LEPTO Dipstick: a rapid and simple method for serodiagnosis of acute leptospirosis.

The LEPTO Dipstick assay is a newly developed test for the diagnosis of leptospirosis and uses a broadly reactive antigen for detecting IgM antibodies. The test was evaluated in the Andaman and Nicobar Islands, using 867 serum samples from known cases of leptospirosis and controls. The efficacy of IgM ELISA was also tested for comparison. The LEPTO Dipstick had a sensitivity of 78.7%, a specificity of 88.3% and a positive predictive value of 91.0%. The test had a good level of agreement with the standard criteria for diagnosis using paired microscopic agglutination tests (kappa = 0.64). These indices were similar to those of IgM ELISA (sensitivity 78.5%, specificity 87.6%, positive predictive value 90.5%, kappa 0.63). Both the LEPTO Dipstick and IgM ELISA had the highest sensitivity during the second, third and fourth weeks of illness (87.6% and 88.2%, respectively). Sensitivities during the first week and after 4 weeks were relatively low but acceptable. The test is very easy to perform and does not require any special skills for its performance. The reagents and dipsticks have a long shelf-life even at room temperature. As the test can be performed without the aid of sophisticated equipment, it is suitable for use at the peripheral level as a rapid screening test for the diagnosis of leptospirosis.     

Evaluation of a passive microcapsule agglutination test for the screening of human leptospirosis

Microcapsules, absorbed with two mixed antigens- each composed of 3 sonicated leptospira serovars — were developed in the past as a microcapsule agglutination test (MCA-LS) for the screening of clinical leptospirosis.Fof this study, fifty serum samples, taken at an earlier and at a later stage of illness from 25 Italian in-patients with clinical symptoms of leptospirosis, were tested with both the MCA-LS one-dilution test and the microscopic agglutination (MA) test, the confirmatory test for leptospirosis, with 18 leptospira strains circulating in Italy. Compared with MA, MCA-LS showed a sensitivity of 91.7%, and a specificity of 92.3%, including leptospiroses not substained by the diagnostic strains used in the MCA-LS kit.     

酶免疫斑点试验用于钩端螺旋体病早期血清学诊断的研究

应用酶免疫斑点试验（ｄｏｔ－ＥＬＩＳＡ）和显微镜凝集试验（ＭＡＴ）对培养钩端螺旋体（钩体）阳性的３０份早期钩端螺旋体病（钩体病）人血清进行了检测，发现ｄｏｔ－ＥＬＩＳ人的阳性率（４０％）明显高于ＭＡＴ的阳性率（１３．３％）．同时对１９８份临床确诊为期期钩体病人的血清进行了ｄｏｔ－ＥＬＩＳＡ、ＭＡＴ和血培养对照研究，证明ｄｏｔ一ＥＬＩＳＡ的阳性率（６４．６％）也明显高于血培养阳性率（１５．２％），同时也高于ＭＡＴ的阳性率（１４．１％）。随着发病时间的延长，血培养阳性率逐渐降低，ｄｏｔ－ＥＬＩＳＡ阳性率变化不明显。     

应用纯化重组外膜脂蛋白LipL32检测钩端螺旋体病抗体

目的 建立以纯化重组外膜脂蛋白LipL32为基础的钩端螺旋体(钩体)病抗体的检测方法 .方法 以摹因重组技术获取重组钩体外膜脂蛋白LipL32,以该蛋白为抗原,分别使用间接法和夹心法ELISA应用于不同人和鼠血清的检测,检测结果 与钩体显微镜凝集试验(MAT)进行比较,同时人血清的检测结果 还与进口试剂盒比较.结果 纯化的重组蛋白检测9例钩体确诊病例双份血清标本,三种ELISA法的检出率与MAT无明显差别.检测45份MAT阳性标本,间接法ELISA敏感性为71.11%(32/45),夹心法ELISA为80.00%(36/45),而进口 ELISA阳性13份,占28.89%(13/45),25份可疑占55.56%(25/45).特异性检测,MAT阴性血清69份,间接和夹心法ELISA特异度均为97.10%(67/69),其中检测门诊体检血清43份,间接和夹心法ELISA均为阴性,进口ELISA检测14份,也为阴性.检测非钩体发热病例血清标本16份,间接和夹心法ELISA共检出2份阳性,进口ELISA则是1份阳性,12份可疑.检测梅毒螺旋体质控阳性血清10份,四种方法 均为阴性.重组蛋白检测鼠血清标本274份,夹心法ELISA的敏感性为86.75%(131/151),特异性为99.19%(122/123),符合率为92.34%(253/274),与MAT检测结果 相符.结论 重组LipL32蛋白具有结合活性,可应用于钩体血清抗体的检测,其中夹心法ELISA对鼠血清钩体抗体的检测显示较好的敏感性和特异性,适用于钩体病现场血清流行病学大样本调查.     

