Epidemiological study of kala-azar by direct agglutination test in two rural communities of eastern Nepal

We conducted a sero-epidemiological study of kala-azar in two endemic communities (Kasaini and Gidhaniya) situated in the Terai (plain) of eastern Nepal. Direct agglutination test (DAT) was used as a serological test for screening. Capillary blood samples were collected by filter paper method from 601 (96%) people of a total population of 628 in Kasaini and from 482 (94%) people of 515 in Gidhaniya. Positive DAT titres (1:2000) were found in 66 (6.09%) of 1083 sera tested. The male-female sero-prevalence ratio was 1.44:1 and the age group of 15 years and above was most affected. Among the bone marrow aspirates collected from 66 DAT seropositive cases, only 19 were positive for Leishmania donovani (LD bodies). Of the 47 DAT seropositive but LD bodies' negative cases, three were clinically active cases of kala-azar. Another nine developed clinical symptoms of kala-azar during 6 months follow-up and 23 were cases that had received prior treatment for kala-azar (within 1 year). The results of this study show the potential of the DAT on filter paper as a screening test for the surveillance of kala-azar at a community level.     

Prospective evaluation and comparison of the direct agglutination test and an rK39-antigen-based dipstick test for the diagnosis of suspected kala-azar in Nepal

The diagnosis of visceral leishmaniasis (kala-azar) remains difficult in rural endemic areas and practical and reliable tests are badly needed. Two serological tests, the Direct Agglutination Test (DAT) and an rK39-antigen-based dipstick test, were compared to parasitological diagnosis in a group of 184 patients presenting at a tertiary care centre in south-eastern Nepal with a history of fever > or = 14 days and splenomegaly; 139 patients had a parasitologically proven kala-azar and 45 patients had a negative parasitological work-up. The rK39 dipstick showed a sensitivity of 97% and a specificity of 71%. The DAT was up to 99% sensitive with a low cut-off titre (1:400) but its specificity did not exceed 82% even with a high cut-off titre (1:51 200). Both tests could be used for screening suspect patients in endemic areas. However, their use as confirmatory tests should be restricted to situations where the proportion of kala-azar among clinical suspect patients is high. The rK39 dipstick is cheaper and easier to use than the DAT and could be used widely provided that both its performance and production remain stable.     

荒漠型黑热病流行区患病影响因素的研究

目的 探讨荒漠型黑热病流行区黑热病患病的影响因素.方法 采用1∶3匹配的病例对照研究方法,以新疆维吾尔自治区伽师县近年发病的21例黑热病患者为病例,按照1∶3的比例在同村选择性别相同、年龄相差不超过1岁、无黑热病病史及黑热病临床症状和体征,且rK39测试结果阴性的63名健康儿童作为对照,对生活习惯、居住环境和行为等因素进行问卷调查,结果用SPSS 17.0软件进行单因素分析和多因素条件Logistic回归分析.结果 单因素分析显示:畜圈与卧室的距离和近两年家庭成员或邻居有无黑热病患者两个因素在两组间存在统计学差异(P＜0.05).多因素分析显示:家畜数量、畜圈与卧室的距离及两者的交互作用具有统计学意义(P＜0.05).结论 在荒漠型黑热病流行区,家畜数量、畜圈与卧室的距离是居民患黑热病的影响因素.     

Comparative evaluation of parasitology and serological tests in the diagnosis of visceral leishmaniasis in India: a phase III diagnostic accuracy study

