加微量EMJH于柯氏培养基对生长欠佳的钩端螺旋体培养观察

<正> 1979年我所在云南牟定县分离出部分钩端螺旋体(下简称钩体)菌株,但其在坷氏培养基内生长欠佳,长期达不到鉴定浓度。经使用荷兰阿姆斯特丹 WHO/FAO 钩体病咨询研究合作中心馈赠的 EMJH(John-son)培养基,虽菌株生长良好,但 EMJH数量有限,且价格昂贵,为此我们采用柯氏(Korthof)培养基内加入微量 EMJH,菌数明显增殖,达到鉴定所需浓度,且保存周期延长,现报告如下。     

平菇双相培养基用于结核杆菌培养的研究

目的　以平菇浸出液为基础成分,研制培养结核分支杆菌的一种新型双相培养基。方法 经用H₃₇R_v基础试验及对351份临床标本用酸性罗氏培养基对照。结果 两种培养基总阳性数为172份(49%),其中平菇双相培养基166份(+),阳性检出率为47.3%。酸性罗氏培养基144份(+),阳性检出率为41.03%。分别占总阳性数的96.51%和83.72%,并且大多数结核杆菌在平菇双相培养基上的初生长时间明显快于酸性罗氏培养基时间。结论 结果表明平菇双相培养基成分来源丰富,价格低廉,制作方便,操作简单,适合于我国广大基层医院和结核院所使用。     

1例宏基因二代测序提示钩端螺旋体感染患者的实验室诊断

目的 对一不明原因发热伴呼吸困难患者进行实验室诊断,探讨钩端螺旋体实验室确诊的检测方法。方法 利用宏基因二代测序技术(mNGS)对一不明原因发热伴呼吸困难患者的肺泡灌洗液进行病原体分析,利用实时荧光定量PCR(Real-time PCR)、显微镜凝集试验(MAT)和传统培养方法对患者进行实验室确诊。结果 mNGS对不明原因发热伴呼吸困难患者的肺泡灌洗液进行病原体分析提示患者可能被问号钩端螺旋体感染,Real-time PCR和传统培养方法对患者的肺泡灌洗液、血液和尿液检测均为阴性,血清抗体检测(MAT法)致病性钩端螺旋体澳洲型滴度为1:800。结论 该患者确诊为致病性钩端螺旋体澳洲型感染,mNGS与实验室传统方法的有机结合有助于对不明原因发热伴呼吸困难患者实验室诊断,特别是对钩端螺旋体的实验室诊断具有重要意义。     

分枝杆菌双相罗氏培养基在结核病诊断中的应用研究

目的 评价分枝杆菌双相罗氏培养基在结核病诊断中的价值,探讨其在我国基层实验室应用的可行性。方法采取多中心试验方法,在我国3个省的3个地市级结核病诊疗机构连续纳入2014年10月至2015年10月期间的初诊肺结核可疑症状者2320例,其中15例因分枝杆菌改良罗氏培养基(简称“罗氏培养基”)和分枝杆菌双相罗氏培养基(简称“双相培养基”)中的1种或2种培养污染或结果缺失,对两种培养基培养结果进行比较时不计入分析。每例患者留取3份痰标本(即时痰、夜间痰、晨痰)进行萋-尼染色法涂片镜检(简称“涂片”),并选取2份性状好的痰标本同时进行罗氏培养基培养、双相培养基培养。使用SPSS 24.0软件,采用配对χ²检验比较两种培养基的阳性检出率,以P<0.05为差异有统计学意义;分别以罗氏培养基培养结果、临床诊断为标准对双相培养基的检测效能进行评价;采用Wilcoxon秩和检验对罗氏培养基与双相培养基检出时间的差异进行统计学分析,以P<0.05为差异有统计学意义。结果罗氏培养基和双相培养基两种培养方法阳性检出率分别为19.65%(453/2305)和22.00%(507/2305),差异有统计学意义(χ²=22.091,P=0.000);以罗氏培养基培养结果为标准,双相培养基的敏感度为91.39%(414/453),特异度为94.98%(1759/1852),一致率为94.27%(2173/2305);以临床诊断为标准,罗氏培养基和双相培养基的ROC曲线下面积分别为0.694(95%CI:0.672~0.715)、 0.707(95%CI:0.685~0.728);罗氏培养基报阳时间中位数(四分位数)[M(Q₁,Q₃)]为28(21,34)d,双相培养基报阳时间M(Q₁,Q₃)为18(14,24)d,差异有统计学意义(Z=15.114,P=0.000)。结论双相培养基较罗氏培养基具有更高的阳性检出率,诊断价值较高,报阳时间明显缩短,是一种可以作为结核病临床诊断的参考方法,在我国基层实验室有一定推广应用价值。     

