Cross-sectional surveys estimating parasite prevalence offer a practical method for
malaria surveillance and are used to monitor changes over time and space 3], 10], 19], 20]. Parasite prevalence is used frequently as a proxy measure of transmission intensity;
however, this measure has limitations as an indicator of malaria burden. Estimates
of parasite prevalence may vary considerably depending on the diagnostic test used
and the age-group being sampled. In addition, these variations may be further modified
by the underlying transmission intensity and temporal factors independent of true
changes in malaria burden. This study, compared estimates of parasite prevalence determined
by microscopy and RDTs, to that determined by PCR (the gold standard) using samples
collected from two consecutive annual community surveys in three areas of varying
transmission intensity. Microscopy had limited but consistent sensitivity, which generally
decreased with increasing age at all three study sites. Specificity of microscopy
was very high, such that relative differences in estimates of parasite prevalence
across different age groups, study sites, and study years followed expected patterns
and were consistent with relative changes in estimates using PCR. The sensitivity
of RDTs was higher than microscopy and also decreased with increasing age. However,
in contrast to microscopy, specificity of RDTs varied considerably from 1Â year to
the next and had a complex relationship to age that varied across the sites. This
resulted in estimates of parasite prevalence that did not follow the same age pattern
as with microscopy and PCR and inaccurately reflected changes in prevalence, as assessed
by PCR, from year to year.
Several diagnostic tests are available for detection of malaria parasitaemia, with
microscopy being the mainstay of diagnosis. Microscopy is relatively inexpensive to
perform, and can be used to differentiate malaria species and quantify parasitaemia,
but has known limitations 21], 22]. In this study, microscopy was highly specific and parasite estimates were consistent
irrespective of the age of the population studied, year and transmission intensity.
These advantages make microscopy, when performed well, a reliable tool for monitoring
disease burden in surveys over time. However, the low sensitivity compared to PCR,
and resulting lower parasite prevalence estimates, need to be taken into account when
interpreting results 23]–25]. RDTs are increasingly being used independently or in combination to microscopy in
surveys 8]. RDTs are attractive as diagnostic tools due to their higher sensitivity compared
to microscopy, ease of use and rapid availability of results 14], 26]. However, specificity of RDTs and parasite prevalence estimates were highly variable
in this study. This variability may affect the interpretation of prevalence estimates.
According to the RDT results, parasite prevalence increased significantly from 2012
to 2013, suggesting an increase in the disease burden. However, these results were
not consistent with estimates from microscopy and PCR, and other study findings conducted
during the same time period 17]. Thus, relying on RDT results would have provided an inaccurate picture of the of
the malaria burden in the study sites.
Malaria surveillance has typically targeted children aged 2–10 years for estimates
of parasite prevalence 27]. The Roll Back Malaria Monitoring and Evaluation group recommends that national surveys
target children under 5Â years for parasitaemia and anaemia testing 8], while other groups have explored alternative target populations, such as school-aged
children who may be more accessible 28], 29]. These results show that the parasite prevalence estimates vary markedly with age,
increasing during childhood and then declining following adolescence. This observed
pattern has been well-described in malaria endemic countries 30], but the shape of the age-parasite prevalence curve is modified by the underlying
transmission intensity 16]. Thus, when selecting a given age group for estimating disease burden, it should
be acknowledged that survey results may over- or under-estimate parasite prevalence,
when compared to the wider population 29]. This also highlights the importance of consistently estimating parasite prevalence
in the same age group when monitoring malaria burden and the impact of control interventions
over time.
In this study, the performance of the diagnostic tests varied with changing transmission
intensity and age. The sensitivity of microscopy was highest in the highest transmission
setting, consistent with the observation that the relative proportion of sub-microscopic
infections, those below the level of detection by microscopy, is higher in lower transmission
settings 12], 31]. The parasite densities of asymptomatic infections will vary with the level of acquired
immunity, which is dependent on the transmission setting and age 32], 33]. For example, in higher transmission settings, recurrent malaria infections lead
to earlier, and greater, age-specific acquired immunity such that individuals are
more likely to tolerate high-density malaria infections without developing symptoms
34]–36]. The lower sensitivity of microscopy in younger children could also be due to a higher
proportion of these children having been treated recently for malaria, resulting in
very low-density parasites remaining from a prior treatment.
Population surveys commonly use traditional diagnostic techniques including microscopy
or RDTs which may miss low-grade infections that are below the level of detection
of these tools (sub-patent infections). Studies in high transmission areas have shown
that as many as two-thirds of microscopy-negative patients may have sub-patent malaria
infections 37]–40]. Molecular techniques, such as PCR, are more sensitive, and thus are more likely
to detect sub-patent infections 41]. However, PCR must be performed by highly trained technicians in sophisticated laboratories,
which makes this method more expensive and less feasible for large-scale surveys.
Recently, loop-mediated isothermal amplification (LAMP) has been optimized for the
rapid amplification and detection of parasite DNA 42], 43]. LAMP testing is highly sensitive and can be performed in minimally equipped laboratories
by technicians after a brief training period 41], 44], which makes it an attractive alternative to PCR for endemic areas, and a potential
option for population-based surveys.
This study was not without limitations. First, a significant variation in the specificity
of RDTs was observed between 2012 and 2013 despite using the same brand of RDTs and
the same survey staff in both surveys, and in the absence of any major control interventions
within the 2Â years. The cause of the variation between the 2Â years could not be established;
however, it is speculated that the performance of the RDTs could have been affected
by transportation or storage conditions, or possibly changes in seasonality. Second,
PCR was not performed on samples that were positive by both microscopy and RDT; however,
it was assumed that PCR would be positive if both microscopy and RDT results were
positive and that this cost-saving measure did not affect the study findings.