In this phase III trial for diagnostics for visceral leishmaniasis (VL) in India, we compared parasitological diagnosis with several serological tests: direct agglutination test (freeze dried; DAT-FD), rK-39 strip test, rK-26 strip test and a latex agglutination test for antigen detection in urine (KAtex) in 452 subjects from the endemic regions of Bihar, India. The subjects were segregated into four categories: 230 confirmed patients, 52 probable cases, 70 non-cases and 100 healthy endemic controls. The first two groups were used for estimating sensitivity, the latter two for specificity. Sensitivity of DAT-FD was 98.9%, rK-39: 98.9%, KAtex: 67.0% and rK-26: 21.3%. Sensitivity of DAT-FD on blood taken on filter paper (DAT-FDF) was 99.3%, which was comparable with that using serum. Specificity of serological tests was comparable and high (DAT-FD and DAT-FDF: 94%, rK-39 strip test: 97%, KAtex: 99% and rK-26 strip test: 100%). The classical 'gold standard' parasitological demonstration in splenic smear performed poorly as it missed 18.4% of cases that benefited from VL treatment. Reproducibility of the serological tests between field and central laboratories was excellent (kappa = 1.0, 0.99, 0.96 and 0.94 respectively for microscopy, DAT-FD, rK-39 strip test and rK-26 strip test). A high degree of agreement was observed between DAT-FD and rK-39 strip test (kappa = 0.986). Although DAT-FD and rK-39 strip test were highly sensitive with excellent specificity, the ease of use of the latter makes it most suitable for the diagnosis of VL in the field conditions.     

Latex based, rapid and easy assay for human leptospirosis in a single test format

Leptospirosis is an often severe disease which requires prompt treatment. Laboratory testing is required to reach a valid diagnosis. An agglutination assay for the detection of Leptospira-specific antibodies consisting of individually wrapped agglutination cards containing a stable, dried detection reagent is evaluated. The assay is simply performed by suspending the dried reagent with a drop of serum. The result is obtained within 30 s. The sensitivity of the assay varied with the stage of the disease and was 72.3% for samples collected during the first 10 days of the illness and 88.2% for samples collected at a later stage. The specificity was 93.9% and 89.8%, respectively. These characteristics make the test ideal for use in areas where the disease is common and where laboratory support is not routinely available.     

陕西省韩城市黑热病流行特征及高危因素分析

目的 了解韩城市黑热病流行病学特征及高危因素,为进一步开展流行病学调查奠定基础。方法 对韩城市历史数据进行描述,回访调查2012-2016年报告病例发病诊治经过,按照1∶4匹配的病例对照研究方法,对生活行为习惯和居住环境等因素进行问卷调查,数据用Excel 2007、R软件描述分析。结果 韩城市1953-2016年共报告病例599例,死亡9例,年平均发病率为14.61/10万,病死率为1.50%;2012年至2016年报告病例18例,死亡2例,发病高峰期是5-9月, 占报告病例数的72.22%;病例大部分分布在北部山区;农民发病较多,男性发病高于女性,成人多于儿童;病例主要症状体征为发热、脾大;利用COX回归分析患病因素,养犬OR值为4.618,95%CI为1.519~14.039,P<0.05,病例组与对照组间存在统计学差异,其余因素病例组与对照组不存在统计学差异。结论 韩城市为黑热病历史疫区, 2012年至2016年报告病例呈点状分布,家中养犬是患病的危险因素,综合分析韩城市可能为山丘型或人犬共患型黑热病流行区。     

Serum erythropoietin concentration in anaemia of visceral leishmaniasis (kala-azar) before and during antimonial therapy

Serum erythropoietin (Epo) concentrations and variables of red cell and iron status were studied in 27 Sudanese patients who were treated with sodium stibogluconate for visceral leishmaniasis (kala-azar). Blood haemoglobin increased from 6.4 (±1.7 SD) to 9.5 (±1.4) g/dl during treatment. Serum ferritin decreased concomitantly. Serum iron levels were unchanged whereas the total iron binding capacity increased slightly. The pre-treatment serum Epo concentration in relation to the blood haemoglobin concentration was not as high as expected from the one in primary haematological diseases, indicating that there is a relative lack of Epo in anaemic kala-azar patients. Serum Epo further decreased during stibogluconate therapy. The normal dependence of the serum Epo level on the blood haemoglobin concentration was lost during mid-term antimonial treatment, but it recovered thereafter. Cell culture studies with the human hepatoma cells HepG2 showed that stibogluconate ( 30 μg/ml) inhibited Epo gene expression. Thus, effective treatment of kala-azar with stibogluconate results in improvement of anaemia, although the drug itself may impair Epo production.     

