A method for detecting microfilaraemia, filarial specific antigens and antibodies and typing of parasites for drug resistance and genotypes using finger prick blood sample

Monitoring and evaluation of programme to eliminate lymphatic filariasis (LF) depends on epidemiological assessment using appropriate indicators. Minimum efforts using reliable tests are necessary to guide the programme managers in decision-making. Impact of Mass Drug Administration (MDA) towards filariasis elimination can be assessed by the detection of microfilariae (mf) or parasite DNA (infective), filarial antigens (infected) and antibodies (exposure). It is also important to monitor drug resistance and variation in genetic structure of parasite populations using molecular markers. We developed a method to carry out parasitological, molecular, immunological and genetic analysis from a minimum volume of blood sample (about 150 μl) drawn from finger tip of an individual residing in LF endemic area. The method involves separation of sera for immunological assays and isolation of mf of Wuchereria bancrofti from the blood clots for counting, which were then used for W. bancrofti specific PCR, screening for albendazole sensitivity/resistance alleles by AS-PCR, RAPD profiling and ITS 2 PCR for genotyping. A protocol is also suggested for the separation of sera for assays to detect antigen and antibodies and isolation of mf from clots for genetic analysis. The protocol developed has shown potential application in monitoring several immunological, parasitological and molecular parameters from a limited amount of blood sample collected by finger prick, in large-scale operations.     

广东省班氏丝虫病防治后期人群和媒介感染情况观察

目的 探索人群微丝蚴率降至1％以下,终止防治措施后人群和媒介感染情况。方法 终止防治措施后,1988～2000年在原广东省班氏丝虫病流行区抽查30％以上流行镇,重点村居民夜间采血120μl血检微丝蚴,在血检点捕集致倦库蚊解剖,观察自然感染情况;选定10个县原丝虫病流行较严重的村,纵向观察人群和媒介感染情况。结果 1988年人群微丝蚴率为0．33％(575／172631),此后人群微丝蚴率和致倦库蚊自然感染率逐年下降,1997年后无微丝蚴血症者;1992．2000年共解剖致倦库蚊248115只,无发现自然感染幼丝虫;1988～1995年纵向观察,1988年人群微丝蚴率和蚊自然感染率分别为0．14％和0.07％,1994年后两者均为0。结论 班氏丝虫病经防治后,人群微丝蚴率降至1％以下则可阻断传播,10年后人群微丝蚴血症消失。     

志愿者感染周期型马来丝虫的临床观察

３名志愿者于１９８１年试验感染人体周期型马来丝虫后，不给以抗丝虫药物及其他任何治疗，连续１０年观察临床表现、病理、免疫和病原生物学等方面的变化结果。２名志愿者分别于感染后４１和４６周首次出现微丝蚴，８年和８年半后自然转阴。本次试验证明血中嗜酸性粒细胞增多主要表现在微丝蚴血症前期和初期，感染后２～１５６周血中淋巴细胞均有不同程度的增高，感染后６周，淋巴结活组织检查显示以嗜酸性粒细胞，淋巴细胞和单核细胞为主的炎细胞浸润。感染后１１周，淋巴管造影显示下肢，骨盆等处淋巴管扩张和淋巴液滞流。感染后２～５周血中首次出现抗体，１２～５６周达高峰，于１０年后２名志愿者抗体自然转阴。感染后１１周总Ｔ玫瑰花环试验结果都低于正常范围，１０年后均已恢复正常。     

不同地区马来丝虫微丝蚴头隙至神经环间体核数的比较

对四川乐山、贵州独山和福建建阳地区的周期型马来丝虫微丝蚴各200条,采用乙酸一地衣红染色压片法进行头隙至神经环间体核数的比较。结果三地区微丝蚴的体核数彼此间无显著性差异(P>0.05)。三地区微丝蚴的体核均数与 Tada 等以同法用朝鲜的周期型马来丝虫微丝蚴的实验结果比较,有显著性差异(P<0.01)。     

Need for a differential criteria to stop mass drug administration,based on an epidemiological perspective of lymphatic filariasis in Thiruvananthapuram,Kerala, India

ObjectiveTo determine the prevalence of any of the clinical manifestations of lymphatic filariasis, parasitological and entomological indices in Thiruvananthapuram district, Kerala, India, prior to launching mass drug administration (MDA) in the district in 2005.MethodsA cross sectional survey was conducted in 7 endemic wards of the district, in a sample of 2 472 individuals above the age of one year. The study consisted of data collection using questionnaire, night blood smear examination and mosquito collection followed by identification and dissection.ResultsThe prevalence of any of the clinical manifestations of filariasis in this endemic area was 3% (73/2 472) (95% CI between 2.3% to 3.7%). The microfilaria rate was found to be 0.38%. Culex quinquefasciatus formed the predominant mosquito species. The vector infection rate was 1.4% and infectivity rate was 0.47%. Half of the population had satisfactory knowledge regarding the disease. In almost an equal number, the knowledge was poor and only about 10% had good knowledge. Most of the people had not heard about the MDA program.ConclusionsThe prevalence of filariasis even in an area considered to be endemic in Kerala, was low. Microfilaraemia was much lower than the clinical manifestations. However, the potential risk of transmission of disease continues. For such areas which had a pre-MDA mf rate less than 1%, there needs to be more stringent criteria for evaluating the effectiveness of the programme, doing transmission assessment surveys and stopping MDA.     

