Epidemiological Analysis of Host Populations with Widespread Sub-Patent Infections: African Trypanosomiasis
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Date
06/2007Author
Cox, Andrew Paul
Metadata
Abstract
The epidemiological study of pathogens largely depends on three technologies,
serology, microscopy and the polymerase chain reaction (PCR). Serological
methods are unable to differentiate between current and past infections.
Microscopy has historically been the mainstay of epidemiological study. In
recent times the use of microscopy has been in decline, as it has been shown to
have an inherent lack of sensitivity and specificity and produces many false
negative results. PCR is now the method of choice for screening samples for the
presence or absence of pathogens. Although PCR is widely regarded as an
extremely sensitive technique, the fact that it assays a very small volume of
sample is often overlooked. If the target pathogen is not present in the tiny
aliquot of sample from an infected host, then a false negative results will occur.
In endemic situations were the pathogen is present at low infection intensities,
then the potential for false negatives results of this type is high. This intensity
related false negative effect can lead to serious underestimation of diagnosed
prevalence and incidence with consequent misinterpretation of the resulting
data. This phenomenon has been reported in the literature for a range of
pathogens and especially for epidemiological study of schistosomiasis. The
extensive occurrence of false negatives during study of schistosomiasis
samples was such an obstacle to epidemiological study it prompted the world
health organisation to repeatedly call for quantitative methods to be employed to
combat the problem.
The main objectives of this thesis are to rationalise and simplify the methods of
diagnosing African trypanosomes in epidemiological studies and to investigate
the consequences of, and methods of dealing with infection intensity related
false negative results that occur as a result of widespread sub-patent infections
in the study population
A new PCR assay was developed that was capable of analysing whole blood
placed onto treated filter paper. The PCR assay was capable of differentiating
between all the important African trypanosome species, producing a unique size
of amplicon for each species of trypanosome. Initial results from repeated
screening of human and cattle samples known to be parasitologically positive
indicated that many false negative results occur. A more extensive analysis of
thirty five bovine blood samples randomly chosen from a collection of field
samples revealed that false negative results occurred regularly. The prevalence of infection after a single screening was 14.3% whereas the cumulative
prevalence after over 100 repeated screenings rose to 85.7%. This showed that
a severe underestimation of prevalence occurs from a single screening of the
samples.
In order to investigate the consequences of, and develop methods of dealing
with this problem, computer based simulations were used to model the
dynamics of screening samples with sub-patent infections. In order to construct
the model the data obtained from repeat screening of the thirty-five bovine blood
samples was fitted to a number of mathematical distributions. A negative
binomial distribution best described the distribution of trypanosomes across the
hosts. Exploration of the phenomenon with the resulting model showed the
extensive underestimation of true prevalence that is possible. The simulations
also showed that it is possible for populations with very different patterns of
infection and true prevalence to all have the same diagnosed prevalence from a
single screening per sample. Statistical comparison of these very different
populations by diagnosed prevalence alone would conclude there was no
significant difference between the populations. It was therefore concluded that
the diagnosed prevalence from a single (or even multiple) screenings is an
inadequate and potentially misleading measure of both infected hosts and
parasite numbers.
In order to deal with these problems new methods were evaluated for use in
epidemiological studies. A simple method of producing quantitative measures of
infection was advocated. The insensitivity of existing screening methods in
detecting significant difference between populations was highlighted and a
greatly improved methodology was shown. Finally, a method for inferring the
true population prevalence from the data obtained from repeat screening of
samples was suggested. Although some of these new methodologies have
limitations, they represent a great improvement on the use of a single diagnostic
test for each host. The work presented in this thesis highlights a serious
potential limitation to our understanding of the epidemiology of pathogens that
exist at sub-patent levels, and develops some possible methods of overcoming
these limitations.