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Publication Date

2008

Document Type

Honors Project

Department

Biochemistry

Abstract

Lymphatic filariasis (LF) is a mosquito transmitted parasitic infection which is a major cause of clinical suffering, disability and handicap in the world. More than 1.3 billion people in 82 countries, approximately 18% of the world's population, live in areas which put them at risk for the infection. The Global Programme to Eliminate Lymphatic Filariasis (GPELF) was launched in the year 2000 with the aim to eliminate LF by the year 2020. The two main goals of the program are to interrupt the transmission of infection and to alleviate and prevent the suffering and disability produced by the disease in a cost-effective and socially responsible manner. About 10% of infections leading to LF are caused by the parasitic nematode Brugia malayi. Currently, PCR-based assays are the primary method of detecting Brugian infection in endemic regions where treatment programs are monitored and evaluated by the Global Programme and funded by the Bill and Melinda Gates Foundation. In a study conducted to assess the sensitivity of a nested PCR assay for B. Malayi using the 320 bp HhaI repeat sequence, Cox-Singh et all (2000), showed that microfilaria-depleted blood spots from microfilaria-positive samples were found to test positive due to the presence of "free DNA" in the samples. During this study, the hypothesis was that this "free DNA", i.e. DNA that is no longer inside the cells of microfilariae, would undergo rapid degradation in the human bloodstream and would not continue to be a source of positives if the same individual's blood was tested at subsequent time intervals. However, the results of this investigation show that when allowed to incubate in blood at 37°C, the HhaI sequence, a highly repeated, tandemly arranged, species-specific sequence can be amplified by conventional PCR up to at least 30 days of incubation. The results also show that the free DNA is not completely degraded by DNase I, and that the samples are PCR amplifiable using the same HhaI repeat after at least 5 days of incubation in blood and DNase I at 37°C. These results have led to the conclusion that the non-coding, tandemly repeated HhaI DNA is packaged as constitutive heterochromatin making it less accessible to nucleases. We believe that DNA packaged as constitutive heterochromatin with histone nucleosomal proteins and other non-histone proteins protect it from degradation by DNase I and allow it to persist for extended periods of time. This investigation has also shown that blood and serum both have endogenous nuclease activities and rapidly degrade highly purified DNA from bacteriophage lambda. In addition this investigation has revealed that neither blood nor serum can inhibit the action of DNaseI. These results directly relate to resolving the question of whether or not the PCR assay to detect parisite infection in mosquitoes currently used by the Global Programme to Eliminate Lymphatic Filariasis, GPELF, is detecting "free DNA" taken up in the mosquitoe blood meal rather than detecting actual parasite infection. This investigation will help to understand whether or not the mosquito infection rate determined by PCR is inflated by the detection of "free parasite DNA" in the blood.

Language

English

Comments

vi, 54 leaves : col ill. Thesis (Honors)--Smith College, Northampton, Mass., 2008. Includes bibliographical references (leaves 49-50)

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