 nucleic acid once he/she had influenza-like symptoms with the epidemiological history in 7 days, and be diagnosed if it was positive. But in the pandemic peak, we made efforts to identify and save severe cases combined with pneumonia or hypoxemia or respiratory failure or septic shock or multiple organ dysfunctions and failure. In general, the prevalence was 2.77/100,000 (2,078/7,510,844); severity rate, 10.44% (217/2,078); fatality rate, 0.48% (10/2,078). The carrier and secondary attack rate were 9.52% (58/612) and 8.66% (53/612), respectively. About 50% of serious cases and 100% of deaths had the basic underlying diseases: cardiovascular diseases, 13.66% (25/217); chronic lung disease, 12.02% (22/217); pregnant, 7.1% (13/217). Of all cases aged from 1 month to 89 years, 52.99% (1,435/2,708) were in the 10-29 years, with most of them distributed in downtown area. The timeline showed two epidemic peaks occurred in September and November 2009, respectively. Furthermore, the hemagglutinin gene remained 99% identical with the American and vaccine strains, but only 70% with the 1947-2008 Chinese strains. In conclusion, Hangzhou pandemic influenza H1N1 was caused by the highly conserved virus, with low prevalence, transmission, and mortality, because we took efficient controlling methods.

Critical role of mac-1 sialyl lewis x moieties in regulating neutrophil degranulation and transmigration.

Leukocyte cell surface sialyl Lewis x (sLe(x)) and related epitopes play an important role in cell rolling and adhesion during diapedesis via interaction with E-selectin. Here, we present evidence that Mac-1 (CD11b/CD18, CR-3) is a major neutrophil glycoprotein decorated with sLe(x) and ligation of these carbohydrate moieties by anti-sLe(x) antibody significantly impairs neutrophil functions. First, Western blot analysis shows that both CD11b and CD18 subunit of purified Mac-1 are decorated with sLe(x) moieties. A significant co-localization of CD11b and sLe(x) moieties is observed at neutrophil secondary granules. With stimulation of formyl-Met-Leu-Phe (fMLP), neutrophil surface labeling with anti-sLe(x) antibody follows an identical up-regulation pattern of Mac-1. Second, protein-binding assays indicate that sLe(x) moieties on Mac-1 are critical for binding interaction of Mac-1 to E-selectin. Removal of sLe(x) moieties completely abolishes Mac-1-E-selectin binding. Finally, ligation of Mac-1 sLe(x) by anti-sLe(x) antibody induces a significant degranulation of neutrophil secondary granules at the absence of chemoattractant stimulation. This "dysregulated" degranulation induced by anti-sLe(x) antibody strongly inhibits neutrophil transmigration in response to fMLP. In summary, Mac-1 sLe(x) moieties play a critical role in regulating beta(2) integrin functions during neutrophil transmigration and degranulation.

Monobutyl phthalate inhibits steroidogenesis by downregulating steroidogenic acute regulatory protein expression in mouse Leydig tumor cells (MLTC-1).

Di-n-butyl phthalate (DBP) and its active metabolite, monobutyl phthalate (MBP), display no binding affinity for the androgen receptor, yet exert antiandrogenic effects by altering steroid biosynthesis. However, the mechanisms underlying this observed effect are not known. The purpose of this study was to determine the site of MBP action on steroidogenesis in vitro using mouse Leydig tumor cells (MLTC-1). Various concentrations of MBP (0, 50, 100, 200, 400, or 800 micromol/L) were added to the medium for 24 h followed by stimulation with some compounds such as human chorionic gonadotrophin (hCG), cholera toxin (CT), cAMP analog 8-Br-cAMP, 22(R)-hydroxycholesterol (22R-HC), and pregnenolone. Data showed that MBP inhibited the increases in progesterone production induced by hCG and CT. In contrast, the levels of intracellular cAMP remained unaltered. In addition, 8-Br-cAMP-stimulated progesterone production was also suppressed by MBP. These results suggested that the site in the steroid biosynthesis pathway affected by MBP occurs downstream of PKA activation in MLTC-1 cells. Moreover, incubation with 22R-HC and pregnenolone as progesterone precursors for P-450 side-chain cleavage enzyme (P450scc) and 3beta-hydroxysteroid dehydrogenase (3betaHSD) respectively resulted in no marked change in progesterone production, indicating that MBP did not influence P450scc and 3betaHSD but did exert an effect on cholesterol transportation into mitochondria, the rate-limiting step. These results were supported by the downregulated StAR expression seen with MBP administration, as StAR is a key factor in this process. Data indicate that MBP interfered with steroid hormone production by affecting StAR expression in MLTC-1 cells.

Antiandrogenic activity of pyrethroid pesticides and their metabolite in reporter gene assay.

