Therapeutic efficacy of artemether-lumefantrine in uncomplicated falciparum malaria in India

<p>Abstract</p> <p>Background</p> <p>Artemisinin-based combination therapy (ACT) is the treatment of choice for uncomplicated falciparum malaria. Artemether-lumefantrine (AL), a fixed dose co-formulation, has recently been approved for marketing in India, although it is not included in the National Drug Policy for treatment of malaria. Efficacy of short course regimen (4 × 4 tablets of 20 mg artemether plus 120 mg lumefantrine over 48 h) was demonstrated in India in the year 2000. However, low cure rates in Thailand and better plasma lumefantrine concentration profile with a six-dose regimen over three days, led to the recommendation of higher dose globally. This is the first report on the therapeutic efficacy of the six-dose regimen of AL in Indian uncomplicated falciparum malaria patients. The data generated will help in keeping the alternative ACT ready for use in the National Programme as and when required.</p> <p>Methods</p> <p>One hundred and twenty four subjects between two and fifty-five years of age living in two highly endemic areas of the country (Assam and Orissa) were enrolled for single arm, open label prospective study. The standard six-dose regimen of AL was administered over three days and was followed-up with clinical and parasitological evaluations over 28 days. Molecular markers <it>msp</it>-<it>1 </it>and <it>msp</it>-2 were used to differentiate the recrudescence and reinfection among the study subjects. In addition, polymorphism in <it>pfmdr</it>1 was also carried out in the samples obtained from patients before and after the treatment.</p> <p>Results</p> <p>The PCR corrected cure rates were high at both the sites viz. 100% (n = 53) in Assam and 98.6% (n = 71) in Orissa. The only treatment failure case on D7 was a malnourished child. The drug was well tolerated with no adverse events. Patients had pre-treatment carriage of wild type codons at positions 86 (41.7%, n = 91) and 184 (91.3%, n = 91) of <it>pfmdr1 </it>gene.</p> <p>Conclusion</p> <p>AL is safe and effective drug for the treatment of acute uncomplicated falciparum malaria in India. The polymorphism in <it>pfmdr</it>1 gene is not co-related with clinical outcome. However, treatment failure can also occur due to incomplete absorption of the drug as is suspected in one case of failure at D7 in the study. AL can be a viable alternative of artesunate plus sulphadoxine/pyrimethamine (AS + SP), however, the drug should be used rationally and efficacy needs to be monitored periodically.</p

Plasmodium vivax: immunological properties of tryptophan-rich antigens PvTRAg 35.2 and PvTRAg 80.6

Need for malaria vaccine necessitates the characterization of potential antigens of the Plasmodium parasite. Recently, we have identified several Plasmodium vivax tryptophan-rich antigens (PvTRAgs). Here, we describe the immunological characterization of hitherto undescribed two such antigens PvTRAg 35.2 and PvTRAg 80.6 which are respective homologue of Plasmodium falciparum merozoite associated tryptophan-rich antigen (PfMaTrA) and P. falciparum tryptophan and threonine rich antigen (PfTryThrA) involved in erythrocyte invasion. Each of the pvtrag genes is comprised of two exons where exon 2 encodes for major part of the protein. PvTRAg 35.2 and PvTRAg 80.6 showed 97.06% and 94.12% (n=34) seropositivity rates, and 92.3% (n=13) and 100% (n=29) lymphoproliferative responses, respectively, among P. vivax exposed individuals. Geometric mean values of IL-12, IFN-γ, TNF-α, IL-4 and IL-10 in PBMC culture supernatants of P. vivax exposed individuals were 182.02, 60.3, 62.84, 196.01 and 177.17 pg/ml against PvTRAg 35.2 and 185.27, 58.15, 64.56, 142.01 and 157.2 pg/ml against PvTRAg 80.6 showing mixed immune response with distinct biased towards anti-inflammatory Th2 phenotype. The pvtrag 35.2 gene was highly conserved in the parasite population whereas pvtrag 80.6 showed minor variations in the N-terminal region but highly conserved in the C-terminal region containing tryptophan-rich domain

