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We recently reported a new rapid screening method for glucose-6-phosphate dehydrogenase (G6PD) deficiency. This method incorporates a new formazan substrate (WST-8) and is capable of detecting heterozygous females both qualitatively and quantitatively. Here, we report its evaluation during field surveys at three malaria centres and in malaria-endemic villages of Myanmar and Indonesia, either alone or in combination with a rapid on-site diagnosis of malaria. A total of 57 severe (45 males and 12 females) and 34 mild (five males and 29 females) cases of G6PD deficiency were detected among 855 subjects in Myanmar whilst 30 severe (25 males and five females) and 23 mild (six males and 17 females) cases were found among 1286 subjects in Indonesia. In all cases, severe deficiency was confirmed with another formazan method but due to limitations in its detection threshold, mild cases were misdiagnosed as G6PD-normal by this latter method. Our results indicate that the novel method can qualitatively detect both severely deficient subjects as well as heterozygous females in the field. The antimalarial drug, primaquine, was safely prescribed to Plasmodium vivax-infected patients in Myanmar. Our new, rapid screening method may be essential for the diagnosis of G6PD deficiency particularly in rural areas without electricity, and can be recommended for use in malaria control programmes.

We established a new, simple and rapid screening method for detection of glucose-6-phosphate dehydrogenase (G6PD)-deficiency by using a new formazan substrate, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium monosodium salt (WST-8) with a hydrogen carrier of 1-methoxyphenazine methosulfate (1-methoxy PMS), instead of a combination of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) and phenazine methosulfate (PMS), as used in many previous formazan methods. WST-8 does not react with haemoglobin, and the formed formazan is highly water-soluble, differing from MTT. Thus, the whole procedure can be performed in aqueous solution in a tube or well without any special equipment other than micropipettes. Within 1 h at room temperature, the strong orange colour of the WST-8 formazan formed in normal blood samples could be distinguished, by naked eye, from G6PD-deficient blood samples with less than 50% residual activity. We also found that reagents in the WST-8/1-methoxy PMS method were more resistant against exposure to sunlight than those in an MTT/PMS method. As the new method is both qualitative and quantitative, it is possible to express G6PD activity as increase of NADPH concentration by reading absorbance at 460 nm after incubation for 30 or 60 min.

The presence of Plasmodium ovale has never been previously reported in Myanmar. Using blood samples obtained in many villages across the country between 1996 and 2000, molecular diagnosis of Plasmodium species was made with semi- or full-nested polymerase chain reaction (PCR) with species-specific primers, followed by agarose gel electrophoresis to detect amplification products. The presence of P. ovale was also confirmed with the another PCR-based diagnosis, the microtiterplate hybridization (MPH) method using species-specific probes. Both methods target the A type of the small subunit ribosomal RNA gene of the four human malaria parasites. Plasmodium ovale DNA was amplified in samples from 65 (4.9%) of 1323 PCR-positive patients, with perfect agreement between results obtained by nested PCR and MPH. Only four P. ovale-infected patients had single-species infection; all others were coinfected with P. falciparum, P. vivax and/or P. malariae. Quadruple infections were observed in six subjects. Parasites with typical P. ovale morphology were found in only 19 patients by conventional microscopy of Giemsa-stained thin smears or fluorescence microscopy of acridine orange-stained thin smears. Plasmodium ovale infections were found in villages situated in the southern, central and western regions of Myanmar, suggesting that P. ovale may be widely distributed in this country.

We performed a field evaluation of polymerase chain reaction (PCR)-based enzyme-linked immuno-sorbent assays (ELISA) for the diagnosis of malaria. A commercially available PCR-ELISA microplate hybridization (MPH) assay was used. Blood specimens were collected from 300 volunteers seeking care at malaria clinics in Thailand. Examination of 200 high power fields by Giemsa-stained thick and thin smear (GTTS) revealed 51 P. falciparum (Pf), 45 P. vivax (Pv), seven mixed Pf-Pv infections. These plus a random sample of 48 GTTS-negative specimens were selected for this study. All 151 specimens were processed for parasite DNA extraction and assayed by PCR-MPH. The target DNA sequence of the 18S small subunit ribosomal RNA (SSUrRNA) gene was amplified by PCR and hybridized with species-specific probes for Pf, Pv, P. malariae (Pm) and P. ovale (Po) immobilized in the wells of the microtiter plate and detected by colorimetric assay. Colour development was assessed at an optical density (OD) of 405 nm. An absorbance reading of > or = 0.1 was used as a positive cut-off. In comparison with GTTS results, PCR-MPH sensitivity was 91.4% (53/58, 95% CI 84.2-98.6) for Pf, 94.2% (49/52, 87.9-100) for Pv and specificity was 95.8% (46/48, 95% CI 90.2-100). There was statistically significant positive correlation between parasite densities < or = 7000/microl blood and absorbance reading, suggestive of PCR-MPH being semiquantitative. PCR-MPH also detected additional Pf and Pv cases as well as Pm and Po.

A rapid single-step screening method for detection of glucose-6-phosphate dehydrogenase (G6 PD) deficiency was evaluated on Halmahera Island, Maluku Province, Indonesia, and in Shan and Mon States, Myanmar, in combination with a rapid diagnosis of malaria by an acridine orange staining method. Severe deficiency was detected by the rapid test in 45 of 1126 volunteers in Indonesia and 54 of 1079 in Myanmar, but it was difficult to distinguish blood samples with mild deficiency from those with normal activity. 89 of 99 severely deficient cases were later confirmed by formazan ring method in the laboratory, but 5 with mild and 5 with no deficiency were misdiagnosed as severe. Of the samples diagnosed as mild and no deficiency on-site, none was found to be severely deficient by the formazan method. Malaria patients were simultaenously++ detected on-site in 273 samples on Halmahera island and 277 samples from Shan and Mon States. In Mon State, primaquine was prescribed safely to G6 PD-normal malaria patients infected with Plasmodium vivax and/or gametocytes of P. falciparum. The new rapid test for G6 PD deficiency may be useful for detecting severe cases under field conditions, and both rapid tests combined are can be useful in malaria-endemic areas, facilitating early diagnosis, prompt and radical treatment of malaria and suppression of malaria transmission.

The prevalence of the four human malaria parasites was investigated among malaria patients at northern, central and southern towns in Thailand along the border with Myanmar between September 1995 and May 1996. Thin smears obtained from 548 Thai and Burmese patients were reviewed by an acridine orange staining method, and many mixed infections with two to four species, including P. malariae and P. ovale, were detected. These diagnostic results were compared with those by two PCR-based diagnoses, microtitre plate hybridization (MPH) and a nested PCR method, both of which targets the same, species-specific regions in the 18S rRNA genes. In both PCR diagnoses, many P. malariae and P. ovale infections were also detected. Detection sensitivity of P. malariae infection was higher in nested PCR than MPH, and a total prevalence of P. malariae infection estimated by nested PCR reached 24.3% (133/548). In 16 of them, the size of PCR products amplified by the P. malariae-specific primer was about 20-bp shorter than the expected size of 115-bp. Four of 16 possessed two different bands with normal and shorter sizes, suggesting that P. malariae isolates may be separated into two types, and that those with shorter products may be new variant form (s) with a nucleotide deletion in the target region. On the other hand, 21 P. ovale infections (3.8%) were detected by nested PCR, but four of them were MPH-negative because of the sequence variation at the probe region. These results indicated that the prevalence of P. malariae and P. ovale along the Thai-Myanmar border may be substantially higher than previously reported.