Amino Acid Sequence of Oligopeptide Causes Marked Difference in DNA Compaction and Transcription.

Compaction of T4 phage DNA (166 kbp) by short oligopeptide octamers composed of two types of amino acids, four cationic lysine (K), and four polar nonionic serine (S) having different sequence order was studied by single-molecule fluorescent microscopy. We found that efficient DNA compaction by oligopeptide octamers depends on the geometrical match between phosphate groups of DNA and cationic amines. The amino acid sequence order in octamers dramatically affects the mechanism of DNA compaction, which changes from a discrete all-or-nothing coil-globule transition induced by a less efficient (K4S4) octamer to a continuous compaction transition induced by a (KS)4 octamer with a stronger DNA-binding character. This difference in the DNA compaction mechanism dramatically changes the packaging density, and the morphology of T4 DNA condensates: DNA is folded into ordered toroidal or rod morphologies during all-or-nothing compaction, whereas disordered DNA condensates are formed as a result of the continuous DNA compaction. Furthermore, the difference in DNA compaction mechanism has a certain effect on the inhibition scenario of the DNA transcription activity, which is gradual for the continuous DNA compaction and abrupt for the all-or-nothing DNA collapse.

Further Molecular Analysis of G6PD Deficiency Variants in Southern Vietnam and a Novel Variant Designated as G6PD Ho Chi Minh (173 A>G; 58 Asp>Gly): Frequency Distributions of Variants Compared with Those in Other Southeast Asian Countries.

We conducted a survey of glucose-6-phosphate dehydrogenase (G6PD) deficiency among newborn babies at Tu Du Hospital, Ho Chi Minh, southern Vietnam. A total of 90 deficient babies were detected, including 85 in the Kinh ethnic group, 4 Chinese, and 1 in the K'Ho minority group. In the Kinh ethnic group, G6PD variants such as G6PD Viangchan (n=32), Kaiping (n=11), Canton (n=8), Chinese-5 (n=7), Union (n=5) and Quing Yuan (n=4) were detected. A variant with silent mutations at 1311 C>T and IVS11 nt 93 T>C was also detected in 17 cases. A novel mutation (173 A>G) in exon 4 with a predicted amino acid change of 58 Asp>Gly was also found in a Kinh newborn girl and her father, and it was designated as G6PD Ho Chi Minh. These findings demonstrated that the Kinh ethnic group in southern Vietnam has 8 different G6PD variants, indicating that the members of this group have many ancestors in terms of G6PD variants from Southeast Asia, China, and Oceania. We compared the frequency distribution of G6PD variants in the Kinh population with those of other Southeast Asian populations, and the Kinh population's distribution was quite similar to that in the Thai population, but differed from it by the absence of G6PD Mahidol.

Specific Conformational Change in Giant DNA Caused by Anticancer Tetrazolato-Bridged Dinuclear Platinum(II) Complexes: Middle-Length Alkyl Substituents Exhibit Minimum Effect.

Derivatives of the highly antitumor-active compound [{cis-Pt(NH3)2}2(µ-OH)(µ-tetrazolato-N2,N3)]2+ (5-H-Y), which is a tetrazolato-bridged dinuclear platinum(II) complex, were prepared by substituting a linear alkyl chain moiety at C5 of the tetrazolate ring. The general formula for the derivatives is [{cis-Pt(NH3)2}2(µ-OH)(µ-5-R-tetrazolato-N2,N3)]2+, where R is (CH2)nCH3 and n = 0 to 8 (complexes 1-9). The cytotoxicity of complexes 1-4 in NCI-H460 human non-small-cell lung cancer cells decreased with increasing alkyl chain length, and those of complexes 5-9 increased with increasing alkyl chain length. That is, the in vitro cytotoxicity of complexes 1-9 was found to have a U-shaped association with alkyl chain length. This U-shaped association is attributable to the degree of intracellular accumulation. Although circular dichroism spectroscopic measurement indicated that complexes 1-9 induced comparable conformational changes in the secondary structure of DNA, the tetrazolato-bridged complexes induced different degrees of DNA compaction as revealed by a single DNA measurement with fluorescence microsopy, which also had a U-shaped association with alkyl chain length that matched the association observed for cytotoxicity. Complexes 7-9, which had alkyl chains long enough to confer surfactant-like properties to the complex, induced DNA compaction 20 or 1000 times more efficiently than 5-H-Y or spermidine. A single DNA measurement with transmission electron microscopy revealed that complex 8 formed large spherical self-assembled structures that induced DNA compaction with extremely high efficiency. This result suggests that these structures may play a role in the DNA compaction that was induced by the complexes with the longer alkyl chains. The derivatization with a linear alkyl chain produced a series of complexes with unique cellular accumulation and DNA conformational change profiles and a potentially useful means of developing next-generation platinum-based anticancer drugs. In addition, the markedly high ability of these complexes to induce DNA compaction and their high intracellular accumulation emphasized the difference in mechanism of action from platinum-based anticancer drugs.