空调对结核分枝杆菌医院内传播的影响

目的　探索医院空调对结核分枝杆菌院内传播的影响。方法 用罗氏固体平板培养基,分别采集医院内中央空调控温区、分体空调控温区和无空调区的空气标本,经36.5℃培养28d。根据菌落形态、抗酸染色特性作出初步鉴别,然后经一系列生化试验确定结核分枝杆菌。结果 结核分枝杆菌检出率中央空调控温区为31.1%;分体空调控温区为15.6%;不用空调控温区为4.4%,相互间有明显差异。结论 医院空调特别是中央空调对结核分枝杆菌院内传播有一定的促进作用,应该引起必要的重视。     

南充地区钩端螺旋体病人及鼠类血清群动态分析

<正> 钩体病是本区多发的人兽共患病,菌群复杂,变化多样,影响流行强度。为了摸清其变化规律,我们收集了 1972～1991 年全区从钩体病人及鼠类分离并鉴定的217株钩体菌,井对其血清群进行动态分析,现报告如下。 一、材料与方法 (一)菌株分离:取早期钩体病人血、鼠肾作常规柯氏培养; (二)鉴定:分离出钩体菌由本站或四川省卫生防     

应用噬菌体裂解法测定吡嗪酰胺药物敏感性试验的方法学研究

目的　探索利用噬菌体裂解法测定吡嗪酰胺药物敏感性试验的更佳实验条件、时间及其临床应用价值。方法 应用已建立的PhaB法;但在Middlebrook 7H9酸性液体培养基中加入0.4%的丙酮酸钠作为探索实验对照组。然后观察原已建立的PhaB法与加入丙酮酸钠后实验对照组这两者间的空白管溶菌斑数量以及药物最佳作用时间,并以此对86例结核分枝杆菌临床分离株进行吡嗪酰胺药物敏感性试验,最后与比例法的药物敏感性试验结果进行比较。结果 在Middlebrook 7H9酸性液体培养基中加入丙酮酸钠后的实验对照组,其空白管溶菌斑平均有71个,而PhaB法空白管溶菌斑平均只有53个,两者间平均有18个溶菌斑的差异;药物与细菌的作用:实验对照组1d时间与比例法药物敏感性试验结果有90.7%的符合率,2d时间为87.2%,PhaB法的符合率是88.4%。结论 在已建立的PhaB法基础上,于Middlebrook 7H9酸性液体培养基中加入丙酮酸钠,可以提高实验的准确性以及缩短药物与细菌的作用时间。     

涂阳肺结核病人发现程序的探讨

目的　探索高发现、操作简便、成本低的病人发现程序。方法 山东省2000年结核病流行病学抽样调查资料。结果 在1715例同时胸透和痰涂片检查的可疑肺结核症状者中,发现涂阳肺结核病人51例,其中胸透诊断正常者15例。结论 对可疑肺结核症状者必须全部查痰。     

首次从喜马拉雅旱獭分离布鲁氏菌与鉴定

目的 从喜马拉雅旱獭抗体阳性的血液分离布鲁氏菌并对其进行种的鉴定。方法 采用血培养法对布鲁氏菌抗体阳性的旱獭血液进行细菌分离,并应用传统方法对该菌进行种的鉴定。结果 经优化后的肝浸液双相培养基培养出布鲁氏菌可疑菌落,经传统方法鉴定为羊种布鲁氏菌。结论 首次证实喜马拉雅旱獭存在羊种布鲁氏菌。     

痰结核菌培养检出时间与肺结核治疗效果关系探讨

目的　探讨痰结核菌培养检出时间 (DTP)和肺结核病人抗结核疗效之间的关系。方法 对 56例住院肺结核病人,于疗程 0、7、14、30及 60d留痰行快速痰结核菌培养,记录培养阳性检出时间并追踪临床疗效以评价两者关系。结果 抗结核治疗后 40例病人对治疗显现良好反应同时其结核杆菌在培养管中的DTP逐渐延长。 16例痰菌仍阳性病人,其结核杆菌在培养管中的DTP没有变化或增长很少。结论 在预测治疗效果上DTP与肺结核治疗反应关系密切,可能是临床预测病人疗效的一种方法。     

