Use of locally isolated saprophytic Leptospira strain for serological testing of human leptospirosis.

A saprophytic Leptospira isolate recovered from tap water was utilized for serological testing. One hundred-twenty Serum samples comprising 55 cases from PUO/febrile jaundice and 65 samples from apparently healthy individuals were tested by MAT and HA using this environmental saprophytic strain and the results compared with that of Leptospira biflexa semaranga patoc, the standard saprophytic strain commonly employed for sero-diagnosis of leptospirosis. The MAT data showed 96.4 per cent correlation between the two strains. Similarly, the HA results were matching to the extent of 94.5 per cent. Results, therefore, suggest that local saprophytic Leptospira strain may serve as a substitute to serovar patoc for serodiagnosis of leptospirosis.     

问号钩端螺旋体检测基因芯片的研制

目的　制备一种用于快速检测、鉴定问号钩端螺旋体的DNA微阵列芯片。方法　从Genbank中选取钩体 2 3SrDNA序列 ,设计一对种特异性引物和DNA探针 ,通过Blast的基因同源性比较 ,验证引物和探针的通用性与特异性。将合成的探针点样到玻片上制备成基因芯片。用Cy3标记引物 ,通过不对称PCR反应获取荧光素标记的靶序列 ,然后与制备的芯片杂交。结果　设计的引物与探针仅同时与问号钩体 2 3srDNA完全同源。该引物可扩增我国 18个血清群的 2 5株钩体 ,均出现单一 4 82bp的扩增产物 ,而双曲钩体及其他螺旋体、病原、空白对照均无任何DNA扩增条带。芯片杂交结果与常规PCR方法结果一致 ,敏感性高于PCR。结论　基因芯片可以快速、灵敏、特异地检测问号钩端螺旋体。     

环介导等温扩增技术检测钩端螺旋体的研究

目的 利用环介导等温扩增技术,建立检测钩端螺旋体的方法。方法 选取钩体LipL32基因,设计了LAMP引物。对27株钩体、25株非钩体样本予以检测,然后进行灵敏度和模拟样本的研究。结果 扩增27株钩体结果为阳性,扩增25株非钩体结果为阴性。灵敏度为200拷贝/μL,模拟样本检测下限为200条钩体/μL(煮沸法)和20条钩体/μL(试剂盒提取法)。结论 LAMP实验操作简单,对设备要求不高,可以作为钩体的快速检测方法。     

DNA diagnosis for the detection of sickle hemoglobinopathies

At this time, the sole generally accepted use for DNA diagnosis in the hemoglobinopathies is for the prenatal detection of disease, which can be identified by these means early in the first trimester of pregnancy. By ascertaining genotype rather than phenotype, the confusion that results from diagnostic errors should be diminished. DNA diagnostics are the future of all genetic disease detection and this future will soon be upon us.     

Accuracy of loop-mediated isothermal amplification for diagnosis of human leptospirosis in Thailand

There is a lack of diagnostic tests for leptospirosis in technology-restricted settings. We developed loop-mediated isothermal amplification (LAMP) specific for the 16S ribosomal RNA gene (rrs) of pathogenic and intermediate group Leptospira species. The lower limit of detection was 10 genomic equivalents/reaction, and analytical specificity was high; we observed positive reactions for pathogenic/intermediate groups and negative reactions for non-pathogenic Leptospira species and other bacterial species. We evaluated this assay in Thailand by using a case-control study of 133 patients with laboratory-proven leptospirosis and 133 patients with other febrile illnesses. Using admission blood, we found that the rrs LAMP showed positive results in 58 of 133 cases (diagnostic sensitivity = 43.6, 95% confidence interval [CI] = 35.0-52.5) and in 22 of 133 controls (diagnostic specificity = 83.5, 95% CI = 76.0-89.3). Sensitivity was high for 39 patients who were culture positive for Leptospira spp. (84.6, 95% CI = 69.5-94.1). The rrs LAMP can provide an admission diagnosis in approximately half of patients with leptospirosis, but its clinical utility is reduced by a lower specificity.     