Genotypes of Candida albicans isolated from healthy individuals and their distribution in patients with oral candidiasis.

For the study of Candida albicans genotypes involved in development of candidiasis, Candida albicans isolates were collected from healthy volunteers and patients with oral candidiasis and genotyped on the basis of 25S rDNA and microsatellite polymorphisms. In the microsatellite analysis using two microsatellite markers (CDC3 and CAI), 63 healthy volunteer isolates were classified into 35 genotypes (allelic relations to CDC3 alleles 1:2/CAI alleles 1:2), among which genotypes II (115:119/23:23), III (115:123/18:27), and V (123:127/32:41) were found at frequencies of 12.7%, 7.9%, and 7.9%, respectively. In 68 oral candidiasis isolates classified into 39 genotypes, genotypes II and III were identified in 4.4% and 20.6% of the isolates, respectively. The frequency of genotype III was higher in the candidiasis isolates than in the healthy isolates (p < 0.05). These results suggest that genotype III C. albicans assigned by CDC3/CAI is related to the development of oral candidiasis.

Preservation of wild isolates of human malaria parasites in wet ice and adaptation efficacy to in vitro culture.

Wild isolates of malaria parasites were preserved in wet ice for 2-12 days and cultivated by a candle jar method. In four isolates of Plasmodium falciparum collected from Myanmar and preserved for 12 days, all failed to grow. In 31 isolates preserved for 5-10 days, nine were transformed to young gametocytes, but 22 isolates grew well. From Ranong, Thailand, nine isolates preserved for 7 days were examined, and six grew well. On the other hand, all of the 59 isolates collected from eastern Indonesian islands failed to establish as culture-adapted isolates, even most of them were preserved only for 2-3 days: 10 isolates stopped to grow, and 49 isolates were transformed to sexual stages by Day 10. These results indicated that a great difference in adaptation to in vitro culture may exist between wild isolates distributed in continental Southeast Asia and in eastern Indonesia and that gametocytogenesis might be easily switched on in Indonesian isolates. In wild isolates of P. vivax, P. malariae and P. ovale preserved for 2-9 days, ring forms or young trophozoites survived, but adaptation to in vitro culture failed. These results indicate that wild isolates can be preserved in wet ice for 9-10 days.

Genotypes of Candida albicans involved in development of candidiasis and their distribution in oral cavity of non-candidiasis individuals.

Genotype characteristics and distribution of commensal Candida albicans should be studied to predict the development of candidiasis, however, extensive genotype analysis of commensal C. albicans has not been made. In this study, 508 C. albicans isolates were collected from patients with/without candidiasis and divided into 4 isolate groups (SG-1, oral cavity of non-candidiasis patients; SG-2, patients with cutaneous candidiasis; SG-3, patients with vaginal candidiasis; SG-4, patients with candidemia). These isolates were characterized to study the relationship between genotypes and pathogenicity using microsatellite analysis. Using CDC3 and CAI, 5 genotypes (I, 111: 115/33: 41; II, 115: 119/23: 23; III, 115: 123/18: 27; IV, 115: 123/33: 40; and V, 123: 127/32: 41) were found in 4.2%, 8.9%, 7.1%, 2.2% and 3.1% of the isolates, respectively. Genotypes II and III were commonly found in all isolate groups. These genotypes were further divided into 28 types by additional HIS3 and CAIII microsatellite markers. In this analysis, C. albicans with type 6 and type 23 was widely distributed as a commensal species in the oral cavity of non-candidiasis patients and found to be related with candidiasis development. Additionally, genotypes I and IV were found in SG-2 and/or SG-4, suggesting that the fungus with those genotypes is also involved in this development. In contrast, genotype V was not identified in any infective isolates.

Highly efficient DNA compaction mediated by an in vivo antitumor-active tetrazolato-bridged dinuclear platinum(II) complex.