Seroepidemiology and serological follow-up of anti-leptospiral IgG in children in Southern Vietnam

A follow-up study was conducted with 23 months interval to investigate the seroepidemiology and persistence of Leptospira IgG antibodies among healthy children in Binh Thuan province, Southern Vietnam. Sera from 262 children (7–13 years of age) were collected and analysed with a commercially available enzyme-linked immunosorbent assay (ELISA) for Leptospira IgG. Seroconversion was observed in 10.4% (22 of 211, 95% CI: 5.6–26.7) of the children, of whom 18 (8.5%) had probably and four (1.9%) had certainly been exposed to Leptospira. Based on the reduction of sero-negatives of 1.9% among children who have been certainly exposed, the annual seroconversion rate, a measure of the incidence rate of Leptospira infections, corresponds to 0.99% (95% CI: 0.39–2.52). In 61% (31 of 51, 95% CI: 47.1–73.0) of the children with past-infection, Leptospira IgG antibodies remain detectable after 2 years. Data from this study indicate that IgG antibody responses against Leptospira may persist at least for 2 years in children without manifestations of leptospirosis. Results of study uncover the true incidence of leptospirosis infection, the dynamics of waxing and waning antibody concentrations and points at a larger burden of clinically non-significant Leptospira infections in Southern Vietnam. This also indicates background reactivity for serological testing and thus serological result of a single serum sample must be carefully interpreted.     

江西省致病性钩端螺旋体血清学和分子流行病学研究

目的 对2013年以来江西省致病性钩端螺旋体进行血清学、基因分型分析,以了解江西省钩端螺旋体血清学和分子流行病学特征。方法 对27株钩端螺旋体进行暗视野显微镜凝集试验确定血清群。PCR扩增16S rRNA基因、测序,确定基因种。利用MLST(multilocus sequence typing)研究进行基因分型分析,并应用BioNumerics (Version5.10)软件进行聚类分析。结果 血清群鉴定:27株菌株隶属于4个血清群,其中黄疸出血群为主要优势血清群,占59.26%,其次依次是爪哇群25.92%、澳洲群7.41%和巴达维亚群7.41%。基因种鉴定:27株菌株隶属于L. interrogans和L. borgpetersenii 2个致病性基因种,L. interrogans为江西省主要优势型别,占77.78%。MLST研究显示27株菌株隶属于5个ST型别,其中ST1为主要基因型,占59.26%。BioNumerics软件分析:27株菌株分为5个Clusters对应于5个ST型,MLST基因型别具有明显的地域性特征,而年代间变化不明显。结论 黄疸出血群为江西省主要流行血清群,L. interrogan为主要致病基因种,ST1为主要基因型,充分了解江西省钩体病血清学和分子流行病学特征将对钩体病防控和疫苗制备有一定的指导意义。     

Epidemiology and characterization of leptospirosis at an urban and provincial site in Thailand

Patients with FUOs at the Children's Hospital in Bangkok and the Chao Phya Abhai Bhu Bejhr Hospital in Prachinburi were screened for leptospirosis by blood and urine culture in addition to microagglutination testing of their serum. Animal populations in urban and periurban areas of Bangkok were surveyed for evidence of leptospira infection. Three rural sites near the Prachinburi Provincial Hospital were also surveyed. The rodents' and domestic animals' blood, urine, and/or kidney cell samples were cultured for leptospira. Sera from these animals were also tested for leptospira antibody. The bataviae serovar was the most commonly detected leptospiral agent in both man and animals. Presenting symptoms varied with age with children showing primarily fever, vomiting, headache, abdominal and generalized muscle pain and diarrhea whereas adults had fever, headache, anorexia, muscle pain and constipation. Blood samples from patients suspected of having leptospirosis were tested for antibody by the MAT and cultured in EMJH media. The following serogroups were identified: bataviae, autumanalis, javanica, hebdomadis, and pyrogens. Leptospirosis incidence in humans was much higher in the rainy/flooding year of 1983 compared to the relatively dry year of 1984. Results of our animal surveillance studies indicate that in addition to rats, which have previously been mentioned, dogs, bandicoots, cattle and pigs could be the source of human leptospirosis infection in both urban and provincial locations in Thailand.     

