4例新型冠状病毒感染病例咽拭子与痰标本病毒核酸检测的比较

目的比较分析新型冠状病毒病例咽拭子与痰标本的病毒核酸检测效果。方法对4例新型冠状病毒确诊病例的咽拭子与痰标本分别进行人体细胞GAPDH管家基因、病毒ORF 1ab基因、N基因及S基因Real time RT-PCR核酸检测与比较。结果4例病例的咽拭子和痰标本中,人体细胞管家基因GAPDH均呈现明显典型的扩增信号曲线;病毒ORF 1ab基因、N基因及S基因核酸检测中,痰标本的扩增曲线信号均比咽拭子强,扩增曲线的CT值均低于咽拭子,在病例1和4表现更加明显,而病例4的咽拭子标本检测中,商品化试剂呈现阴性结果,而痰标本则呈现明显的阳性结果。结论在开展新型冠状病毒实验室核酸检测中,痰标本的病毒含量高于咽拭子标本,其检测效果优于咽拭子标本。     

两种新型冠状病毒核酸试剂检测结果的对比研究

目的 比较两种新型冠状病毒核酸试剂检测结果 的一致性.方法 回顾性分析深圳市第三人民医院确诊新型冠状病毒患者痰标本、鼻拭子标本和肛拭子标本共计539份核酸检测结果 ,采用一致性检验进行统计分析.并从两种试剂检测的核酸Ct值均>36的样本中随机抽取4份进行测序验证.结果 两种试剂对痰标本、鼻拭子标本和肛拭子标本一致性检验...     

毛细管电泳片段分析法、荧光定量PCR法在ALRTI患儿咽拭子标本病原体诊断中的应用对比观察

目的 毛细管电泳片段分析法、荧光定量PCR法在急性下呼吸道感染患儿咽拭子标本病原体诊断中的应用。方法 急性呼吸道感染患儿65例（65份咽拭子标本）,分别采用毛细管电泳片段分析法、荧光定量PCR法检测患儿咽拭子病原体,比较两种方法病原体检出率,并分析两种检测方法与金标准（Sanger测序法）检测结果的一致性。结果 毛细管电泳片段分析法病原体检出率为52.3%（呼吸道合胞病毒21份、副流感病毒4份、衣原体病毒2份、腺病毒2份、鼻病毒2份、偏肺病毒3份、博卡病毒1份,其中1份为合胞病毒与鼻病毒混合感染）,荧光定量PCR法病原体检出率为45.0%（呼吸道合胞病毒20份、副流感病毒3份、衣原体病毒2份、腺病毒1份、鼻病毒1份）,两种方法病原体检出率比较,P>0.05。毛细管电泳片段分析法与Sanger测序法检测结果阳性一致率为100.0%,阴性一致率为100.0%,准确率为100.0%;荧光定量PCR法与Sanger测序法检测结果阳性一致率为86.2%,阴性一致率为93.5%,准确率为90.0%。结论 毛细管电泳片段分析法与荧光定量PCR方法病原体检出率无差异,但毛细管电泳片段分析法能检测...     

SARS-CoV-2核酸复阳患者临床特点及不同标本核酸检测结果分析

目的了解SARS-CoV-2核酸复阳的2019新型冠状病毒肺炎(coronavirus disease 2019,COVID-19)患者临床特征,为实行针对性管理提供参考依据。方法分析SARS-CoV-2核酸复阳COVID-19患者的临床表现和实验室检查结果,对该类患者住院期间和出院后呼吸道及粪便标本病毒核酸持续时间进行分析评估。结果43例患者中有15例在住院期间SARS-CoV-2核酸检测短暂转阴后又出现“复阳”(复阳组),复阳比例为34.9%,均为普通型患者。与病毒核酸未出现复阳患者(未复阳组)相比,复阳组在入院时多表现为轻度发热,咳痰持续时间更短,ESR、CRP、血清淀粉样蛋白A水平更低(P均<0.05);2组患者鼻咽拭子标本病毒核酸初始转阴时间差异无统计学意义,但与未复阳组相比,复阳组鼻咽拭子病毒最长脱落时间显著延长,痰液中病毒脱落时间显著延长,粪便核酸阳性率更高(P均<0.05)。呼吸道标本病毒核酸复阳和粪便标本中病毒核酸持续阳性患者均未发现胸部CT较前改变。结论SARS-CoV-2核酸复阳COVID-19患者多表现为轻症感染和病毒持续阳性的特征,同时该类患者存在痰液标本病毒脱落时间延长和粪便标本病毒核酸持续阳性的特点,因而对病毒核酸复阳患者应给予特别关注和更长时间的医学观察,以预防和控制疫情的再次传播。     

