This paper has evaluated HRP-2 test performance in people of distinct age groups living in an area of intense P. falciparum transmission. The age pattern of parasitaemia and the high prevalence of P. falciparum infection in the childhood population indicate the intense malaria transmission in our study area.
HRP-2 test sensitivity was consistently high in the younger age groups, in keeping with the findings of previous studies in similar settings [7, 9, 11, 12]. There was no evidence that the lower sensitivity in people aged 25 years or more was due to lower parasite density in older people. Numerous studies have shown that HRP-2 tests might fail to detect low-level parasitaemia [6, 8, 9, 13–15]. In the current study, two cases of P. falciparum infection with high parasite density were undetected by the test. While similar reports exist [20–22], the cause has not been elucidated. It has been suggested that the HRP-2 test sensitivity could be affected by the variability of the target antigen in specific settings  but this hypothesis is unlikely to explain the findings in the current study as antigenic polymorphism is geographically based. An alternative hypothesis is that age dependent immune status might lower HRP-2 test sensitivity, independently of parasite density .
With respect to the HRP-2 test specificity, over one third of the individuals with a positive HRP-2 test result had a negative blood slide. The overall HRP-2 test specificity in our study is substantially lower than that found by all but one previous study sharing a similar methodology in similar malaria transmission settings [7–12, 14, 17, 24]. In addition, the current study found that the test specificity varied with age, and was inversely related to the prevalence of positive blood slides in different age groups, i.e. the specificity decreased as malaria prevalence increased. These findings corroborate the results of a recent study where the investigators found an overall specificity of 65%, and a decrease in specificity with decreasing age and increasing malaria prevalence in symptomatic patients . Another study reported similarly low HRP-2 test specificity (52%) in symptomatic children under five years of age in an area of intense transmission .
These striking findings have to be interpreted with some caution as this difference may have arisen for various methodological reasons. Although the microscopic examination of blood slides was subject to quality control, its use as "gold standard" might have been problematic, as it does not have perfect sensitivity. In areas of intense malaria transmission, a high proportion of the population carries low level of parasites at any given time. It is well established that PCR has a sensitivity superior to microscopy when the parasite density is low . Previous studies suggest that a proportion of false positive HRP-2 results are due to sub-patent parasitaemia undetectable by microscopy [17, 27].
The vast majority of comparable published studies on HRP-2 performance using microscopy as a "gold standard" in areas of intense malaria transmission recruited symptomatic participants at health facilities. The samples in this study were from mainly asymptomatic participants. Therefore, it is possible that the proportion of individuals with sub-patent parasitaemia was higher than in other studies. This hypothesis is also supported by the relatively low mean parasite density in this study's sample.
It has been reported that the RDT can stay positive for more than five weeks after successful treatment, because of prolonged persistence of HRP-2 antigen in the circulation after parasite death . Information on recent past anti-malarial treatment was not elicited for the majority of the study population, and a high proportion of false positives may have been the result of recent treated infections, and these are likely to have been more frequent in younger age groups. This effect may be exaggerated if enhanced immunity acquired with age hastens the clearance of HRP-2 antigens. In this respect, rapid diagnostic tests based on detection of the parasite's lactate dehydrogenase (pLDH) are less likely to suffer the variable specificities in different age groups that we document because p-LDH is cleared from the circulation rapidly on effective treatment of infection.
HRP-2 tests are attractive because they are rapid and relatively easy to perform, and do not require equipment or electricity. However, our results suggest that their interpretation needs caution. As the risk of malaria infection depends mostly on the intensity of malaria transmission, the use of protective measures against infection and age-dependent immunity against P. falciparum, it seems reasonable to deduce that the different age groups in our study reflect distinct categories of likelihood of infection. As the HRP-2 assay's positive predictive value varies widely with the likelihood of current or recent infection at the time the test is performed, its use for the comparison of malaria prevalence between different age groups in a given malaria transmission setting, or for the comparison of malaria prevalence between populations living in different transmission settings, might be misleading. In populations with a high likelihood of infection, in whom test specificity is expected to be relatively low, the use of an HRP-2 test to evaluate the impact of a protective intervention will tend to under-estimate the effect of the intervention. Such comparisons may be more robust if based on blood slide or molecular data.
When HRP-2 tests are used for primary diagnosis in area of intense transmission, our results suggest a risk of malaria over-diagnosis in children, which might have serious consequences as the diagnosis of non-malaria febrile illnesses might be missed or delayed. This concern may not be so important if the variation in specificity with age is less marked in symptomatic individuals, who will tend to have a higher parasitaemia, but warrants further investigation. In practice, clinicians should bear in mind that their therapeutic and case-management decisions should not exclusively be based on the HRP-2 test result but should take into consideration the age of the patient, the clinical presentation (suggesting a non-malaria pathology) and a detailed history of recent drug treatment.