Safety evaluation of an antimalarial herbal product from Andrographis paniculata (AS201-01) in healthy volunteers.

OBJECTIVES: Andrographis paniculata tablets (AS201-01) have previously been shown to have potent bioactivity as an antimalarial and to produce no unwanted side effects in animal models. Here, we present the phase 1 clinical trial conducted to evaluate the safety of AS201-01 tablets in healthy volunteers. METHODS: The study was a randomized, double-blind controlled cross-over, a placebo-controlled design consisting of a 4-day treatment of AS201-01 tablets. A total of 30 healthy human volunteers (16 males and 14 females) were divided into two groups, and each group was given 4 tablets, twice daily for 4 days. Group 1 received AS201-01, while group 2 received placebo tablets. Volunteers were given a physical examination before the treatment. The effects of AS201-01 on random blood glucose, biochemical, and hematological as well as urine profiles were investigated. RESULTS: There were no changes in observed parameters as a result of AS201-01 being administered. Statistical analysis showed no significant difference (p>0.05) between the test and control group regarding hematology profile, biochemical profile, and random blood glucose. Increased appetite and better sleep, which categorized as grade 1 adverse event was reported after treatment with AS201-01 tablet. CONCLUSIONS: The outcome supports our previous observation that the AS201-01 tablet, given twice a day for 4 days, is safe and nontoxic.

Distribution of G6PD deficiency genotypes among Southeast Asian populations.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a group of X-linked, hereditary genetic disorders caused by mutations in the G6PD gene and results in functional variants of about 400 biochemical and clinical phenotypes. Among them, more than 215 genotypes have been identified so far. In this review, specific features of the genotype distribution in different communities and countries are discussed based on multiple reports and our molecular epidemiological studies of Southeast Asian countries. Particularly, in Indonesia, the frequency distribution of G6PD deficiency variants was distinct between western and eastern Indonesian populations, suggesting two different gene flows during Indonesian expansions.

Human migration and the spread of malaria parasites to the New World.

We examined the mitogenomes of a large global collection of human malaria parasites to explore how and when Plasmodium falciparum and P. vivax entered the Americas. We found evidence of a significant contribution of African and South Asian lineages to present-day New World malaria parasites with additional P. vivax lineages appearing to originate from Melanesia that were putatively carried by the Australasian peoples who contributed genes to Native Americans. Importantly, mitochondrial lineages of the P. vivax-like species P. simium are shared by platyrrhine monkeys and humans in the Atlantic Forest ecosystem, but not across the Amazon, which most likely resulted from one or a few recent human-to-monkey transfers. While enslaved Africans were likely the main carriers of P. falciparum mitochondrial lineages into the Americas after the conquest, additional parasites carried by Australasian peoples in pre-Columbian times may have contributed to the extensive diversity of extant local populations of P. vivax.

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.

Extremely low Helicobacter pylori prevalence in North Sulawesi, Indonesia and identification of a Maori-tribe type strain: a cross sectional study.

BACKGROUND: Sulawesi in Indonesia has a unique geographical profile with assumed separation from Sundaland. Studies of Helicobacter pylori in this region are rare due to the region's rural location and lack of endoscopy equipment. Indirect methods are, therefore, the most appropriate for measuring H. pylori infection in these areas; with the disposable gastric brush test, we can obtain gastric juice as well as small gastric tissue samples for H. pylori culture. We investigated the prevalence of H. pylori infection and evaluated human migration patterns in the remote areas of North Sulawesi. METHODS: We recruited a total of 251 consecutive adult volunteers and 131 elementary school children. H. pylori infection was determined by urine antibody test. A gastric brush test was used to culture H. pylori. We used next-generation and polymerase chain reaction based sequencing to determine virulence factors and multi-locus sequence typing (MLST). RESULTS: The overall H. pylori prevalence was only 14.3% for adults and 3.8% for children, and 13.6% and 16.7% in Minahasanese and Mongondownese participants, respectively. We isolated a single H. pylori strain, termed -Manado-1. Manado-1 was East Asian type cagA (ABD type), vacA s1c-m1b, iceA1 positive/iceA2 negative, jhp0562-positive/ß-(1,3) galT-negative, oipA "on", and dupA-negative. Phylogenetic analyses showed the strain to be hspMaori type, a major type observed in native Taiwanese and Maori tribes. CONCLUSIONS: Our data support that very low H. pylori infection prevalence in Indonesia. Identification of hspMaori type H. pylori in North Sulawesi may support the hypothesis that North Sulawesi people migrated from north.