Incidence of leptospirosis in a select population in Nepal

The geographic distribution of leptospirosis is widespread but no national surveillance program exists in Nepal to establish the incidence of leptospirosis or the disease burden. This study reports the incidence of symptomatic leptospirosis in military personnel participating in an efficacy study of a hepatitis E virus vaccine in Nepal. Among the 1566 study volunteers who completed follow-up, we evaluated 271 illnesses over 2.2 years for the presence of leptospira IgM antibodies by ELISA. Positive ELISA results were confirmed by the microscopic agglutination test. The annual incidence of disease was between 3.5 and 6.1 cases/1000. The prevalence of confirmed leptospirosis was 9% among hepatitis cases and 8% among febrile cases. The most reactive serovars were Bratislava, Autumnalis, Icterohaemorrhagiae, and Sejroe. Leptospirosis should be considered in the differential diagnosis of febrile illnesses and icteric syndromes in Nepal. Additional studies are needed to establish the broader distribution and the spectrum of disease in Nepal.     

聚合酶链反应和地高辛标记的核酸杂交技术用于钩端螺旋体的检测

目的为钩端螺旋体快速诊断和流行病学调查建立一种比较理想的方法.方法根据钩端螺旋体赖株DNA合成一对flaB引物,用PCR技术对钩端螺旋体菌株、疫区现场动物标本等进行flaB基因扩增,用地高辛（DIG）标记flaB基因探针,用琼脂糖凝胶电泳和斑点杂交技术进行检测.结果纯化钩端螺旋体DNA 5pg经flaB-PCR扩增后,琼脂糖凝胶电泳可以目测.用DIG标记的flaB探针可以检测到5fg及以下的DNA扩增产物.疫区70份蛙肾标本,分离细菌8株,阳性率11.43%.flaB扩增阳性14份,阳性率20%;DIG标记探针斑点杂交检测,阳性19份,阳性率为27.14%.结论PCR-斑点杂交是一种灵敏、特异、快速的钩端螺旋体检测方法,既可用于快速检测和早期诊断,也可用于疫情监测和流行病学调查.     

Peripheral nerve palsy in a case of leptospirosis

We describe an unusual case of leptospirosis in a 54-year-old man presenting peripheral nerve palsy. The diagnosis of leptospirosis was confirmed by ELISA IgM and the microscopic agglutination test. Electrophysiological studies showed that no response could be obtained from the right fibular nerve. At 7 months after the initiation of treatment, additional electrophysiological studies and a neurological examination showed, respectively, a chronic axonal lesion of right fibular nerve with signs of re-innervation and a nearly complete clinical recovery. We feel that this case may serve to remind clinicians that peripheral nerve palsy is a potential clinical feature of leptospirosis.     

Leptospirosis in Kolenchery, Kerala, India: Epidemiology, prevalent local serogroups and servoars and a new serovar

Leptospirosis is the most widespread zoonosis in the world. It is mostly an occupational disease especially in rural areas. Kolenchery is in the midlands of Kerala. The landscape consists of marshy land and dry land interspersed by rivulets and ponds. Irrigation of dry lands for cultivation during the summer months started in mid 1980s after the irrigation projects were commissioned. Cases of leptospirosis were rarely diagnosed in Kolenchery before 1987. Since then a yearly increase in incidence is observed. No major study on either epidemiology or clinical picture has been done on human leptospirosis in Kerala so far. The aim of this study was to define the major clinical presentations, epidemiology of the infection and demonstrate the predominant serogroups and serovars causing the illness in this limited geographical area. The study involves 976 cases of leptospirosis confirmed by culture and/or serological tests. Serogroups Autumnalis, Australis and Icterohaemorrhagiae were the commonest. Mortality rate was 5.32%. The increase in incidence is probably due to the geographical characteristics, continuous moisture of the soil due to irrigation in summer and year-round cultivation making food and cover available to host rodents. Close interaction of humans, animals, soil and water in this region make the spread of leptospirosis to humans easy. Confirmation of cases has been done by enzyme linked immunosorbent assay (ELISA) and/or microscopic agglutination test (MAT) and/or by culture. Serovar identification has been done in 21 cultures isolated from patients. One new serovar belonging to serogroup Australis has been identified from a patient and named serovar Australis bharathy.     