Evaluation of the direct agglutination test based on freeze-dried Leishmania donovani promastigotes for the serodiagnosis of visceral leishmaniasis in Sudanese patients

The direct agglutination test (DAT) based on freeze-dried (FD) Leishmania donovani antigen was evaluated for the serodiagnosis of kala-azar in a rural setting in eastern Sudan. The performance of the FD-DAT was compared with standard liquid antigen (LQ) by testing serum samples and blood samples collected on filter paper of microscopically and PCR-confirmed VL patients, apparently healthy endemic controls and patients with other relevant infectious diseases for the region. In the present study, the FD-DAT had a sensitivity of 96.8% and a specificity of 96.2%. The LQ-DAT had a sensitivity of 91.0% and a specificity of 96.6%. A high degree of agreement (97.3%; r-value 0.94) was observed between the FD-DAT and the LQ-DAT, as well as between the FD-DAT performed on serum samples and corresponding blood samples collected on filter paper (agreement 97.8%; r-value 0.79). The FD-DAT is very suitable as diagnostic test for kala-azar in remote rural conditions as it is sensitive, specific and stable. The antigen is affordable, reproducible and available, which contributes to the sustainability of the DAT as a diagnostic test for VL.     

An original method to study autoantibody specificity in haemoglobin stained eluates by the column agglutination techniques

Summary When studying autoantibody specificity by the indirect antiglobulin test with column agglutination techniques ether and xylene elution techniques result in haemoglobin stained eluates which give a red colouration to the gel or glass beads and do not allow the identification of positive reactions. Xylene eluates were incubated with commercially available group 0-test red cell panels at 37°C for 45 min in the wells of a microtitre plate in a 3:1 eluate:red cell ratio. After washing with normal saline, sensitized red cells, resuspended in low ionic strength solution (LISS), were applied onto the microtubes containing the antiglobulin serum and positive reactions were recorded after centrifugation. We studied the specificity of 35 autoantibody containing eluates from 12 patients with lymphoproliferative disorders (six having autoimmune haemolysis) and 23 HIV patients without autoimmune haemolysis. All patients had a gel or column positive (IgG) direct antiglobulin test while the tube direct antiglobulin test failed to show red cell bound IgG. We found a reactive indirect antiglobulin test in 20/23 eluates from HIV infected patients (with a panreactive specificity), in all patients with autoimmune haemolysis (one with anti-C, two with anti-E, one with anti-K and two with a panreactive specificity) and in all patients with positive direct antiglobulin test but without immune mediate haemolysis (in all cases with panreactive specificity). The method proposed is a promising tool for the study of the specificity of antibody containing haemoglobin stained eluates; in this study it allowed us to confirm that some HIV patients have specific binding of IgG on their RBC and to identify the specificity of tube test non-reactive eluates.     

Comparison of an rK39 dipstick rapid test with direct agglutination test and splenic aspiration for the diagnosis of kala-azar in Sudan

We compared an rK39 dipstick rapid test (Amrad ICT, Australia) with a direct agglutination test (DAT) and splenic aspirate for the diagnosis of kala-azar in 77 patients. The study was carried out under field conditions in an endemic area of north-east Sudan. The sensitivity of the rK39 test compared with splenic aspiration was 92% (46/50), the specificity 59% (16/27), and the positive predictive value 81% (46/57). Compared with the diagnostic protocol used by Médecins sans Frontières, the sensitivity of the rK39 test was 93% (50/54), the specificity 70% (16/23), and the positive predictive value 88% (50/57). Compared with splenic aspirates, the sensitivity of a DAT with a titre > or =1:400 was 100% (50/50), but its specificity only 55% (15/27) and the positive predictive value was 80% (50/62). Using a DAT titre > or =1:6400, the sensitivity was 84% (42/50), the specificity 85% (23/27) and the positive predictive value 91% (42/46). All four patients with DAT titre > or =1:6400 but negative splenic aspirate were also rK39 positive; we consider these are probably 'true' cases of kala-azar, i.e. false negative aspirates, rather than false DAT and rK39 seropositives. There were no false negative DATs (DAT titre < or =1:400 and aspirate positive), but there were four false negative rK39 tests (rK39 negative and aspirate positive). The rK39 dipstick is a good screening test for kala-azar; but further development is required before it can replace the DAT as a diagnostic test in endemic areas of the Sudan.     