痰标本检测p53基因突变及其在肺癌早期临床诊断中的意义

目的　评价痰标本检测p53基因点突变方法及其作为肺癌早期临床诊断监测指标的真实性和可靠性。方法　用聚合酶链反应（PCR）-单链多肽性（SSCP）-银染法检测54例原发性肺癌患者和114例良性肺疾病患者痰标本中p53基因第5～8外显子的点突变,同时进行痰涂片细胞学检查。结果　p53突变在肺癌组检出率为55.56%(30/54),非肺癌组检出率为1.75%(2/114),P<0.001。痰标本检测p53基因突变作为肺癌临床诊断监测指标的灵敏性为55.56%,特异性为98.25%,阳性似然比为31.75。肺癌组p53阳性检出率（55.56%）与痰涂片瘤细胞阳性检出率（35.19%）比较,差异有显著性（P＜0.05）,关联性有极显著意义（P＜0.01）。肺癌组p53检出率与性别、吸烟指数、病理分型、疾病分期均无明显关系,但与年龄有密切关系,高龄患者（≥60岁）检出率高（P=0.02）。结论　PCR-SSCP-银染法检测痰标本p53突变可以在可疑肺癌患者（如吸烟并慢性肺疾患者）中作为一项随访监测指标,并将有助于肺癌的早期临床诊断。     

河南省濮阳市一起人感染H5N6禽流感流行病学及病原学特征分析

目的 分析2022年3月河南省濮阳市首例人感染H5N6禽流感流行病学特征，探索感染来源，为防控人感染禽流感提供科学依据。方法 对人感染H5N6禽流感病例、密切接触者和涉禽外环境开展流行病学调查，采集病例和密切接触者的呼吸道标本、环境标本、禽类生物样本和禽类产品，运用实时荧光定量PCR方法检测禽流感病毒核酸。结果该病例肺泡灌洗液、咽拭子和鼻拭子样本均检出H5N6亚型禽流感病毒核酸，病例有宰杀禽类职业暴露史。1份屠宰厂的案板涂抹拭子检出H5N6亚型禽流感病毒核酸，9份鸭头类产品的口咽拭子检出H5亚型禽流感病毒核酸，确诊为人感染H5N6禽流感，为单个散发病例。结论 濮阳市首例人感染H5N6禽流感病例为单个散发病例，病原甲型H5N6亚型禽流感病毒可能来自宰杀的鸭子；应加强禽流感日常监测及健康科普。     

甲型流感病毒快速基因分型POCT方法的建立

目的建立一种无需核酸提取,直接检测鼻咽拭子中甲型流感病毒H1HA pdm09、H3HA亚型和季节性流感病毒H1HA non-pdm09的POCT快速分型方法。方法针对3种流感病毒的血凝素(hemagglutinin,HA)基因序列保守区设计高度特异性的引物和HyBeacon探针,同时以人类RNaseP基因作为内参。优化多重PCR反应条件,建立用熔解曲线分析甲型流感病毒基因分型的方法,并将其应用到ParaDNA核酸POCT检测仪,对50例甲型流感病毒抗原初筛阳性且经过实时荧光定量PCR检测的临床鼻咽拭子标本进行检测。结果该方法可在1.5 h内完成对3种甲型流感病毒亚型HA基因的特异性扩增和分型,与其他7种呼吸道病原微生物无交叉反应,其核酸检测下限为50 copies/μl。对临床50例甲型流感病毒抗原阳性的鼻咽拭子标本进行直接检测,检出H1HA pdm09阳性标本48例,H3HA阳性标本2例,未检出季节性流感H1HA nonpdm09阳性标本。结论基于HyBeacon探针和ParaDNA核酸POCT检测仪所建立的甲型流感病毒快速分型方法能同时检测H1HA pdm09、H3HA和季节性流感病毒H1HA non-pdm09。该方法无需核酸提取,操作简便,检测时间短,适于对甲型流感病毒抗原初筛阳性的鼻咽拭子开展现场基因分型检测,可为流感的诊治和防控提供及时快速的实验室依据。     