钩端螺旋体C-70培养基的研制

本文报道C-70培养基对四个型的钩端螺旋体菌种的培养与检定。所培养的菌种,形态正常,运动活泼。以1％种子量(菌液浓度10亿/ml)接种时,培养7天,即能达到生长高峰期。生长浓度比原4·1培养基提高4.5～11.6倍(由原来1.5～2亿/ml提高到10亿/ml左右)。反向炭凝试验测定钩体抗原效价也高出4～64倍(由原来1:128～256++提高到1:1024～8196++)。制成菌苗后,物理性状良好,安全试验符合钩体菌苗制检规程合格标准,抗原经30倍稀释(含菌0.33亿/ml)作保护力试验,免疫动物100％保护。     

Leptospirosis in Istanbul, Turkey: a wide spectrum in clinical course and complications

Patients with high fever and multiorgan involvement were investigated for the determination of frequency, clinical course and complications of leptospirosis in Istanbul. Leptospirosis was determined in 22 cases among the 35 hospitalized patients that were pre-diagnosed as leptospirosis according to 'Probable Leptospirosis Diagnosis and Follow-up' form. Among the leptospirosis cases 19 were male and 16 were military staff. Mean age was 35.6 y. Dark field examination (DFE), latex agglutination test (LAG), ELISA IgM, leptospirosis culture (LC) and microscopic agglutination test (MAT) were performed to confirm the diagnoses. The most frequent initial symptoms and findings were fever, fatigue, headache, nausea-vomiting and increased muscle sensitivity. Jaundice was noted only in 2 cases. A 74-y-old female patient died after the recurrence of the disease with severe rhabdomyolysis and pulmonary failure. Sagittal sinus thrombosis, perimyocarditis and chronic renal failure were major complications in another 3 patients. ELISA IgM, LC, DFE, LAG and MAT tests were positive in 68, 72, 82, 100 and 100% of the patients, respectively. As a conclusion, diagnosis of leptospirosis is usually overlooked. Clinical awareness, use of probable leptospirosis diagnosis forms and the application of different laboratory methods in the diagnosis of suspected cases may offer the chance to diagnose the leptospirosis accurately.     

Comparison of serology and isolates for the identification of infecting leptospiral serogroups in Hawaii, 1979-1998

Laboratory confirmation of leptospirosis is usually accomplished serologically, without isolates, using the microscopic agglutination test (MAT). However, optimal performance of the MAT is dependent on the knowledge of enzootic serogroups and serovars so that an appropriate MAT antigen testing battery can be established. Infecting leptospiral serogroups can be identified serologically without isolates, using the MAT, or by serogrouping of isolates, but little information is available regarding the correlation between these methods. The identification of infecting serogroups for 53 culture-confirmed leptospirosis cases, diagnosed in Hawaii between 1979 and 1998, using serology and culture isolates were compared. The overall agreement between the two methods was good (kappa = 0.71, 95% CI: 0.56, 0.86). However, the agreement varied between serogroups from 0 to 100%. In establishing the prevalence of serogroups, results obtained via MAT serology (in the absence of serogrouped isolates) should be considered presumptive rather than definitive.     

Introduction of a rapid dipstick assay for the detection of Leptospira-specific immunoglobulin m antibodies in the laboratory diagnosis of leptospirosis in a hospital in Makassar, Indonesia

An easy, rapid and robust dipstick assay for detection of leptospira-specific immunoglobulin M (IgM) antibodies was evaluated on 403 patients admitted for hospitalization because of fever. The clinical symptoms and signs of 35 patients were consistent with leptospirosis. The final diagnosis for the remaining patients was as follows: 136 with typhoid fever, 82 with hepatitis, 74 with malaria, 48 with infections of the respiratory tract, and 20 with fever of unknown origin. The clinical diagnosis of leptospirosis was confirmed for 24 (68.6%) patients by the combined results of the microscopic agglutination test (MAT), the reference test for leptospirosis, and of IgM ELISA, a standard laboratory test for the serodiagnosis of leptospirosis. In addition, serum specimens from 8 (2.2%) patients with a final clinical diagnosis other than leptospirosis were found to be positive in MAT and/or IgM ELISA. Compared with the results of MAT and IgM ELISA a sensitivity of 91.6% and specificity of 93.6% was calculated for the dipstick assay. Most of the serum samples from the laboratory confirmed patients gave a moderate to strong staining intensity of the antigen band of the dipstick and were easy to read. The results demonstrate that the dipstick assay is convenient to use and allows the rapid and accurate confirmation of patients with clinical suspicion of leptospirosis in areas where the disease is endemic.     