We investigated the effects of antitumor-active tetrazolato-bridged dinuclear platinum(II) complexes [{cis-Pt(NH(3))(2)}(2)(µ-OH)(µ-tetrazolato-N(1),N(2))](2+) (1) and [{cis-Pt(NH(3))(2)}(2)(µ-OH)(µ-tetrazolato-N(2),N(3))](2+) (2) on the higher-order structure of a large DNA molecule (T4 phage DNA, 166 kbp) in aqueous solution through single-molecule observation by fluorescence microscopy. Complexes 1 and 2 cause irreversible compaction of DNA through an intermediate state in which coil and compact parts coexist in a single DNA molecule. The potency of compaction is in the order 2 > 1 ≫ cisplatin. Transmission electron microscopic observation showed that both complexes collapsed DNA into an irregularly packed structure. Circular dichroism measurements revealed that the dinuclear platinum(II) complexes change the secondary structure of DNA from the B to C form. These characteristics of platinum(II) complexes are markedly different from those of the usual condensing agents such as spermidine(3+) and [Co(III)(NH(3))(6)](3+). The ability to cause DNA compaction by the platinum(II) complexes is discussed in relation to their potent antitumor activity.

Microsatellite-based genotyping of Candida albicans isolated from patients with superficial candidiasis.

This study aimed to examine the genotype distribution of Candida albicans and the major genotypes involved in superficial candidiasis. The genotypes of C. albicans isolated from the infection sites of patients with superficial candidiasis (referred to as infection isolates) were analyzed by fragment analysis using 4 microsatellite markers (HIS3, CDC3, CAI and CAIII). Genotypes of the infection isolates were compared with those of C. albicans isolated from oral mucosa of non-candidiasis patients (referred to as oral isolates). Isolates of C. albicans showed 4 major genotypes for HIS3/CAI (" a " for 148 : 148 / 23 : 23," b " for 148 : 160 / 33 : 41," c " for 148 : 164 / 32 : 41 and " d " for 152 : 152 / 18 : 27). The genotypes " a "," b " and " d " were commonly found in oral (4.7, 8.8 and 7.6%, respectively) and infection (6.6, 9.2 and 15.4%, respectively) isolates. No isolates of genotype " c " were isolated from infection sites. The genotype " a " was found in the isolates from patients with genitalia candidiasis. Genotyping of multiple isolates from an individual patient showed that C. albicans from infection sites was genetically homogenous as compared with that of oral isolates, even in the same patient with candidiasis.

Salt has a biphasic effect on the higher-order structure of a DNA-protamine complex.

We observed single DNA molecules by fluorescence microscopy to clarify the effect of protamine on their higher-order structure. With an increase in the protamine concentration, the conformation of DNA molecules changes from an elongated coil state to a compact state through an intermediate state. Furthermore, the long-axis length of DNA gradually decreases while maintaining a distribution profile with a single peak. Such behavior is markedly different from the conformational transition of DNA induced by small polyamines such as spermidine and spermine, where individual DNA molecules exhibit an all-or-none transition from a coil to a globule state and the size distribution is characterized by twin peaks around the transition region. Next, we examined the effect of salt on the conformation of the DNA-protamine complex. Interestingly, at a fixed concentration of protamine, DNA tends to shrink with an increase in the NaCl concentration up to 300 mM, and then swells with a further increase in the NaCl concentration, that is, biphasic behavior is generated depending on the salt concentration. For comparison, we examined the effect of salt on DNA compaction induced by the trivalent polyamine spermidine. We confirmed that salt always has an inhibitory effect on spermine-induced compaction. To clarify this biphasic effect of salt on protamine-induced DNA compaction, we performed a numerical simulation on a negatively charged semiflexible polyelectrolyte in the presence of polycations with relatively large numbers of positive charges by taking into account the effect of salt at different concentrations. The results showed that salt promotes compaction up to a certain concentration and then tends to unfold the polyelectrolyte chain, which reproduced the experimental observation in a semiquantitative manner. This biphasic effect is discussed in relation to the specific shielding effect that depends on the salt concentration.

Incidence and mutation analysis of glucose-6-phosphate dehydrogenase deficiency in eastern Indonesian populations.