Evaluation of the one-point microcapsule agglutination test for diagnosis of leptospirosis.

We have developed a one-point microcapsule agglutination test (MCAT) for the serodiagnosis of leptospirosis. The MCAT kit was evaluated for use in humans by six WHO Collaborating Centres for Reference and Research on Leptospirosis. The laboratories classified their serum samples on the basis of the microscopic agglutination test (MAT) and the following screening tests: enzyme-linked immunosorbent assay (ELISA), macroscopic (slide) agglutination test, or the complement fixation test. The MCAT may in some instances give a positive result earlier in the course of the disease than MAT or the ELISA IgM; on the other hand, it did not detect antibodies against some serovars, for example, those of the Sejroe or Australis serogroup in Slovakia. In contrast, however, the MCAT detected antibodies to serovar hardjo (the same serogroup as Sejroe) in patients from the United Kingdom and the Russian Federation.     

Diagnosis of leptospirosis: evaluation of a solid-phase enzyme immunoassay in different stages of the disease]

OBJECTIVE: To develop a solid-phase enzyme immunoassay (ELISA) for genus-specific immunoglobulin G (IgG) determination with leptospirosis and to evaluate the ELISA in different stages of the disease. METHODS: A total of 1,077 serum samples from 812 patients with suspected leptospirosis were analyzed. The samples had come from diagnoses done in the laboratory of the National Institute of Respiratory Diseases (Instituto Nacional de Enfermedades Respiratorias), in the city of Santa Fe, Argentina, between 1999 and 2005. Included in the study were 182 confirmed cases (267 samples), 167 negative cases (293 samples), and 40 probable cases (60 samples) (based on case definitions based on the results from the microscopic agglutination test (MAT), leukocyte counts, and neutrophilia values). Each sample was classified, according to the days of the natural history of disease, into one of three stages: first (< 10 days), second (10-25 days), or third (> 25 days). The antigen used in the ELISA was an extract of a mixture of pyrogenes and tarassovi serovars cultivated in a liquid medium, treated with ultrasound, and immobilized by adsorption on polystyrene plates. As a secondary antibody, a peroxidase-conjugated goat anti-human IgG monoclonal antibody was used. The cutoff value, sensitivity, and specificity of the ELISA were determined using the definitions of confirmed cases and of negatives cases as the standard. In order to determine the optimal cutoff value, the area under the receiver operating characteristic curve was calculated. RESULTS: The sensitivity of the evaluated test was much higher in the second stage (93.2%) than in either the first stage (68.1%) or the third stage (78.8%). The specificity increased gradually from 96.3% in the first stage to 100% in the third stage. CONCLUSIONS: Our results indicate that this ELISA test can be a very useful complement to the MAT for the diagnosis of leptospirosis in all the stages and, in particular, in order to diagnose acute disease sooner.     

Occupational risks of zoonotic infections in Dutch forestry workers and muskrat catchers

Lyme borreliosis (LymeB), Hemorrhagic fever with renal syndrome (HemoFRS), Lymphocytic choriomeningitis (LymphoCM), Tick-borne encephalitis (TickBE), Q Fever (QFever), and Weil's leptospirosis (Lepto) are known to be occupational diseases for rural outdoor workers. We investigated the occupational infection risks for these diseases in greater detail. Antibodies to these agents were assessed in 312 forestry workers and muskrat catchers, and in 356 matched office workers. Three levels of occupational exposure were distinguished: high for active forestry workers, low for supervisory forestry staff and muskrat catchers and zero for office workers. At high exposure the prevalence odds ratios (with 95% CI between brackets) were: LymeB 15 (5.5–42), HemoFRS 11 (1.3–501), LymphoCM 5.4 (1.0–50), TickBE 1.0 (0.3–3.0), QFever 1.0 (0.4–2.1), and Lepto 0 (0.0–33). No significant risk of infection was found at low exposure. Part of the present group had also been studied in 1989 and 1990. Within this cohort the conversion rates to Borrelia burgdorferi have been estimated for the periods 1989–1990 and 1990–1993 as 0.23 and 0.066 year^-1, respectively, and the reversion rate as 0.44 year^-1. The corresponding values for HemoFRS were 0.031, 0.018 and 1.42 year^-1. Active forestry workers in the Netherlands are at high risk of infection by LymeB, HemoFRS and LymphoCM. Prevalence of antibodies to LymeB and HemoFRS appeared to reflect the levels of these infection hazards in recent history.     