Indirect immunoperoxidase test for the diagnosis of leptospirosis

Leptospirosis is a widespread zoonotic disease that affects all mammals, including humans, in different parts of the world. Clinical recognition of leptospirosis is challenging, and the definitive serologic diagnosis assay, the microscopic agglutination test (MAT), is time-consuming and difficult to conduct. In this study, an indirect immunoperoxidase (IIP) test to detect Leptospira-specific antibodies in human serum samples was developed. The efficacy of the IIP was compared with the indirect immunofluorescent assay (IFA) and MAT. A total of 368 human serum samples were analyzed by MAT, IFA, and IIP. Using a MAT titer of > or = 1:100 as the gold standard, the sensitivities for the detection of Leptospiral antibodies at a titer of 1:200 were 94.7% by IFA and 93.6% by IIP; specificities were 95.3% by IFA and 94.9% by IIP; and accuracies were 95.1% by IFA and 94.6% by IIP. With a titer of 1:400, the sensitivity, specificity, and accuracy were 86.2%, 98.9%, and 95.7% by IFA, respectively; whereas, for the IIP, the sensitivity was 85.1%, specificity 98.5%, and accuracy 95.1%. A further evaluation of this test with 80 unknown-febrile-disease sera was also included. We found that the sensitivity and specificity of this test were 100% and 76.8%, respectively. Therefore, the IIP test is a potentially valuable tool for the diagnosis of leptospirosis.     

Accuracy of a commercial IgM ELISA for the diagnosis of human leptospirosis in Thailand

The Leptospira immunoglobulin M enzyme-linked immunosorbent assay (IgM ELISA) has been recommended for the rapid diagnosis of leptospirosis in endemic areas. We conducted a retrospective case-control study of 218 patients (109 leptospirosis cases confirmed by Leptospira culture and/or microscopic agglutination test and 109 control patients with acute febrile illness) to evaluate the diagnostic accuracy of a commercial IgM ELISA (Panbio) in northeast Thailand. Paired serum samples taken on admission and at least 10 days after the onset of symptoms were tested. Using the cutoff value recommended by the manufacturer (11 Panbio units), sensitivity and specificity of IgM ELISA on paired sera were 90.8% and 55.1%. A receiver operating characteristic curve was used to determine the optimal cutoff value. This was 20 Panbio units, which gave a sensitivity and specificity of 76.1% and 82.6%, respectively, on paired sera. We conclude that using either cutoff value, the accuracy of IgM ELISA is limited in our setting.     

Immunodiagnosis of human leptospirosis by ELISA-IgM, employing different antigenic preparations from prevalent serovars of Leptospira interrogans

A comparative study among different serovars of Leptospira interrogans was performed in order to prepare antigens to detect IgM antibodies by ELISA in early and late phase of human leptospirosis. Ten serovars were chosen among the most prevalent detected by microscopic seroagglutination (SAM) in S?o Paulo city. Using ELISA-IgM five of them showed better results (canicola, hebdomadis, icterohaemorrhagiae, cynopteri and brasiliensis). These ones were also studied in a pool. The non-treated antigens showed higher reactivity than the Triton X-100 (4%/50 degrees C/4h). ELISA-IgM using individually or pool of non-treated antigens proved to be reliable with high sensitivity and should be used for an earlier diagnosis of leptospirosis, as a trial test. Faster diagnostic elucidation can be useful to detect epidemic situations, so, allowing epidemiological surveillance interventions.     

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.     

Gold immunoblot analysis of IgM-specific antibody in the diagnosis of human leptospirosis.