2017—2018年北京市海淀区流行性感冒监测数据病原学分析

目的　对2017年1月—2018年12月北京市海淀区流行性感冒（流感）监测数据进行分析,了解海淀区流感的流行情况和趋势,为海淀区流感防控工作提供科学依据。方法　采集北京市海淀区国家级流感监测哨点医院2017—2018年的流感样病例咽拭子标本,采用实时荧光定量PCR方法进行流感病毒核酸检测。结果　2017—2018年共采集1743例咽拭子标本,检出流感病毒核酸阳性样本262例,阳性检出率为15.03%;2017年与2018年流感病毒核酸阳性检出率差异无统计学意义（P＞0.05）;男性流感病毒核酸阳性检出率高于女性（P＜0.05）;60岁及以上年龄组流感病毒核酸阳性检出率最高（P＜0.05）;冬、春季流感病毒核酸阳性检出率高于夏、秋季（P＜0.05）。结论　北京市海淀区在2017年与2018年流感流行强度较高,冬、春季为高峰季节,但2年流行的流感病毒优势株有所不同,老年病例流感病毒核酸阳性检出率最高,是流感防控重点人群,学校和托幼机构是聚集性疫情重点防控场所。     

肺炎衣原体PCR、套式PCR评价与临床应用

目的评价PCR和套式PCR检测肺炎衣原体(Chlamydia pneumoniae,Cpn)的特异性和灵敏度。方法按Campbell等建立PCR反应体系,按Tong等与秦玲等建立套式PCR反应体系,本文并以Cpn 16SrRNA基因(16SrDNA)为靶基因自行建立套式PCR反应体系,对Cpn、沙眼衣原体、鹦鹉热衣原体、大肠埃希稀菌、肺炎克雷伯菌、流感嗜血杆菌、粘质沙雷菌、洛菲不动杆菌、鲍曼不动杆菌、嗜肺军团杆菌、巨细胞病毒、单纯疱疹病毒进行检测以评价各方法的特殊性异性;以纯化的Cpn-DNA和1例阳性临床标本作梯度稀释后分别参与各反应体系检测以评价各方法的灵敏度。结果1、PCR和套式PCR均只能检出Cpn TW-183株和CWL-29株,而其它衣原体、支原体、细菌、病毒均不能检出;2、套式PCR检测Cpn较PCR敏感100～1000倍;3、经以16SrRNA基因为靶基因套式PCR检测新生儿肺炎(非吸入性肺炎)和儿童肺炎者咽拭子标本Cpn阳性率分别为16.2％和22.0％,肺结核者痰标本为18.9％,男性STD者尿道拭子标本为0％。结论 PCR和套式PCR检测Cpn均有较高的特异性,其中套式PCR比PCR敏感100～1000倍,在肺部炎症者咽拭子或痰标本中Cpn有较高的检出率。     

噬菌体生物扩增法快速检测结核分枝杆菌的临床应用附182例分析

目的评价噬菌体生物扩增法检测痰、胸水、腹水中结核分枝杆菌的阳性检出率及临床应用价值。方法采用噬菌体生物扩增法对68例痰标本,63例胸水标本,17例腹水标本等进行检测。并对60例痰标本同时进行涂片抗酸染色对比。结果噬菌体生物扩增法检测182例各类标本中,其阳性检出率分别为痰38%（26/68）胸水46%（29/63）腹水52%（9/17）脓汁1例阳性（1/4）,脑脊液19例和尿11例均为未检出阳性。60例痰标本同时做涂片抗酸染色,两者的阳性检出率有显著性差异（χ2=11.25P〈0.01）。结论噬菌体生物扩增法具有快速、灵敏、特异等优点。对痰菌阴性患者提高阳性检出率具有明显价值。同时对检测胸腹水中结核分枝杆菌具有较高的敏感性和特异性,无需特殊设备适宜在基层医院推广。     

Application of vaginal tampon as an alternative to nasal swabs for higher recovery of DNA from sheep and goats for PCR based diagnosis of bovine tuberculosis

A study was conducted to find the applicability of vaginal tampons as an alternative to regular cotton swabs as a nasal secretion collection tool for the higher recovery of DNA. Nasal secretions were collected from sheep and goats using regular cotton swab and tampon swab. The mean yield and purity of the DNA extracted from tampon were significantly higher than that of the DNA extracted from cotton swab. The tampon swabs resulted higher DNA recovery than the cotton swabs after they were allowed to absorb M. bovis culture. The tampon swab was also found to be more sensitive in detecting M. bovis by PCR. This study concluded that vaginal tampons are having a higher absorption capacity with more DNA yield and can be used as a nasal swab in the diagnosis of bovine tuberculosis.     