We conducted a field survey of glucose-6-phosphate dehydrogenese (G6PD) deficiency in the eastern Indonesian islands, and analyzed G6PD variants molecularly. The incidence of G6PD deficiency in 5 ethnic groups (Manggarai, Bajawa, Nage-Keo, Larantuka, and Palue) on the Flores and Palue Islands was lower than that of another native group, Sikka, or a nonnative group, Riung. Molecular analysis of G6PD variants indicated that 19 cases in Sikka had a frequency distribution of G6PD variants similar to those in our previous studies, while 8 cases in Riung had a different frequency distribution of G6PD variants. On the other hand, from field surveys in another 8 ethnic groups (Timorese, Sumbanese, Savunese, Kendari, Buton, Muna, Minahasa, and Sangirese) on the islands of West Timor, Sumba, Sulawesi, Muna and Bangka, a total of 49 deficient cases were detected. Thirty-nine of these 49 cases had G6PD Vanua Lava (383T＞C) of Melanesian origin. In our previous studies, many cases of G6PD Vanua Lava were found on other eastern Indonesian islands. Taken together, these findings may indicate that G6PD Vanua Lava is the most common variant in eastern Indonesian populations, except for Sikka.

Genotyping of Candida albicans by fragment analysis of microsatellites combined with 25S rDNA and RPS-based strategies.

Because of its high discriminatory potential, fragment analysis of microsatellites has been frequently used for genotyping of Candida albicans at the strain level. In order to evaluate a genotyping system based on the fragment analysis of microsatellites combined with PCRs targeting 25S rDNA and RPS, 456 independent strains of C. albicans were subjected to genotype analysis using 4 microsatellite markers (CDC3, HIS3, CA I and CA III), followed by 25S rDNA and RPS-based genotyping. The fragment analysis using CA I showed the highest discriminatory potential (DP=0.9782), followed by HIS3 (DP=0.8780). Using combined microsatellite markers, 456 C. albicans strains were divided into 384 genotypes (DP=0.9984). PCRs targeting 25S rDNA and RPS were performed to differentiate the strains that showed identical genotypes in the fragment analysis, resulting in 434 genotypes (DP=0.9996). The combined genotyping system showed high discriminatory power at the strain level, and therefore is useful for rapid genotyping in molecular epidemiological studies of candidiasis.

Improvement of the repetitive sequence-based identification and genotyping of Candida albicans using ALT-specific primers.

The nucleotide sequences of the inner repeats of the repetitive sequence (RPS), termed ALTs, of Candida albicans and its related species C. albicans var. stellatoidea and C. dubliniensis, were analyzed. ALT sequences were grouped into 4 types for C. albicans (Aa, Ab, Ac and Ad) and C. albicans var. stellatoidea (Sa1, Sa2, Sb, Sc and Sd), and 3 types for C. dubliniensis (Da, Db and Dc). In addition to the primer set P-II (specific to RPS), 2 primer sets (AS-I and AiR-I) specific to the nucleotide sequences of C. albicans ALT were designed and tested for their potential for RPS-based identification/genotyping of C. albicans. PCRs using AS-I and AiR-I clearly distinguished C. albicans from both C. albicans var. stellatoidea and C. dubliniensis. Furthermore, the strains of C. albicans that showed similar electrophoretic patterns in the PCR using P-II were discriminated at the subtype level. These results indicate that the PCRs using RPS- and ALT-specific primer sets are useful as simple and rapid systems for the specific identification and genotyping of C. albicans.

Rhodovastum atsumiense gen. nov., sp. nov., a phototrophic alphaproteobacterium isolated from paddy soil.

A photoorganotrophic alphaproteobacterium designated strain G2-11(T) was isolated from submerged paddy soil. This bacterium had relatively large, oval to rod-shaped cells (2.0-3.0x3.0-10 microm). Cells were motile by means of single polar flagella. The color of phototrophically growing cultures was reddish-brown. The cell extract had absorption maxima at 375, 465, 492, 529, 592, 804, and 844 nm, indicating the presence of bacteriochlorophyll a and carotenoides of the spirilloxanthin series. Vesicular intracytoplasmic membranes were present. The main component of cellular fatty acids was C(18:1)omega7c. Ubiquinone-10 and rhodoquinone-10 were the major quinones. A 16S rRNA gene sequence analysis revealed that the isolate is closest to the acidophilic aerobic photosynthetic bacterium Acidisphaera rubrifaciens strain HS-AP3(T) (93.3% similarity). The G+C content of genomic DNA is 67.8 mol%. The name Rhodovastum atsumiense gen. nov., sp. nov. is proposed for the novel isolate. The type strain is strain G2-11(T) (=NBRC 104268(T)=KCTC 5708(T)).

Rhodoplanes serenus sp. nov., a purple non-sulfur bacterium isolated from pond water.