Current status of bancroftian filariasis in rural communities of the lower cross river basin,Nigeria: parasitological and clinical aspects

Background and objectives  There is a dearth of information on the geographical distribution of bancroftian filariasis in Africa on which to establish elimination programmes. The aim of this study is to assess the prevalence and density of microfilaraemia and the prevalence of clinical manifestations of bancroftian filariasis in six rural communities of Lower Cross River Basin, Nigeria. Methods  A total of 829 finger prick blood smear samples were collected from volunteers between 22.00 and 02.00 h and were examined for presence of helminth parasite Wuchereria bancrofti using standard parasitological method of diagnosis. All the volunteers were also screened for clinical manifestations of lymphatic filariasis. Results  Forty-six (5.6%) of the 829 samples collected were found to be microscopically positive for W. bancrofti. The prevalence of microfilaraemia was significantly (P<0.05) higher in females (8.4%) than males (3.6%). There is a significant variation (P<0.05) in the age-specific prevalence, with the age group 41–60 years being the most affected (10.2%). The overall geometric mean microfilarial density of the infected persons was 9 mf/50 μl. It was higher in males (11 mf/50 μl) than females (7 mf/50 μl) (P>0.05). The prevalence of infection and geometric mean density showed large variations; there was a trend towards increasing prevalence and density with increase in age in both sexes. The overall disease prevalence was 9.2%; the most important clinical manifestation was hydrocele (10.5%) and lymphoedema (2.9%). These chronic disease manifestations increased with age (P<0.05). Conclusion  These baseline data would be useful in planning for the elimination of lymphatic filariasis in Africa as per the WHO goal to eliminate lymphatic filariasis by the year 2020.     

宿主对周期型马来丝虫微丝蚴细胞毒作用的体内研究

以我国流行的周期型马来丝虫为研究为对象，研究宿主体内清除虫体，减轻虫体负荷、控制丝早感染的免疫效应机制。方法提取纯净的周期型马来丝虫微丝蚴装入微孔实验盒，然后植入ＳＤ大鼠体内，在不同时间观察机体免疫系统对ｍｆ的杀伤作用。结果含ｍｆ的３．０μｍ微孔盒内，迁入的细胞量随植入时间延长而逐渐增多。迁入的细胞种类及百分率，各实验组之间无明显差异。免疫血清组ｍｆ被效应细胞粘附、杀伤率均明显高于正常血清组。免疫     

Transforming growth factor-β1 variant Leu10Pro is associated with both lack of microfilariae and differential microfilarial loads in the blood of persons infected with lymphatic filariasis

Antigen testing and ultrasound detection have shown that many persons are infected with Wuchereria bancrofti even though they do not have microfilariae (Mf) in the blood. To ascertain the role of human host immunogenetics on the lack of circulating Mf in the blood, 152 lymphatic filariasis (LF)-infected patients comprising 118 patients with microfilaremic (Mf+, patent) infection and 34 patients with latent (Mf-, antigen-positive) infection were recruited and genotyped for association of single nucleotide polymorphisms of TGF-β1 and differential Mf load and/or lack of Mf in the blood from infected persons in Ghana. An association was found between the TGF-β1 Leu10Pro variant and lack of Mf in the blood. Patients with latent infection had a higher frequency of the Leu/Leu genotype than patients with patent infection (p = 0.03). Secondary analysis revealed an association among the three possible Leu10Pro genotypes and different Mf loads in the blood. In conclusion, the differential Mf loads and the lack of Mf in the blood of patients is likely to have a genetic basis. Because the adult worms are responsible for pathology, these results underscore the need for a review of using only Mf detection in blood smears for diagnosis of LF infection in endemic areas. This information is also important for the mapping and surveillance activities of national and global programs for elimination of LF.     

Standard karyotyping concentrates microfilaria and can be a valid concentrating technique for their detection

During karyotype preparation from the bone marrow aspirates of 209 haematological malignancy cases, microfilaria were detected in four samples, whereas routine marrow and peripheral blood smears of these four cases did not show any parasite. The patients were recalled, and their peripheral blood was processed by karyotyping and standard concentration techniques. Karyotype preparation from peripheral blood was performed with and without addition of colchicine. When the blood was processed for karyotyping with colchicine, microfilaria were detected in the peripheral blood of all four patients. In samples without added colchicine, no parasite was observed. The same samples were processed by Knott’s concentration technique, which showed microfilariae only in one of the four patients. Routine thick and thin smears of these patients showed no parasite. It seems that the standard karyotype preparation technique with colchicine concentrates the microfilariae in samples where parasite load is small and not demonstrable with standard techniques. Serological tests are available for W. bancrofti and costly, whereas no regular serodiagnosis is available for B. malayi. In a country like India, both parasites are endemic and patients are treated on clinical suspicion when parasitaemia could be low. Low parasitaemia is common because of repeated infection and partial immunity. In such circumstances, a cost‐effective concentration technique like this may be useful.