Abstract
BackgroundNucleic acid amplification provides the most sensitive and accurate method to detect and identify pathogens. This is primarily useful for epidemiological investigations of malaria because the infections, often with two or more Plasmodium species present simultaneously, are frequently associated with microscopically sub-patent parasite levels and cryptic mixed infections. Numerous distinct equally adequate amplification-based protocols have been described, but it is unclear which to select for epidemiological surveys. Few comparative studies are available, and none that addresses the issue of inter-laboratory variability.MethodsBlood samples were collected from patients attending malaria clinics on the Thai-Myanmar border. Frozen aliquots from 413 samples were tested independently in two laboratories by nested PCR assay. Dried blood spots on filter papers from the same patients were also tested by the nested PCR assay in one laboratory and by a multiplex PCR assay in another. The aim was to determine which protocol best detected parasites below the sensitivity level of microscopic examination.ResultsAs expected PCR-based assays detected a substantial number of infected samples, or mixed infections, missed by microscopy (27 and 42 for the most sensitive assay, respectively). The protocol that was most effective at detecting these, in particular mixed infections, was a nested PCR assay with individual secondary reactions for each of the species initiated with a template directly purified from the blood sample. However, a lesser sensitivity in detection was observed when the same protocol was conducted in another laboratory, and this significantly altered the data obtained on the parasite species distribution.ConclusionsThe sensitivity of a given PCR assay varies between laboratories. Although, the variations are relatively minor, they primarily diminish the ability to detect low-level and mixed infections and are sufficient to obviate the main rationale to use PCR assays rather than microscopy or rapid diagnostic tests. The optimal approach to standardise methodologies is to provide PCR template standards. These will help researchers in different settings to ensure that the nucleic acid amplification protocols they wish to use provide the requisite level of sensitivity, and will permit comparison between sites.
Highlights
Nucleic acid amplification provides the most sensitive and accurate method to detect and identify pathogens
Sample collection Samples were collected from febrile patients presenting at clinics of the Shoklo Malaria Research Unit (SMRU) and screened as part of treatment trials approved by the Oxford University Tropical Research Ethics committee (OXTREC) and the Faculty of Tropical Medicine Ethics Committee
The remaining case, where the assay conducted at the Shoklo Malaria Research Unit in Thailand (SMRU) failed to detect a substantial P. falciparum parasitaemia (52,501 P/μl of blood), was considered to represent the only true discordance. These results strongly suggested that the sensitivity of the nested polymerase chain reaction (PCR) assay conducted at the SMRU did not equal that of the same assay conducted at the Muséum National d’Histoire Naturelle in Paris (MNHN)
Summary
Nucleic acid amplification provides the most sensitive and accurate method to detect and identify pathogens. This is primarily useful for epidemiological investigations of malaria because the infections, often with two or more Plasmodium species present simultaneously, are frequently associated with microscopically sub-patent parasite levels and cryptic mixed infections. The number of endemic residents with sub-microscopic infections often exceeds that of persons with microscopically patent parasitaemia. This is because the untreated malaria infection is predominantly a low-grade often sub-patent chronic infection that usually persists for many months [2]. Neither microscopy nor rapid diagnostic tests are suited for the detection and identification of parasites in the mosquito
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