Field evaluation of a one-step dipstick assay for the diagnosis of human leptospirosis in the Seychelles

OBJECTIVE AND METHOD: To compare the response of a dipstick assay (DSA) detecting Leptospira-specific immunoglobulin M (IgM) antibodies with that of an enzyme-linked immunosorbent assay (ELISA), an indirect haemagglutination assay (IHA), the microagglutination test (MAT) and a polymerase chain reaction assay (PCR) in patients with leptospirosis confirmed by MAT alone or by MAT and/or PCR (MAT/PCR). RESULT: In 75 patients with acute leptospirosis diagnosed by MAT (respectively, 90 patients diagnosed by MAT/PCR), the response in paired early and convalescent sera was positive in 78.9% (67.9%) by DSA, 76.0% (67.8%) by ELISA, 58.7% (55.6%) by IHA, 44.0% (53.3%) by PCR, and 100% (90.0%) by MAT. In early serum only, the response in patients diagnosed by MAT (respectively by MAT/PCR) was positive in 36.0% (38.9%) by DSA, 36.0% (37.8%) by ELISA, 14.7% (18.9%) by IHA, 39.2% (48.3%) by PCR, and 53.3% (58.9%) by MAT titre > or =1:100. DSA detected the main serogroups implicated in human leptospirosis in Seychelles and demonstrated sensitivity comparable to ELISA. In 124 single sera from control subjects without overt disease, the response was positive in 4.8% by DSA, 3.2% by ELISA, 3.2% by IHA, 13.8% by PCR, 37.9% by MAT titre > or =1:100, and 2.4% by MAT titre > or =1:800, giving evidence of the frequency of both past and current subclinical infection in Seychelles and that DSA was less sensitive than MAT to detect moderate levels of leptospiral antibodies. CONCLUSION: DSA is a simple and reproducible assay well adapted to field conditions and could usefully contribute to the evaluation of leptospirosis in areas devoid of serological laboratory facilities.     

Evaluation of two enzyme-linked immunosorbent assay methods for detection of immunoglobulin M antibodies in acute leptospirosis

Leptospirosis is a common zoonosis of worldwide distribution. Diagnosis of leptospirosis is usually accomplished by serology, but the microscopic agglutination test (MAT) generally requires paired sera for detection of seroconversion and is considered too complex for routine use. A number of rapid assays have been developed in recent years. In the present study, 2 immunoglobulin (Ig) M enzyme-linked immunosorbent assay (ELISA) methods were evaluated for the early diagnosis of acute leptospirosis in Barbados. A total of 103 patients admitted to the Queen Elizabeth Hospital for diagnosis of suspected leptospirosis were investigated. A case of leptospirosis was confirmed by a 4-fold rise in titer between 2 sera tested by MAT, an initial titer of > or = 800 in the MAT, or by isolation of leptospires from blood or urine. A total of 48 cases of leptospirosis were confirmed. In 33 cases, both commercial assays were positive in the first sample, taken at admission, a mean of 6.7 days after onset of symptoms, whereas seroconversion was detected in a further 9 cases. Both assays were negative in 5 cases, and the remaining case gave discordant results in the 2 assays. False-positive IgM results were detected in 4 patients without leptospirosis. The sensitivity of the 2 assays was 89.6 and 97.5%, respectively, and specificities were 92.7 and 96.4%, respectively. The positive predictive values were 87.8 and 95.5%, and the negative predictive values were 90.7 and 89.5%, respectively. Either of these assays can be used for early diagnosis of leptospirosis, particularly in laboratories that cannot perform more specialized leptospiral serology.     

Comparison of a slide agglutination test, LeptoTek Dri-Dot, and IgM-ELISA with microscopic agglutination test for Leptospira antibody detection

A slide agglutination test (SAT), LeptoTek Dri-Dot and IgM-ELISA were compared with a microscopic agglutination test (MAT) for the detection of Leptospira antibodies. Paired sera from 10 patients whose leptospirosis was clinically suspected and diagnosed by MAT, were evaluated in this study. Our data, especially from acute samples, demonstrate the SAT and Dri-Dot were more sensitive as initial screening tests than MAT. IgM-ELISA has an advantage over MAT, SAT, and Dri-Dot since the results can be interpreted from a single serum testing if the results of the test are positive. Eight of the ten cases could be diagnosed by IgM-ELISA. Our data suggest that IgM-ELISA may be used for the diagnosis of leptospirosis. However, the agglutination test is useful for screening and for secondary infection cases for which IgM antibodies may be undetectable. MAT can be performed as a reference test and when information regarding the causative serovar is required.