Malaria Journal (Apr 2010)

Sub-microscopic malaria cases and mixed malaria infection in a remote area of high malaria endemicity in Rattanakiri province, Cambodia: implication for malaria elimination

  • Socheat Duong,
  • Chou Monidarin,
  • Hewitt Sean,
  • Chy Sophy,
  • Incardona Sandra,
  • Duval Linda,
  • Jeanne Isabelle,
  • Okell Lucy,
  • Rogers William O,
  • Steenkeste Nicolas,
  • Babin François-Xavier,
  • Ariey Frédéric,
  • Rogier Christophe

DOI
https://doi.org/10.1186/1475-2875-9-108
Journal volume & issue
Vol. 9, no. 1
p. 108

Abstract

Read online

Abstract Background Malaria microscopy and rapid diagnostic tests are insensitive for very low-density parasitaemia. This insensitivity may lead to missed asymptomatic sub-microscopic parasitaemia, a potential reservoir for infection. Similarly, mixed infections and interactions between Plasmodium species may be missed. The objectives were first to develop a rapid and sensitive PCR-based diagnostic method to detect low parasitaemia and mixed infections, and then to investigate the epidemiological importance of sub-microscopic and mixed infections in Rattanakiri Province, Cambodia. Methods A new malaria diagnostic method, using restriction fragment length polymorphism analysis of the cytochrome b genes of the four human Plasmodium species and denaturing high performance liquid chromatography, has been developed. The results of this RFLP-dHPLC method have been compared to 1) traditional nested PCR amplification of the 18S rRNA gene, 2) sequencing of the amplified fragments of the cytochrome b gene and 3) microscopy. Blood spots on filter paper and Giemsa-stained blood thick smears collected in 2001 from 1,356 inhabitants of eight villages of Rattanakiri Province have been analysed by the RFLP-dHPLC method and microscopy to assess the prevalence of sub-microscopic and mixed infections. Results The sensitivity and specificity of the new RFLP-dHPLC was similar to that of the other molecular methods. The RFLP-dHPLC method was more sensitive and specific than microscopy, particularly for detecting low-level parasitaemia and mixed infections. In Rattanakiri Province, the prevalences of Plasmodium falciparum and Plasmodium vivax were approximately two-fold and three-fold higher, respectively, by RFLP-dHPLC (59% and 15%, respectively) than by microscopy (28% and 5%, respectively). In addition, Plasmodium ovale and Plasmodium malariae were never detected by microscopy, while they were detected by RFLP-dHPLC, in 11.2% and 1.3% of the blood samples, respectively. Moreover, the proportion of mixed infections detected by RFLP-dHPLC was higher (23%) than with microscopy (8%). Conclusions The rapid and sensitive molecular diagnosis method developed here could be considered for mass screening and ACT treatment of inhabitants of low-endemicity areas of Southeast Asia.