Abstract
BackgroundHonduras is a tropical country with more than 70% of its population living at risk of being infected with either Plasmodium vivax or Plasmodium falciparum. Laboratory diagnosis is a very important factor for adequate treatment and management of malaria. In Honduras, malaria is diagnosed by both, microscopy and rapid diagnostic tests and to date, no molecular methods have been implemented for routine diagnosis. However, since mixed infections, and asymptomatic and low-parasitaemic cases are difficult to detect by light microscopy alone, identifying appropriate molecular tools for diagnostic applications in Honduras deserves further study. The present study investigated the utility of different molecular tests for the diagnosis of malaria in Honduras.MethodsA total of 138 blood samples collected as part of a clinical trial to assess the efficacy of chloroquine were used: 69 microscopically confirmed P. falciparum positive samples obtained on the day of enrolment and 69 follow-up samples obtained 28 days after chloroquine treatment and shown to be malaria negative by microscopy. Sensitivity and specificity of microscopy was compared to an 18 s ribosomal RNA gene-based nested PCR, two single-PCR reactions designed to detect Plasmodium falciparum infections, one single-PCR to detect Plasmodium vivax infections, and one multiplex one-step PCR reaction to detect both parasite species.ResultsOf the 69 microscopically positive P. falciparum samples, 68 were confirmed to be P. falciparum-positive by two of the molecular tests used. The one sample not detected as P. falciparum by any of the molecular tests was shown to be P. vivax-positive by a reference molecular test indicating a misdiagnosis by microscopy. The reference molecular test detected five cases of P. vivax/P. falciparum mixed infections, which were not recognized by microscopy as mixed infections. Only two of these mixed infections were recognized by a multiplex test while a P. vivax-specific polymerase chain reaction (PCR) detected three of them. In addition, one of the day 28 samples, previously determined to be malaria negative by microscopy, was shown to be P. vivax-positive by three of the molecular tests specific for this parasite.ConclusionsMolecular tests are valuable tools for the confirmation of Plasmodium species and in detecting mixed infections in malaria endemic regions.
Highlights
Honduras is a tropical country with more than 70% of its population living at risk of being infected with either Plasmodium vivax or Plasmodium falciparum
Honduras is a tropical country with more than 70% of its population living at risk of malaria infections
In Honduras, P. vivax is responsible for the largest number of malaria cases while P. falciparum accounts for 10-15% of the cases reported [1] and causes a more severe clinical presentation
Summary
Sample collection A total of 138 blood samples (about 50 μl each) were collected and stored on Whatman filter paper number 3 for molecular testing. Reactions were performed in 25 μL total volume containing 1X buffer, 2.5 mM MgCl2, 200 μM dNTPs, 200 nM primers, and 1.25 units of Taq Polymerase (New England Biolabs, Ipswich, MA, USA), and 1-3 μl of DNA template. Un-nested multiplex and single-tube species-specific PCRs Various primers used for the different tests are provided in Table 1 and were based on a previous study [15]. Test 3 was a multiplex unnested PCR to detect both P. falciparum and P. vivax using specific primers (AL7178/AL7142 and AL7175/ AL7074, respectively). If a discordant result was obtained, the experiment was repeated a third time and the final result was determined by two concordant tests
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