A bright pink to red-coloured, phototropic, purple non-sulfur bacterium, designated strain TUT3530(T), was isolated from pond water. Cells of the novel isolate were found to be Gram-negative, motile, budding rods. Cell extracts from phototrophically grown cultures had absorption maxima at 378, 482, 512, 550, 590, 800 and 850 nm, indicating the presence of bacteriochlorophyll a and carotenoids of the spirilloxanthin series. The intracytoplasmic membrane system was of the lamellar type. Anaerobic photo-organotrophy with simple organic acids such as pyruvate was the preferred mode of growth. Aerobic growth at full atmospheric oxygen tension and anaerobic denitrifying growth in darkness were also possible. Photolithotrophic growth occurred with thiosulfate, but not with sulfide or hydrogen, as the electron donor. No growth factors were required. The major whole-cell fatty acid was C(18 : 1)omega7c. The major quinones were ubiquinone-10 and rhodoquinone-10. A phylogenetic analysis based on 16S rRNA gene sequences and studies involving DNA-DNA hybridization demonstrated that strain TUT3530(T) occupies a distinct taxonomic position within the genus Rhodoplanes. On the basis of these data, strain TUT3530(T) represents a novel species of the genus Rhodoplanes, for which the name Rhodoplanes serenus sp. nov. is proposed. The type strain is TUT3530(T) (=DSM 18633(T)=NBRC 102049(T)).

Molecular approaches in the diagnosis of dermatophytosis.

Dermatophytosis is one of the most common infectious diseases in the world and can be caused by several dermatophyte species. These species are closely related in genetic structure in spite of different phenotypic and ecological features. The morphological similarity, variability, and polymorphism of dermatophytes have meant that species identification for dermatophytes is time consuming and requires a significant degree of knowledge and technological expertise. Molecular biology-based techniques have solved problems concerning the morphology-based identification of dermatophytes and have improved our knowledge on the epidemiology of dermatophytosis. Further development of molecular diagnosis of dermatophytosis requires the investigation of additional molecular markers for diagnostic tools targeting multiple loci as well as the improvement of techniques.

A case of oral geotrichosis caused by Geotrichum capitatum in an old patient.

Geotrichosis is an uncommon fungal infection. Geotrichum capitatum is commonly acknowledged as an opportunistic fungal pathogen that causes systemic geotrichosis in immunocompromised patients, especially patients with acute leukemia and severe neutropenia. Here, we report a case of oral geotrichosis caused by G. capitatum in an old patient with no hematological malignancies. Fungal cells were detected in clinical specimens obtained with oral swabs using the KOH technique. Yeast colonies with peripheral hairs were exclusively isolated as fungi from the oral mucosa and feces of the patient. The isolates were identified as G. capitatum by morphological findings, sugar-assimilation tests, and the nucleotide sequences of the ITS regions of the rDNA. Effective treatment of the patient was achieved with amphotericin B syrup in accord with the results of in vitro susceptibility tests. G. capitatum should be recognized as a fungal pathogen involved in superficial infections of older persons, as should Candida spp., even in the absence of hematological malignancies.

Seven different glucose-6-phosphate dehydrogenase variants including a new variant distributed in Lam Dong Province in southern Vietnam.

We conducted a survey for glucose-6-phosphate dehydrogenase (G6PD) deficiency using blood samples from male outpatients of a local hospital in southern Vietnam. Most of the samples were from the Kinh (88.9%), the largest ethnic group in Vietnam, with a small number (11.1%) coming from the K'Ho, Chauma, Nung, and Tay minorities. We detected 25 G6PD-deficient cases among 1,104 samples (2.3%), and read the open reading frame of G6PD. A novel mutation (352T>C) predicting an aminoacid change of 118Tyr>His was found in a 1-year-old Kinh boy. His G6PD activity was estimated to be less than 10% residual activity, although he did not show chronic hemolytic anemia. Thus, we categorized this variant as Class II and named it G6PD Bao Loc. In the Kinh population, G6PD Viangchan (871G>A, 1311C>T, intron 11 nt93T>C), one of the most common variants in continental Southeast Asian populations, was the highest (6/19), followed by variants originating from the Chinese such as G6PD Canton (1376G>T) (5/19), G6PD Kaiping (1388G>A) (3/19), G6PD Gaohe (95A>G) (1/19), and G6PD Quing Yuan (392G>T) (1/19). In addition, G6PD Union (1360C>T) (2/19), which originated from the Oceania, was also detected. These findings suggest that the Kinh people are derived from various ancestries from continental Southeast Asia, China, and Oceania. In contrast, all of the 5 deficient cases in the K'Ho population were G6PD Viangchan, suggesting that they were very close to Southeast Asian populations such as the Khmer in Cambodia and the Lao in Laos. It is interesting that G6PD Mahidol (487G>A), another common variant in continental Southeast Asian populations in Myanmar, Thailand, and Malaysia, has not been detected from the Vietnamese.