PCR diagnosis of visceral leishmaniasis in an endemic region, Mymensingh district, Bangladesh

Detection of Leishmania parasites in a clinical sample is necessary to confirm a suspected case of leishmaniasis. We compared the sensitivity of internal transcribed spacer 1-PCR (ITS 1-PCR) assay for parasite diagnosis with that of microscopic detection in clinical samples from kala-azar (KA) or post-kala-azar dermal leishmaniasis (PKDL) suspects in Mymensingh. Of 39 specimens collected from 35 KA and four PKDL suspects, 26 were positive by microscopic examination of smears from bone marrow and skin exudates; 38 specimens spotted on filter paper and 27 of the 28 Giemsa-stained slides tested by PCR proved positive by ITS1-PCR.     

塔里木盆地荒漠型黑热病宿主动物调查研究

目的调查并查找新疆塔里木盆地荒漠型黑热病自然疫源地的宿主动物。方法参考世界卫生组织(WHO)黑热病宿主动物的5个标准,采用流行病学方法查明荒漠型黑热病患者被感染的主要生境;采集疫区不同生境优势动物,以特异性抗体检测方法筛查抗体阳性动物;调查疫区不同生境白蛉密度和传播媒介吴氏白蛉自然感染利什曼原虫的感染率;采用特殊培养基和敏感动物从患者、阳性动物和吴氏白蛉中分离利什曼原虫,开展分子遗传学相似性鉴定;以吴氏白蛉开展优势动物吸血实验,查找吴氏白蛉的供血宿主;对照黑热病宿主动物的5个标准,根据上述实验调查结果,采用排除法确认天然宿主动物,同时确认荒漠型黑热病疫区的核心区;黑热病暴发时在核心区及其周边以杀虫剂灭蛉,与对照区比较,根据两个区的实际控制效果和时间,最后确定宿主动物。结果荒漠型黑热病患者被感染的生境是胡杨柽柳生境及其相邻农田;胡杨柽柳生境的优势动物为塔里木兔(Lepus yarkandensis)、子午沙鼠(M.meridianas)、毛脚三趾跳鼠(D.sagitta)和科氏三趾矮跳鼠(S.crassicada);共获得16种动物1374份样品(野生动物1137份、家畜237份),只有塔里木兔(45/485)检出抗利什曼原虫阳性个体,从塔里木兔分离出4株利什曼原虫(4/485)。在疫区的所有生境中,只有胡杨柽柳生境中的塔里木兔的抗体阳性率和吴氏白蛉的利什曼原虫感染阳性率最高;吴氏白蛉只能吸到塔里木兔和大耳猬的血,吸血率分别为9.5%和3.3%;塔里木兔的利什曼原虫分离株与当地黑热病患者和传播媒介吴氏白蛉分离株的乙酰氨基葡萄糖磷酸转移酶(NAGT)核基因鉴定结果显示相似性100%,属婴儿利什曼病2型;自2008年黑热病暴发以来,实验区飞机喷洒杀虫剂灭蛉一次,连续7年无流行,对照区流行2次,每次流行持续2年。结论所有动物中只有塔里木兔与WHO规定的黑热病宿主动物的5个标准最接近,传播媒介吴氏白蛉的供血宿主实验是验证天然宿主动物的重要方法;荒漠型黑热病患者被感染的生境是查找黑热病宿主动物的关键区域,即胡杨柽柳核心区及其相邻农田;核心区灭蛉是长期有效控制黑热病流行的重要措施,能为证明荒漠型黑热病宿主动物提供重要证据。     