Wide variation in microsatellite sequences within each Pfcrt mutant haplotype

Flanking microsatellites for each of the Pfcrt mutant haplotype of Plasmodium falciparum remain conserved among geographical isolates. We describe here heterogeneity in the intragenic microsatellites among each of the Pfcrt haplotype. There were fourteen different alleles of AT repeats of intron 2 and eight alleles of TA repeats of intron 4 of the pfcrt gene among Indian isolates. This resulted in 33 different two-locus (intron 2 plus intron 4) microsatellite genotypes among 224 isolates. There were 15 different two-locus microsatellite genotypes within the South American Pfcrt haplotype (S<SUB>72</SUB>V<SUB>73</SUB>M<SUB>74</SUB>N<SUB>75</SUB>T<SUB>76</SUB>S<SUB>220</SUB>) and 11 genotypes in the southeast Asian haplotype (C<SUB>72</SUB>V<SUB>73</SUB>I<SUB>74</SUB>E<SUB>75</SUB>T<SUB>76</SUB>S<SUB>220</SUB>) in these isolates. Indian isolates with Pfcrt haplotype C<SUB>72</SUB>V<SUB>73</SUB>I<SUB>74</SUB>E<SUB>75</SUB>T<SUB>76</SUB>S<SUB>220</SUB> shared one of its two-locus microsatellite genotype with southeast Asian P. falciparum parasite lines from Thailand (K1) and Indochina (Dd2 and W2). Conversely, Indian isolates containing S<SUB>72</SUB>V<SUB>73</SUB>M<SUB>74</SUB>N<SUB>75</SUB>T<SUB>76</SUB>S<SUB>220</SUB> Pfcrt haplotype did not share any of their two-locus microsatellite genotype with South American parasite line 7G8 from Brazil. Significantly, large number of newer two-locus microsatellite genotypes were detected in a 2-year time period (P &lt; 0.05). Microsatellite variation was more prominent in the areas of high malaria transmission. It is concluded that the genetic recombination in the intragenic microsatellites continues in the parasite population even after microsatellites flanking the pfcrt gene had already been fixed. Presence of various Pfcrt haplotypes and a variety of intragenic microsatellites indicates that there is a wide spectrum of chloroquine resistant parasite population in India. This information should be useful for malaria control programs of the country

Emergence of an unusual sulfadoxine-pyrimethamine resistance pattern and a novel K<SUB>540</SUB>N mutation in dihydropteroate synthetase in Plasmodium falciparum isolates obtained from Car Nicobar Island, India, after the 2004 Tsunami

Background: Enormous amounts of drugs were used to contain the outbreak of infectious diseases in areas of India affected by the tsunami in December 2004. The impact of this drug use on the Plasmodium falciparum population needs to be investigated. Methods: The nucleotide sequence of the pfcrt, pfdhps and pfdhfr genes was determined for 229 clinical P. falciparum isolates collected from patients on Car Nicobar Island at 6 different time points between May 2004 and May 2008. Results: Over time, there was an increase in the proportion of the P. falciparum population that had mutations in the pfcrt, pfdhps and pfdhfr genes associated with higher levels of chloroquine, sulfadoxine, and pyrimethamine resistance, respectively. However, the parasites collected during October 2005 had mutations associated with a lower level of pyrimethamine resistance and a higher level of sulfadoxine resistance (a rare combination), as well as a novel K540N mutation in P. falciparum dihydropteroate synthetase (PfDHPS). The emergence of this parasite population coincided with the widespread use of an additional antifolate drug, trimethoprim-sulfamethoxazole, to treat other infections during January-March 2005. Molecular modeling revealed that the sulfadoxine binding affinity of the new PfDHPS triple mutant A<SUB>436</SUB> G<SUB>437</SUB> N<SUB>540</SUB>A<SUB>581</SUB>A<SUB>613</SUB> was similar to that of A<SUB>436</SUB> G<SUB>437</SUB> E<SUB>540</SUB>A<SUB>581</SUB>A<SUB>613</SUB> (bold type indicates mutated amino acids). Conclusions: The use of 2 antifolate drugs in combination should be avoided to prevent the selection of parasites with newer mutations and altered drug susceptibilities

Genetic variation in the Plasmodium falciparum circumsporozoite protein in India and its relevance to RTS,S malaria vaccine

Contains fulltext : 108006.pdf (publisher's version ) (Open Access)RTS,S is the most advanced malaria vaccine candidate, currently under phase-III clinical trials in Africa. This Plasmodium falciparum vaccine contains part of the central repeat region and the complete C-terminal T cell epitope region (Th2R and Th3R) of the circumsporozoite protein (CSP). Since naturally occurring polymorphisms at the vaccine candidate loci are critical determinants of the protective efficacy of the vaccines, it is imperative to investigate these polymorphisms in field isolates. In this study we have investigated the genetic diversity at the central repeat, C-terminal T cell epitope (Th2R and Th3R) and N-terminal T cell epitope regions of the CSP, in P. falciparum isolates from Madhya Pradesh state of India. These isolates were collected through a 5-year prospective study aimed to develop a well-characterized field-site for the future evaluation of malaria vaccine in India. Our results revealed that the central repeat (63 haplotypes, n = 161) and C-terminal Th2R/Th3R epitope (24 haplotypes, n = 179) regions were highly polymorphic, whereas N-terminal non-repeat region was less polymorphic (5 haplotypes, n = 161) in this population. We did not find any evidence of the role of positive natural selection in maintaining the genetic diversity at the Th2R/Th3R regions of CSP. Comparative analysis of the Th2R/Th3R sequences from this study to the global isolates (n = 1160) retrieved from the GenBank database revealed two important points. First, the majority of the sequences (~61%, n = 179) from this study were identical to the Dd2/Indochina type, which is also the predominant Th2R/Th3R haplotype in Asia (~59%, n = 974). Second, the Th2R/Th3R sequences in Asia, South America and Africa are geographically distinct with little allele sharing between continents. In conclusion, this study provides an insight on the existing polymorphisms in the CSP in a parasite population from India that could potentially influence the efficacy of RTS,S vaccine in this region