A study of leptospirosis among animals in Barbados W.I.

Microscopic agglutination tests performed during 1971 and 1972 on Barbadian livestock showed positive serological reactions for leptospirosis as follows: cattle 51%, pigs 13%, sheep 18%, horses 64% and goats 19%. The serogroup Autumnalis predominated in all but horses in which the reactions to the Pyrogenes group appeared slightly higher. After the isolation of a strain of Leptospira serotype fort-bragg (Barbadian strain No. 119) of the Autumnalis serogroup in 1973, and its subsequent inclusion in our test battery of leptospiral antigens, high percentages of cattle, dogs, mongooses and man showed sero-positive reactions against the strain. Attention is called to the wide dissemination of the fort-bragg serotype on the island and to the role of rodents and the mongoose in its spread. 74 clinically affected Barbadian cattle from three herds were studied during 1975-76; 60 (81%) were serologically positive and nine different serogroups were represented. Predominant sero-positive reactions to Hebdomadis, Autumnalis, Ballum and Pyrogenes antigens were recorded. The highest titres were against Autumnalis, Hebdomadis and Pomona. Hebdomadis and Pomona serogroup antibodies may be due to many importations of cattle with those infections into Barbados.     

Evaluation of the Standard Diagnostics Leptospira IgM ELISA for diagnosis of acute leptospirosis in Lao PDR

The diagnostic utility of the Standard Diagnostics Leptospira IgM ELISA for detection of acute leptospirosis was assessed in febrile adults admitted in Vientiane, Laos. Using the cut-off suggested by the manufacturer [optical density (OD) ≥0.75], the assay demonstrated limited diagnostic capacity with a sensitivity of 95% and a specificity of 41% compared with the Leptospira microscopic agglutination test, which is the serological gold standard. However, re-evaluation of the diagnostic cut-off to an OD of 1.7 demonstrated improved diagnostic accuracy overall (sensitivity 70%; specificity 78%).     

Pre- and post-treatment antibody levels in visceral leishmaniasis

Enzyme-linked immunosorbent assay (ELISA) and the direct agglutination test (DAT) were employed to test sera obtained from a visceral leishmaniasis (VL) endemic area, the Aba-Roba focus in south-west Ethiopia. Thirty sera of untreated VL patients, 37 sera 6-90 months after treatment, 18 sera from endemic controls, 8 sera from non-endemic controls and 23 sera from patients with other diseases (schistosomiasis, tuberculosis, cutaneous leishmaniasis) were tested. Based on ELISA, the percentages negative were found to be 50% up to one year and 89% from 2 to 8 years after treatment. In contrast, these rates based on DAT were 0% in one year and 33% from 1-8 years after treatment. The relevance of persisting antibodies in the kinetics and profile of antibody production during treatment is discussed. The use of ELISA in the evaluation of clinical prognosis and patient follow-up is recommended. Serum from a diffuse cutaneous leishmaniasis patient cross-reacted with the DAT and ELISA for VL.     