Further investigations of glucose-6-phosphate dehydrogenase variants in Flores Island, eastern Indonesia.

We conducted field surveys for malaria and glucose-6-phosphate dehydrogenase (G6PD) deficiency in the eastern part of Flores Island, East Nusa Tenggara Province, Indonesia. A total of 1,108 volunteers (642 males and 466 females) belonging to three ethnic groups (Sikka, Ende and Bajo) were examined, and 55 G6PD-deficient individuals (38 males and 17 females) were detected. Among them, 50 samples were analyzed molecularly, in addition to three deficient cases in a Bajo family. In the Sikka population, G6PD Kaiping (1388G>A), one of the two common variants in the Chinese population, was unexpectedly found as the most dominant variant (11/22, 50.0%), followed by G6PD Chatham (1003G>A, 36.4%), G6PD Coimbra (592C>T, 9.1%) and G6PD Vanua Lava (383T>C, 4.5%). Frequency of G6PD Kaiping in the Sikka might be the highest among non-Chinese populations reported so far. In the Ende population, G6PD Vanua Lava (9/14, 64.3%) was the highest, followed by G6PD Kaiping (14.3%), G6PD Chinese-5 (1024C>T, 14.3%) and G6PD Chatham (7.1%). In the Bajo population, a total of 18 deficient cases were analyzed, and a novel mutation (844G>T) in exon 8 with a predicted amino acid change of 282 Asp>Tyr was found in a 7-year-old boy at a Bajo village near Maumere. This new Class II (mild type) variant was also confirmed in his mother and sister, and designated as G6PD Bajo Maumere. The missense mutation at the same nucleotide 844 has been known as G6PD Seattle/Lodi/Modena/Ferrara II, but this mutation is caused by a G>C substitution (282 Asp>His). In the Bajo population, G6PD Viangchan (871G>A, IVS 11 nt93 T>C, 1311C>T), the most common variant in continental Southeast Asian populations, was found to be the dominant (11/18, 61.1%), followed by G6PD Vanua Lava and the new variant (each 16.7%), and G6PD Coimbra (5.6%). These results strongly suggest that the Bajo peoples may have different ancestors from those for Sikka and Ende, and may be much closer to continental Southeast Asian populations. It is interesting that G6PD Canton (1376G>T), another common variant in Chinese, was not seen in the Flores population.

Sequence variation in the T-cell epitopes of the Plasmodium falciparum circumsporozoite protein among field isolates is temporally stable: a 5-year longitudinal study in southern Vietnam.

In an effort to decipher the nature and extent of antigen polymorphisms of malaria parasites in a setting where malaria is hypomesoendemic, we conducted a 5-year longitudinal study (1998 to 2003) by sequencing the Th2R and Th3R epitopes of the circumsporozoite protein (CSP) of 142 Plasmodium falciparum field isolates from Bao Loc, Vietnam. Samples were collected during the high-transmission season, September through December 1998 (n = 43), as well as from July 2000 to August 2001 (n = 34), September 2001 to July 2002 (n = 33), and August 2002 to July 2003 (n = 32). Marked sequence diversity was noted during the high-transmission season in 1998, but no significant variation in allele frequencies was observed over the years (chi(2) = 70.003, degrees of freedom = 57, P = 0.116). The apparent temporal stability in allele frequency observed in this Bao Loc malaria setting may suggest that polymorphism in the Th2R and Th3R epitopes is not maintained by frequency-dependent immune selection. By including 36 isolates from Flores Island, Indonesia, and 19 isolates from Thaton, Myanmar, we investigated geographical patterns of sequence polymorphism for these epitopes in Southeast Asia; among the characterized isolates, a globally distributed variant appears to be predominant in Vietnam (75 of 142 isolates, or 52.8%) as well as in Myanmar (15 of 19 isolates, or 78.9%) and Indonesia (31 of 36 isolates, or 86.1%). Further analyses involving worldwide CSP sequences revealed distinct regional patterns, a finding which, together with the unique mutations observed here, may suggest a possible role for host or local factors in the generation of sequence diversity in the T-cell epitopes of CSP.