Resistance to antimalarial drugs is a public health problem worldwide. Molecular markers for drugresistant malaria, such as pfcrt and pfmdr1 polymorphisms, could serve as useful surveillance tools. To evaluate this possibility, sequence polymorphisms in pfcrt (position 76) and pfmdr1 (positions 86, 184, 1034, 1042, and 1246) and in vitro drug sensitivities were measured for 65 Plasmodium falciparum isolates from Thailand, Myanmar, Vietnam, and Bangladesh. The pfcrt Thr76 polymorphism was present in 97% of samples, consistent with observations that chloroquine resistance is well established in this region. Polymorphisms in pfmdr1 clustered into four specific patterns: the wild type (category I), a Tyr86 polymorphism only (category II), a Phe184 polymorphism only (category III), and Phe184 in combination with Cys1034 and/or Asp1042 (category IV). Isolates in categories I and III were more sensitive to chloroquine and more resistant to mefloquine, artesunate, and artemisinin than isolates in categories II and IV (P < 0.01). Mefloquine resistance was significantly more common in category I and III isolates than in category II and IV isolates, with a prevalence ratio of 14.95 (95% confidence interval, 3.88 to 57.56). These categories identified mefloquine resistance with a sensitivity and a specificity of 94 and 91%, respectively. The pfmdr1 gene copy number was measured by real-time PCR as a ratio of the amount of pfmdr1 DNA to the amount of lactate dehydrogenase (ldh) DNA. Eight samples had pfmdr1 DNA/ldh DNA ratios >3. The isolates in all 8 samples fell into categories I and III and were significantly more resistant to mefloquine, quinine, artemisinin, and artesunate and more sensitive to chloroquine than the isolates in the 57 samples with <3 copies of the gene (P < 0.001). Thus, measurement of pfmdr1 mutations and gene copy number may be useful for surveillance of mefloquine-resistant malaria in Southeast Asia.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a heterogeneous enzyme abnormality with high frequency in tropical areas. We performed population screening and molecular studies of G6PD variants to clarify their distribution and features in Southeast Asia. A total of 4317 participants (2019 males, 2298 females) from 16 ethnic groups in Myanmar, Lao in Laos, and Amboinese in Indonesia were screened with a single-step screening method. The prevalence of G6PD-deficient males ranged from 0% (the Akha) to 10.8% (the Shan). These G6PD-deficient individuals and 12 G6PD-deficient patients who had been diagnosed at hospitals in Indonesia and Malaysia were subjected to molecular analysis by a combination of polymerase-chain-reaction-based single-strand conformation polymorphism analysis and direct sequencing. Ten different missense mutations were identified in 63 G6PD-deficient individuals (50 hemizygotes, 11 heterozygotes, and 2 homozygotes) from 14 ethnic groups. One missense mutation (1291 G-->A) found in an Indonesian Chinese, viz., G6PD Surabaya, was previously unknown. The 487 G-->A (G6PD Mahidol) mutation was widely seen in Myanmar, 383 T-->C (G6PD Vanua Lava) was specifically found among Amboinese, 871 G-->A (G6PD Viangchan) was observed mainly in Lao, and 592 C-->T (G6PD Coimbra) was found in Malaysian aborigines (Orang Asli). The other five mutations, 95 A-->G (G6PD Gaohe), 1003 G-->A (G6PD Chatham), 1360 C-->T (G6PD Union), 1376 G-->T (G6PD Canton), and 1388 G-->A (G6PD Kaiping) were identified mostly in accordance with distributions reported previously.

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SummaryThe 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 speciesspecific 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.

SummaryThe 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 ThaiMyanmar border may be substantially higher than previously reported.keywords Plasmodium malariae, Plasmodium ovale, Thailand, Myanmar, acridine orange staining, PCR diagnosis correspondence Dr F. Kawamoto,