洪雅县钩端螺旋体病流行特征分析

[目的]了解洪雅县钩端螺旋体病流行特征及防治效果。[方法]收集1964年至今洪雅县钩端螺旋体病的人群发病及死亡情况、血清学及病原学检测结果、免疫接种资料,将资料整理后导入SPSS11.5进行统计分析。[结果]洪雅县1964年至今各年均有钩体病发生,1964￣1993年期间年发病率波动在5.9￣290.1/10万之间(年均发病率56.3/10万)。1970￣1977年的流行高峰在每年的8￣10月,病人数占全年的88.6%,与水稻收割时间一致;人群分布以农民为主,占82.4%;年龄组以10￣39岁组为主,占88.5%。1979￣1980年,5种鼠(226只)肾组织培养钩体阳性率为5.3%,其中黑线姬鼠钩体阳性率为18.1%。1979￣1984年,132例患者血培养6例阳性(阳性率4.5%)。1979￣1985年,206例患者血清显凝试验,阳性率24.3%,收集86份阳性血清进行显凝试验:有黄疸出血群、秋季群、澳洲群等7个血清群。[结论]我县钩体病的流行趋势与全省流行趋势基本一致,主要流行菌群为黄疸出血群,钩体病疫苗接种对降低发病率有一定效果。     

Persistence of anti-leptospiral IgM,IgG and agglutinating antibodies in patients presenting with acute febrile illness in Barbados 1979-1989

The persistence of anti-leptospiral IgM and IgG antibodies and agglutinating antibodies was studied in serologically confirmed cases of severe leptospirosis during the acute illness and over periods of several years after recovery. The antibody response in non-leptospirosis patients presenting to hospital with similar symptoms over the same period of time was used to estimate the background antibody level to leptospirosis in the community. All patients enrolled in the study had blood samples collected twice in the acute stage of illness, once during convalescence and then annually from the time of initial hospitalisation until the end of the study period. Six hundred and thirty-eight patients presented to hospital with acute febrile illness, of whom 321 were diagnosed with leptospirosis. Patients who had severe leptospirosis commonly remained seropositive, with IgM, IgG and agglutinating antibodies detectable for several years after infection. A significant proportion of cases had high titres of agglutinating antibody detectable by the microscopic agglutination test ( 800). There were marked differences in the magnitude and duration of persistence of agglutinating antibodies directed against different serogroups. More than 20% of cases with evidence of infection with serogroup Autumnalis retained titres of > 800, 4 years after the acute illness. In one case a titre of 800 was detected 11 years after infection. Persistence of agglutinating antibody titres can create problems in interpretation of serological results and make it impossible to estimate the time of infection, given a specific titre. This study demonstrates that in endemic areas where seroprevalence is high, use of a single elevated titre is not reliable to define a current infection.     

The problem of human leptospirosis in Barbados.

The results of a retrospective study of human leptospirosis cases occurring in Barbados during the period 19-68-74, and of serological findings among high risk occupational groups, fever cases and health centre patients monitored for the disease during 1975-77, indicate that infection by serotype fort-bragg of the Autumnalis group is widespread in this Caribbean island. The occupational groups most affected are sanitation workers and sugar-cane workers. Results of serological tests conducted at the principle hospital are comparable with the results obtained by the microscopic agglutination reference test procedure conducted at the Pan American Zoonosis Centre (CEPANZO).     

2008-2009年峨眉山市健康人群钩端螺旋体血清学监测结果分析

目的了解2008-2009年峨眉山市钩体疫源地健康人群钩体抗体水平及菌群分布,为钩端螺旋体病的防治提供科学依据。方法采用显微凝集试验(MAT)对采集的健康人血清进行抗体检测。结果 2008年、2009年采集健康人血清202份,钩端螺旋体抗体阳性率为51.5%(104/202),2年监测的阳性率分别为81.4%(83/102)、21%(21/100)。抗体阳性率与性别、年龄无关,健康人群含12群钩体血清抗体,澳洲群为41.58%,黄疸出血群为23.76%,七日热群为8.91%,流感伤寒群为3.47%,犬群、秋季群、波摩纳群均为0.99%,赛罗群、明尼群、蛮耗群为0.49%。结论峨眉山市健康人群钩端螺旋体抗体阳性率水平较低。     

甲型肝炎病人粪便排出甲肝病毒的规律

本研究用EIA对62例甲肝病人的82份粪便进行了检测。发病前1~15天8份粪便,7份HAV阳性(87.50%),发病前16天以上2份粪便均为阴性。发病后1~7天阳性率为42.86%(6/14),8~15天为13.33%(6/45),16天以上13份,无一份阳性。对4名病人的19份系列粪便标本检测HAV表明,发病前1~15天粪便中HAV滴度最高(P/N=16.2±0.60),发病后急剧下降,至发病后第2周刚刚高于EIA的灵敏度(P/N=4.1±0.78)。