Sub-microscopic malaria cases and mixed malaria infection in a remote area of high malaria endemicity in Rattanakiri province, Cambodia: implication for malaria elimination.
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.
Steenkeste
Nicolas
N
Unité d'Epidémiologie Moléculaire, Institut Pasteur du Cambodge, Phnom Penh, Cambodia. nicolas.steenkeste@gmail.com
Rogers
William O
WO
Okell
Lucy
L
Jeanne
Isabelle
I
Incardona
Sandra
S
Duval
Linda
L
Chy
Sophy
S
Hewitt
Sean
S
Chou
Monidarin
M
Socheat
Duong
D
Babin
François-Xavier
FX
Ariey
Frédéric
F
Rogier
Christophe
C
D002160:Cambodia; D002851:Chromatography, High Pressure Liquid; D003430:Cross-Sectional Studies; D045303:Cytochromes b / Q000235:genetics; D005260:Female; D006801:Humans; D008288:Malaria / Q000175:diagnosis* / Q000235:genetics / Q000469:parasitology; D008297:Male; D008853:Microscopy; D008969:Molecular Sequence Data; D018512:Parasitemia / Q000175:diagnosis* / Q000235:genetics; D010961:Plasmodium / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013045:Species Specificity
D012337:RNA, Ribosomal, 18S; D045303:Cytochromes b
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