An immunoblot for the detection of leptospirosis was developed in our laboratory. Antigen prepared from Leptospira interrogans serovar bataviae was dotted onto nitrocellulose paper and blocked with skim milk. Test and control sera diluted 1:20 were applied to the dot, incubated, and washed. Anti-human IgM colloidal gold conjugate was added and the dots were washed. A positive reaction was shown by the development of a pink dot against a white background. The test was performed on 62 sera that tested positive for leptospirosis by a microagglutination (MA) test, on 40 sera that were positive by an indirect hemagglutination (IHA) test, and on sera from forty healthy blood donors. Four sera from the blood donors showed a faint pink dot, but the remainder showed a colorless reaction. All 62 sera that tested positive by MA were positive by this new test, while 95% of the 20 sera that tested positive by IHA were positive. Tests for IgG antibody were performed on 20 sera positive by MA using protein A-colloidal gold conjugate, and all showed weak reactivity. The results confirmed previous findings that most antibodies present in leptospirosis patients are of the IgM type. The ELISA takes three hours to perform, but the gold immunoblot can be completed in 30 min. In addition, the test blot can be kept as a permanent record, and is a significant improvement over existing tests.     

Polymerase chain reaction in comparison with serological tests for early diagnosis of human leptospirosis

The aim of this study was to compare the sensitivity and specificity of polymerase chain reaction (PCR) using two primer pairs and combined with blood culture, immunoglobulin M enzyme-linked immunosorbent assay (IgM ELISA), microscopic agglutination test (MAT) and slide agglutination test (SAT) in the diagnosis of human leptospirosis. We analysed 124 serum samples: 60 from patients with confirmed leptospirosis, 20 from patients with other diseases and 44 from healthy individuals. Analysing the first serum sample collected during the first 3-8 days of disease, the sensitivities of the four tests MAT, IgM ELISA, SAT and PCR were, respectively, 69.0%, 79.3%, 72.4% and 62%. In subsequent samples, those same sensitivities were, respectively, 95.4%, 100%, 100% and 72.7% in samples collected from days 9 to 14 and 88.9%, 88.9%, 77.8% and 44.4% in those collected from days 15 to 42. The most specific method (at 100%) was PCR and the least specific (at 89.1%) was IgM ELISA. Although we found PCR to be less sensitive than the serological tests over the course of the disease, our data indicate that PCR was the most sensitive in those initial serum samples presenting no specific antibodies detectable by any of the serological methods tested. We also recommend that PCR can be used in combination with serological tests as we found that this improves the sensitivity of the diagnosis of leptospirosis in the first phase of the disease (93.1-96.5%).     

Tapered optical fiber DNA biosensor for detecting Leptospira DNA

Objective: To establish a DNA detection platform based on a tapered optical fiber to detect Leptospira DNA by targeting the leptospiral secY gene.Methods: The biosensor works on the principle of light propagating in the special geometry of the optical fiber tapered from a waist diameter of 125 to 12 μm. The fiber surface was functionalized through a cascade of chemical treatments and the immobilization of a DNA capture probe targeting the secY gene. The presence of the target DNA was determined ...     

An evaluation of the ELISA-IgM test in the early diagnosis of human leptospirosis]

Thirty-seven sera samples from patients with leptospirosis icterohaemorrhagic form were studied with a time interval of 2 to 12 days between the beginning of the symptoms and the collection blood samples. It was isolated leptospira of 5 patients' hemocultures (13.5%) and from 4 of these the etiological agent pertained to the serogroup Icterohaemorrhagiae serovar copenhageni. Thirty-five of them (94.6%), including the four patients whose the etiological agent was isolated, showed reactivity in the enzyme linked immunosorbent (ELISA) IgM test. By this way, it was demonstrated that this test is important for a rapid diagnosis of human leptospirosis, even in the beginning of the disease, when there is still leptospiraemic phase.     

The serovars of Leptospira interrogans isolated from cases of human leptospirosis in S?o Paulo, Brazil]

Eighteen strains of L. interrogans isolated from human cases were serotyped by the agglutinin-absorption test at Instituto Adolfo Lutz in S?o Paulo, Brazil. Fourteen were identified as serovar copenhageni (icterohaemorrhagiae serogroup), 2 as canicola (canicola serogroup), 1 as castellonis (Ballum serogroup) and 1 as pomona serogroup (serovar not yet defined). The frequency of serovar copenhageni in 100% of the isolates in icterohaemorrhagiae serogroup is emphasized and more studies to verify the real serovars prevalence as subsidy to the epidemiology of this infection are suggested by the authors.