Many pesticides possess hormonal activity and have thus been classified as endocrine disruptors. Pyrethroids are commonly used pesticides worldwide, but little has been done to characterize their antiandrogenic activity potential. We tested three frequently encountered pyrethroids (fenvalerate, cypermethrin, permethrin) and their metabolite 3-phenoxybenzoic acid (3-PBA) for antiandrogenic and androgenic activity using a human androgen receptor (AR) mediated luciferase reporter gene assay in CV-1 African green monkey kidney cell. The assay displayed appropriate response to the known AR agonist 5alpha-dihydrotestosterone and AR antagonist nilutamide and flutamide. At 0.1mM, all the three tested pyrethroids significantly suppressed the luciferase expression. Further, their metabolite 3-PBA also showed antagonist activity. None of the test chemicals showed androgenic activity. Through the antiandrogenic pathways, exposure to certain pyrethroids may contribute to the damage of reproductive system. In conclusion, pyrethroid pesticides can act as antiandrogen in vitro, and metabolizing to 3-PBA cannot eliminate the antagonist activity. This result provides useful information for risk assessment of pyrethroid pesticides.

Effects of terephthalic acid on rat lipid metabolism.

OBJECTIVE: To study the effect of terephthalic acid (TPA) on lipid metabolism in Sprague-Dawley (SD) rats. METHODS: Five groups of SD rats that ingested 0%, 0.04%, 0.2%, 1%, and 5% TPA, respectively, were included in a 90-day subchronic feeding study. Effects of TPA on levels of serum protein, total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL), total antioxidative capability (T-AOC), superoxide dismutase (SOD) and malondialdehyde (MDA) were observed. Urine samples were collected and analyzed for concentration of ion. RESULTS: TPA decreased the level of serum T-AOC in a dose dependent manner. The contents of serum and bladder MDA significantly decreased in 1% and 5% TPA ingestion groups. Serum CuZn superoxide dismutase (CuZnSOD) lowered in groups of 0.2%, 1%, and 5% TPA. TPA subchronic feeding had no significant influences on serum TC, LDL or HDL, but increased serum TG, TP and ALB after administration of 0.04% and/or 0.2% TPA. Concentrations of urinary Ca2+, Mg2+, Na+, and K+ were elevated in 1% and 5% TPA groups. CONCLUSION: Antioxidative potential decreased after TPA exposure. MDA increase in serum and bladder tissues was one of the most important reactions in rats which could protect themselves against TPA impairment. The decrease of serum CuZnSOD was related to the excretion of Zn2+.

Metabolism of terephthalic acid and its effects on CYP4B1 induction.

OBJECTIVE: To investgate the metabolism of terephthalic acid (TPA) in rats and its mechanism. Methods Metabolism was evaluated by incubating sodium terephthalate (NaTPA) with rat normal liver microsomes, or with microsomes pretreated by phenobarbital sodium, or with 3-methycholanthrene, or with diet control following a NADPH-generating system. The determination was performed by high performance liquid chromatography (HPLC), and the mutagenic activation was analyzed by umu tester strain Salmonella typhimurium NM2009. Expression of CYP4B1 mRNA was detected by RT-PCR. Results The amount of NaTPA (12.5-200 micromol x L(-1)) detected by HPLC did not decrease in microsomes induced by NADPH-generating system. Incubation of TPA (0.025-0.1 mmol x L(-1)) with induced or noninduced liver microsomes in an NM2009 umu response system did not show any mutagenic activation. TPA exposure increased the expression of CYP4B 1 mRNA in rat liver, kidney, and bladder. CONCLUSION: Lack of metabolism of TPA in liver and negative genotoxic data from NM2009 study are consistent with other previous short-term tests, suggesting that the carcinogenesis in TPA feeding animals is not directly interfered with TPA itself and/or its metabolites.

Induction of bladder lesion by terephthalic acid and its mechanism.

OBJECTIVE: To provide more information for rational evaluation of potential risks of terephthalic acid (TPA), we studied the effects of TPA on rats' bladders in 90 days after TPA exposure. METHODS: Sprague Dawley rats were subdivided into five groups, ingesting 0%, 0.04%, 0.2%, 1%, and 5% TPA respectively for a sub-chronic feeding study lasting for 90 days. Urine, serum and samples of brain, liver, lung, kidney, bladder, etc. were collected and analyzed. RESULTS: TPA ingesting decreased the value of urinary pH, and increased the contents of Ca2+, Zn2+, Mg2+, Na+, K+ in urine. The volume of 24 h urine was significantly increased in male rats in the 1% and 5% TPA groups. Urinary white sediment was found in both sexes, and its formation in male rats seemed more susceptible than that in female rats. Alpha 2u-globulin (AUG) in serum and urine of male rats was markedly increased in a dose-dependent manner. Fifteen cases of hyperplasia (simple or atypical) were determined in the 5% TPA ingesting group, 14/52 in male rats and 1/23 in female rats. Among them 3 male rats had no stone or calculus. Those with either bladder stones or hyperplasia were accompanied with urinary white sediments. CONCLUSION: White sediment accompanied with elevated urine AUG is the basis of TPA induced urolith formation, and is also associated with TPA induced bladder epithelial cell proliferation. It can act as an early biomarker for the potential toxic effect of TPA.