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

Malar J. 2010 Apr 22:9:108. doi: 10.1186/1475-2875-9-108.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cambodia
  • Chromatography, High Pressure Liquid
  • Cross-Sectional Studies
  • Cytochromes b / genetics
  • Female
  • Humans
  • Malaria / diagnosis*
  • Malaria / genetics
  • Malaria / parasitology
  • Male
  • Microscopy
  • Molecular Sequence Data
  • Parasitemia / diagnosis*
  • Parasitemia / genetics
  • Plasmodium / classification*
  • Plasmodium / genetics
  • Plasmodium / isolation & purification*
  • Polymerase Chain Reaction / methods*
  • Polymorphism, Restriction Fragment Length
  • RNA, Ribosomal, 18S / genetics
  • Sensitivity and Specificity
  • Sequence Analysis, DNA
  • Species Specificity

Substances

  • RNA, Ribosomal, 18S
  • Cytochromes b