Comparison of throat swabs and nasopharyngeal suction specimens in non-sputum-producing patients with cystic fibrosis

Both throat swabs and nasopharyngeal suction (NPS) specimens are used for microbiological assessment in non-sputum-producing patients with cystic fibrosis (CF), but studies comparing their diagnostic yield are lacking. We, therefore, conducted a prospective study in young CF patients, in which both techniques were performed in random order. Forty-seven consecutive CF children aged 6 months to 10 years were studied during routine visits to the clinic. CF relevant pathogens were found in the majority of patients with no significant differences in the rate of positive cultures for Staphylococcus aureus, Haemophilus influenzae, or Pseudomonas aeruginosa. A statistically significant difference was observed in the rate of detection of other organisms with only 9/47 (19%) of throat swab specimens and 27/47 (57%) of NPS specimens being positive (P = 0.0004). This included 12 positive cultures for Streptococcus pneumoniae and 11 cultures that were positive for Moraxella catarrhalis, both of which are frequent colonizers of the upper airway. Therefore, the most common bacterial pathogens affecting the CF lung appear to be detected in similar frequency by throat swab as by nasopharyngeal suction. There is evidence that nasopharyngeal suction yields more specimens of Streptococcus pneumoniae and Moraxella catarrhalis, which may reflect upper airway colonization rather than lower airway infection. We conclude that nasopharyngeal suction is not routinely warranted as there is no benefit over throat swab in detection of CF pathogens in infants and young children with CF.     

An infant with a mild SARS-CoV-2 infection detected only by anal swabs: a case report

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China and has spread rapidly worldwide. We present a mild SARS-CoV-2 infection in a baby with non-productive cough and normal chest computed tomography, in whom only anal swabs tested positive by real-time PCR testing for SARS-CoV-2. She was given atomization inhalation therapy with recombinant human interferon alfa-1b for 10 days. Her anal swabs remained positive for eight days, whereas her throat swabs were persistently negative by real-time PCR testing. Mild and asymptomatic cases, especially in children, might present with PCR negative pharyngeal/nasal swabs and PCR positive anal swabs. Those patients are potential sources of infection via fecal–oral transmission for COVID-19.     

Epigenetic age predictions based on buccal swabs are more precise in combination with cell type-specific DNA methylation signatures

Aging is reflected by highly reproducible DNA methylation (DNAm) changes that open new perspectives for estimation of chronological age in legal medicine. DNA can be harvested non-invasively from cells at the inside of a person''s cheek using buccal swabs – but these specimens resemble heterogeneous mixtures of buccal epithelial cells and leukocytes with different epigenetic makeup. In this study, we have trained an age predictor based on three age-associated CpG sites (associated with the genes PDE4C, ASPA, and ITGA2B) for swab samples to reach a mean absolute deviation (MAD) between predicted and chronological age of 4.3 years in a training set and of 7.03 years in a validation set. Subsequently, the composition of buccal epithelial cells versus leukocytes was estimated by two additional CpGs (associated with the genes CD6 and SERPINB5). Results of this “Buccal-Cell-Signature” correlated with cell counts in cytological stains (R² = 0.94). Combination of cell type-specific and age-associated CpGs into one multivariate model enabled age predictions with MADs of 5.09 years and 5.12 years in two independent validation sets. Our results demonstrate that the cellular composition in buccal swab samples can be determined by DNAm at two cell type-specific CpGs to improve epigenetic age predictions.     

Results of a pilot study using self‐collected mid‐turbinate nasal swabs for detection of influenza virus infection among pregnant women

Background

We evaluated the feasibility of asking pregnant women to self-collect and ship respiratory specimens.