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.

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.

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.

Synergistic enhancement of a copper chelator, bathocuproine disulphonate, and cysteine on in vitro growth of Plasmodium falciparum in glucose-6-phosphate dehydrogenase-deficient erythrocytes.

In vitro growth of Plasmodium falciparum is restricted in glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes (RBC), as a result of oxidative stress. Bathocuproine disulphonate (BCS), a copper chelator, as well as cysteine have been shown to synergistically stimulate the in vitro growth of various mammalian cells and Trypanosoma under oxygenated conditions. We examined the effects of these two chemicals on the in vitro growth of P. falciparum in G6PD-deficient RBC, and found that addition of BCS and cysteine synergistically enhanced the growth of the P. falciparum FCR-3 strain in these RBC to the same level as in normal RBC. However, BCS or cysteine alone had no stimulatory effect. To explain this synergistic enhancement, changes in thiol, NADPH and glutathione contents were investigated. After addition of cysteine alone, thiol content in the medium decreased rapidly, but when BCS was added, it was maintained at about 35% at 24 hours after incubation, suggesting that BCS stimulates parasite growth in G6PD-deficient RBC by inhibiting copper-mediated oxidation of cysteine in the medium. In these RBC, no increase in NADPH level, but a slight increase in glutathione, was observed in the presence of both BCS and cysteine. The slight increase of glutathione, was probably due to incorporation of cysteine from the medium, although this could not fully explain the synergistic growth enhancement. These findings taken together suggest that cysteine incorporated into G6PD-deficient RBC may help maintain the thiol groups in many proteins, such as membrane proteins, hemoglobin and enzymes, and plays an important role in maintaining an appropriate culture state necessary for parasite growth. We also examined the effects of BCS and cysteine on adaptation of wild isolates of P. falciparum to in vitro cultivation using the candle jar method. Although there was no drastic effect on growth enhancement, the presence of BCS and cysteine accelerated the appearance of schizonts in many isolates.

An improved, simple screening method for detection of glucose-6-phosphate dehydrogenase deficiency.

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.

Sequence diversity in the amino-terminal region of the malaria-vaccine candidate serine repeat antigen in natural Plasmodium falciparum populations.

The amino-terminal region of the serine repeat antigen (SERA) of Plasmodium falciparum is a major malaria-vaccine candidate. Variation in this molecule is essentially dimorphic and alleles may be grouped into the types FCR3, K1 and Honduras1. The Honduras1-type is thought to be the product of homologous recombination between FCR3 and K1 alleles. Here we have examined patterns of sequence diversity in exon II of SERA gene, which encodes most of the amino-terminal region of the antigen, in wild P. falciparum isolates from Indonesia (n=60), Myanmar (n=10) and Thailand (n=14). Among the Indonesian isolates the FCR-3 type predominated (56/60), twenty of which we characterized as novel alleles. A new K1-type allele was also found. In Myanmar, however, all isolates displayed K1-type SERA sequences, which included one new allele. The Honduras1-type was not detected in both countries. In contrast, the 14 isolates from Thailand displayed all three allelic types, with one new Honduras1-type and three new K1-type alleles. On examining the global distribution of SERA alleles by combining previously published sequence data with our results, the FCR3-type alleles predominated in Indonesia, Brazil, and Solomon Islands, but were not found in wild isolates from Myanmar and Africa. Brazil was the only area where K1-type alleles were not found. The distribution of Honduras1-type alleles seems to be mostly restricted to parasite populations from Vietnam, Thailand and Africa. In the allelic families FCR3 and K1, most diversity resulted from variation in sequence and number of octamer repeat units and of allotypes encoding the stretch of serine residues. Sequence analysis indicated that both insertions and deletions of repetitive motifs (creating variation within dimorphic allelic families) and homologous recombination between alleles belonging to different allelic families (creating Honduras1-type alleles) play a role in generating new SERA alleles. Since repeat motifs in the amino-terminal region of SERA contain epitopes recognized by parasite-inhibitory antibodies, sequence variation in exon II may represent one of the parasite's immune-evasion strategies.