PCR reveals significantly higher rates of Trypanosoma cruzi infection than microscopy in the Chagas vector, Triatoma infestans: high rates found in Chuquisaca, Bolivia.
PCR reveals significantly higher rates of Trypanosoma cruzi infection than microscopy in the Chagas vector, Triatoma infestans: high rates found in Chuquisaca, Bolivia.
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DOI：
10.1186/1471-2334-7-66
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发表时间：
2007-06-27
影响因子：
3.7
通讯作者：
Stevens, Lori
中科院分区：
文献类型：
--
作者：
Carlos Pizarro, Juan;Lucero, David E.;Stevens, Lori
玻利维亚的安第斯山谷是该国恰加斯病的主要病媒——森林型斑鼻虫的唯一报告地点，据推测，该地区克氏锥虫感染的高人类流行率是由于病媒在国内、周边和森林环境中持续存在的能力所致。确定锥虫病在其锥虫病媒中的感染率是旨在减少人类感染的一个重要因素。传统上，克氏锥虫是通过挤压粪便的直接显微镜检查或整个昆虫的解剖和分析在昆虫媒介中检测到的。尽管该技术已被证明是有用的，但其敏感性存在一些缺陷，特别是在小虫龄的情况下，以及对大量昆虫和死亡标本的适用性，促使研究人员寻找一种基于聚合酶链反应（PCR）的分子测定方法，作为克氏锥虫感染载体寄生虫检测的替代方法。在这里提出的工作中，我们比较了PCR检测和直接显微镜观察在玻利维亚丘基萨卡5个地点和4个栖息地的实地收集的感染T.克氏体感染的诊断。比较了不同稚虫期、不同地点和不同生境的防治效果。我们检测了从玻利维亚丘基萨卡省农村地区采集的152只若虫和成虫。显微镜观察时，用生理盐水稀释经腹部挤压获得的几滴直肠内容物，并将其压缩在载玻片和盖玻片之间。用400倍放大镜记录了50个显微场中有活动寄生虫的存在。分子分析采用TCZ1（5′- CGA GCT CTT GCC CAC ACG GGT GCT - 3′）和TCZ2（5′- CCT CCA AGC AGC GGA TAG TTC AGG - 3′）引物解剖每只昆虫腹部后部，提取DNA并进行PCR扩增。扩增子在2%琼脂糖凝胶上以100 bp的标准尺寸进行层析，溴化乙啶染色，紫外荧光观察。对于显微镜和PCR检测，我们计算了灵敏度（一种方法的阳性数除以两种方法的阳性数）和差异（一种方法为阴性，另一种方法为阳性）在地点，生命阶段和栖息地水平。通过计算Kappa (k)值（95%置信区间）来确定PCR与显微镜之间的一致程度。我们观察到在感染T.大肠杆菌中有较高的克氏体感染率（PCR为81.16%，显微镜为56.52%），并且发现PCR的敏感性明显高于显微镜观察。两种方法的总体一致性为中等（Kappa = 0.43±0.07）。社区间感染水平存在显著差异；然而，不同生境和生命阶段的患病率相似。在玻利维亚丘基萨卡的所有生境、发育阶段和地点，PCR的敏感性明显高于显微镜。总体而言，我们观察到玻利维亚该地区感染克氏t型虫的流行率很高；然而，显微镜检查低估了所有水平的感染。
The Andean valleys of Bolivia are the only reported location of sylvatic Triatoma infestans, the main vector of Chagas disease in this country, and the high human prevalence of Trypanosoma cruzi infection in this region is hypothesized to result from the ability of vectors to persist in domestic, peri-domestic, and sylvatic environments. Determination of the rate of Trypanosoma infection in its triatomine vectors is an important element in programs directed at reducing human infections. Traditionally, T. cruzi has been detected in insect vectors by direct microscopic examination of extruded feces, or dissection and analysis of the entire bug. Although this technique has proven to be useful, several drawbacks related to its sensitivity especially in the case of small instars and applicability to large numbers of insects and dead specimens have motivated researchers to look for a molecular assay based on the polymerase chain reaction (PCR) as an alternative for parasitic detection of T. cruzi infection in vectors. In the work presented here, we have compared a PCR assay and direct microscopic observation for diagnosis of T. cruzi infection in T. infestans collected in the field from five localities and four habitats in Chuquisaca, Bolivia. The efficacy of the methods was compared across nymphal stages, localities and habitats. We examined 152 nymph and adult T. infestans collected from rural areas in the department of Chuquisaca, Bolivia. For microscopic observation, a few drops of rectal content obtained by abdominal extrusion were diluted with saline solution and compressed between a slide and a cover slip. The presence of motile parasites in 50 microscopic fields was registered using 400× magnification. For the molecular analysis, dissection of the posterior part of the abdomen of each insect followed by DNA extraction and PCR amplification was performed using the TCZ1 (5' – CGA GCT CTT GCC CAC ACG GGT GCT – 3') and TCZ2 (5' – CCT CCA AGC AGC GGA TAG TTC AGG – 3') primers. Amplicons were chromatographed on a 2% agarose gel with a 100 bp size standard, stained with ethidium bromide and viewed with UV fluorescence. For both the microscopy and PCR assays, we calculated sensitivity (number of positives by a method divided by the number of positives by either method) and discrepancy (one method was negative and the other was positive) at the locality, life stage and habitat level. The degree of agreement between PCR and microscopy was determined by calculating Kappa (k) values with 95% confidence intervals. We observed a high prevalence of T. cruzi infection in T. infestans (81.16% by PCR and 56.52% by microscopy) and discovered that PCR is significantly more sensitive than microscopic observation. The overall degree of agreement between the two methods was moderate (Kappa = 0.43 ± 0.07). The level of infection is significantly different among communities; however, prevalence was similar among habitats and life stages. PCR was significantly more sensitive than microscopy in all habitats, developmental stages and localities in Chuquisaca, Bolivia. Overall we observed a high prevalence of T. cruzi infection in T. infestans in this area of Bolivia; however, microscopy underestimated infection at all levels examined.
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影响因子：
2.1
作者：
Cortez, Mirko Rojas;Pinho, Ana Paula;Jansen, Ana Maria
通讯作者：
Jansen, Ana Maria
影响因子：
2.8
作者：
Dorn, PL;Flores, J;Monroy, C
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Monroy, C
影响因子：
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作者：
MOSER, DR;KIRCHHOFF, LV;DONELSON, JE
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DONELSON, JE
影响因子：
1.9
作者：
LANDIS, JR;KOCH, GG
通讯作者：
KOCH, GG
DOI：
10.4269/ajtmh.1999.60.740
发表时间：
1999-05-01
影响因子：
3.3
作者：
Dorn, PL;Engelke, D;Monroy, C
通讯作者：
Monroy, C