Methods

In a preliminary laboratory study, we compared the RT-PCR cycle threshold (CT) values of influenza A and B viruses incubated at 4 storage temperatures (from 4 to 35°C) for 6 time periods (8, 24, 48, 72, and 168 hours and 30 days), resulting in 24 conditions that were compared to an aliquot tested after standard freezing (−20°C) (baseline condition). In a subsequent pilot study, during January–February, 2014, we delivered respiratory specimen collection kits to 53 pregnant women with a medically attended acute respiratory illness using three delivery methods.

Results

CT values were stable after storage at temperatures <27°C for up to 72 hours for influenza A viruses and 48 hours for influenza B viruses. Of 53 women who received kits during the pilot, 89% collected and shipped nasal swabs as requested. However, 30% (14/47) of the women took over 2 days to collect and ship their specimen. The human control gene, ribonuclease P (RNase P), was detected in 100% of nasal swab specimens. However, the mean CT values for RNase P (26·5, 95% confidence interval [CI] = 26·0–27·1) and for the 8 influenza A virus positives in our pilot (32·2, 95% CI = 28·9–35·5) were significantly higher than the CTs observed in our 2010–2012 study using staff-collected nasal pharyngeal swabs (P-values < 0·01).

Discussion

Self-collection of respiratory specimens is a promising research method, but further research is needed to quantify the sensitivity and specificity of the approach.     

Detection of human papillomavirus DNA in self-administered vaginal swabs as compared to cervical swabs

The primary risk factor for cervical cancer is infection with high-risk genotypes of human papillomavirus (HPV). This study compared HPV DNA detection between cervical swabs (CX) and self-administered vaginal swabs (SV). Phase I participants were 199 women chosen from a study comparing the detection of Chlamydia trachomatis from various anogenital sites. Phase II participants were 135 women from either the Colposcopy or HIV Outpatient Clinic. HPV DNA testing was performed using polymerase chain reaction and Roche reverse line blot hybridization. In Phase I samples, more CX samples amplified and more HPV genotypes (P < 0.05) were detected in CX. Genotype 52 were seen more in the cervix, whereas genotype 82 (MM4) was detected solely in the vagina. The presence of high-risk HPV genotypes in the cervix was a predictor of an abnormal Papanicolaou (Pap) smear. In Phase II samples, CX samples amplified more, but similar rates of HPV genotypes were seen in SV and CX samples. Higher concordance rates of high-risk genotypes were seen in Phase II compared to Phase I samples. Phase II demonstrated the feasibility of utilizing SV sampling to reflect cervical status. If validated, a self-vaginal swab method to detect cervical HPV DNA status could be utilized to triage women with indeterminate Pap smears and be a useful method to collect epidemiological data from large populations.     

Comparison of nasopharyngeal aspirate and nasal swab specimens for detection of respiratory syncytial virus in different settings in a developing country

OBJECTIVE: To compare detection of respiratory syncytial virus (RSV) for diagnostic purposes using nasopharyngeal aspirate (NPA) and nasal swabs (NS) in different clinical settings in a community study in Guinea-Bissau. METHOD: During 1996-98 paired specimens were obtained from 635 children under 5 years of age (median: 274 days; interquartile range: 144-453 days) with symptoms of lower respiratory infections (LRI). The specimens were analysed by an enzyme-linked immunosorbent assay for RSV antigen in Guinea-Bissau and re-analysed in Denmark using the same assay. The gold standard for RSV antigen detection was defined as any test being positive. RESULTS: RSV antigen was detected in 84 (13%) children, the prevalence being 19% (41/219) among infants aged < 6 months, 12% (22/184) in infants aged 6-11 months, and 9% (21/230) in older children. Sensitivity of antigen detection was higher in NPA (92% in analyses in Guinea-Bissau and 98% in Denmark) than in NS (63% in analyses in Guinea-Bissau, 71% in Denmark). Specificity of RSV antigen detection was equally high in NPA and NS (99-100%). Time since onset of symptoms was significantly shorter in RSV antigen positive than negative samples. Sensitivity did not depend on clinical setting or age of the child. CONCLUSION: Using NS samples was associated with a 27-31% reduction in sensitivity compared with NPA specimens. As NPAs are costly and considered a nuisance by the population, it might be cost-effective in larger epidemiological studies to lose 25-30% in sensitivity but be able to collect samples from a much larger population.