International multi-centre evaluation of a dipstick assay for human leptospirosis

A dipstick assay for the detection of Leptospira-specific immunoglobulin M (IgM) antibodies in human sera was evaluated in 27 laboratories in 23 countries. 873 serum samples from 711 patients including 329 laboratory-confirmed leptospirosis case patients, 239 noncase patients and 69 patients with viral infections causing heamorrhagic fever were tested. Relative to the results of the reference leptospirosis test, the sensitivity of the dipstick assay was 84.5% for serum samples collected during the first 10 days of the disease and 92.1% for serum samples collected 10-30 days after the onset of disease. The specificity was 87.5% and 94.4%, respectively. Similar to viral haemorrhagic fevers, leptospirosis may cause bleeding. A small number of serum samples from patients with haemorrhagic viral infections gave a weak (1 +) stain. All other samples were negative. In conclusion, the dipstick assay is sensitive and specific and reacts well with serum samples from patients infected with a range of leptospiral strains. It is also easy to use and does not require special equipment or refrigeration. Therefore the assay is ideal for use in developing countries and rural settings.     

Serious underreporting of visceral leishmaniasis through passive case reporting in Bihar, India

OBJECTIVES: Visceral leishmaniasis (VL) is a major public health problem in Bihar, India. Unfortunately, accurate data on the incidence or prevalence of the disease are not available. This longitudinal study was undertaken to determine the incidence of VL in a Community Development Block area of the state of Bihar. Survey results were compared with official reports of the disease to assess the extent of underreporting by the Government health system. METHODS: Three health subcentre areas in Kanti Block, consisting of 14 villages with a total population of 26 444, were selected. Active surveillance was performed every month from January 2001 to December 2003 by house to house survey to detect cases of fever for more than 15 days. Patients clinically suspected of suffering from VL were subjected to parasitological examination for confirmation. Analysis of records of the reporting agencies in the district was undertaken to compare and assess the extent of underreporting. RESULTS: A total of 202 cases of VL were identified in 3 years giving an average annual incidence rate of 2.49/1000 population (95% CI = 2.15-2.83). As identification data of patients was not available with the official reporting agencies for 2001 and 2002, extent of underreporting could be assessed for 2003 only. In the study population, 65 cases of VL were detected during 2003 providing an annual incidence rate of 2.36/1000 population. Only eight (12.30%) cases were reported officially, resulting in underreporting by a factor of 8.13. In 2003, the official incidence rate of VL for Kanti Block was 0.31/1000 against the actual rate of 2.36/1000. As the constraints for official reporting at the block and the district levels are similar, the underreporting at district level was also assumed to be similar. This finding has significance in the preparation for elimination programme.     

Bancroftian filariasis in the Varanasi region of north India: an epidemiological study

The age- and sex-specific distributions of human infections with Wuchereria bancrofti were investigated at two sites in the Varanasi region of north India: one a rural, agricultural area (Chiraigaon) and the other an urban-slum area (Sunderpur). A random clinical and parasitological survey revealed that the prevalence of microfilaraemia and elephantiasis in the urban area (14% and 7.3%, respectively) were both higher than in the rural area (9% and 3.1%, respectively). In both areas, prevalence of microfilaraemia generally increased with age, to a maximum in those aged 20-29 years, and then declined. Within most age-groups, the prevalences of microfilaraemia and elephantiasis were higher in males than females. However, the prevalence of microfilaraemia in females from Chiraigaon who were aged > 30 years was higher than in their male counterparts. Though individual microfilarial intensities varied greatly, the geometric mean microfilarial intensity was higher in Sunderpur than in Chiraigaon (214 v. 196 microfilariae/ml). All 83 subjects with elephantiasis, except one in Sunderpur, were amicrofilaraemic. The present results indicate that bancroftian filariasis is one of the major public-health problems in the study area.