Progressive increase in point mutations associated with chloroquine resistance in Plasmodium falciparum isolates from India

Background: Effective malaria control programs require continuous monitoring of drug pressure in the field, using molecular markers. Methods: We used sequence analysis to investigate the pfcrt and pfmdr1 mutations in Indian Plasmodium falciparum isolates. To evaluate the chloroquine drug pressure in the field, isolates were collected from 5 different areas at 2 time points, with an interval of 2 years. Results: In 265 P. falciparum isolates, pfcrt mutations were observed at codons 72, 74, 75, 76, and 220, resulting in 8 different genotypes: SMNTS (61.89%), CIETS (12.08%), CMNKS (0.38%), CMNTA (2.64%), CMNTS (4.91%), SMNTA (0.38%), CIDTS (2.26%), and wild-type CMNKA (15.47%). During the 2-year period, there was a significant decrease in the number of isolates with the SMNTS genotype and an increase in the number of isolates with the highly chloroquine-resistant pfcrt genotype CIETS (P&lt;.05). The N86Y mutation was less prevalent (30.13%) than the Y184F mutation (99.16%) in the pfmdr1 gene in 239 isolates, but the number of isolates with the N86Y mutation increased significantly during the 2-year period (P&lt;.05). The number of isolates with higher total numbers of pfcrt and pfmdr1 2-loci mutations, therefore, increased significantly during this period. There was a regional bias in the mutation rate of these genes, because isolates from areas where chloroquine resistance was high had higher numbers of 2-loci mutations, and areas where chloroquine resistance was low had isolates with lower numbers of 2-loci mutations. Conclusion: There was a temporal increase in the number of pfcrt and pfmdr1 2-loci mutations, and this led to the higher level of chloroquine resistance. This is a cause for concern for the antimalarial drug policy in India

Sequence diversity in the central repeat region of PfCSP.

<p>(<b>A</b>) Representation of the variation in sequence repeats in the central region of the CSP in 161 samples. The sequences of eight laboratory-adapted <i>P. falciparum</i> strains [Dd2 (Indochina), K1 (Thailand), MAD20 (Papua New Guinea), Wellcome (West Africa), 7G8 (Brazil), HB3 (Honduras), 3D7 (The Netherlands) and RO33 (Ghana)] are shown here for comparison. The NANP repeats are indicated as “1 with gray shade", NVDP repeats are indicated as “2 with black shade" and all other repeats are un-shaded. Numbers on the right indicate numbers of samples belonging to that particular haplotype. Numbers above the alignment are amino acid position with reference to 3D7 sequence. Dots represent amino acid positions identical to the 3D7 haplotype, whereas those different are indicated. Dashes have been inserted for maximum alignment. C, community cohort; H, hospital cohort; T, total; WC, Wellcome. (<b>B</b>) Distribution of repeats in the central region of the CSP in 161 samples.</p

Global population structure of csp gene.

<p>A minimal spanning tree (MST) generated using BioNumerics software version 6.6 showing the relationship among all the 117 haplotypes based on the Th2R/Th3R sequences of the CSP from worldwide isolates [Asia, n = 974; South America, n = 181 and Africa, n = 184)]. Each circle represents an individual haplotype and the size of the circle is proportional to the number of isolates belonging to that haplotype (also shown as pie). The lines connecting the circles are branch length and are red if two haplotypes differ by only one mutation, blue if differ by 2 mutations, solid black if differ by 3 mutations, dashed black if differ by 4 mutations and gray if they differ by more than 4 mutations. Numbers outside the circles indicate haplotypes H1 to H117. The Dd2, 3D7, HB3, 7G8 and MAD20 type sequences are highlighted in bold. The haplotype pairs H55 & H58; H57 & H87; H60 & H106 and H97 & H98 are identical at amino acid level; but have one synonymous mutation. H1 to H24 are the same haplotypes we observed in our study sites and shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043430#pone-0043430-g003" target="_blank"><b>Fig </b><b>3B</b></a>. Please refer to <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043430#pone.0043430.s004" target="_blank">Table S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043430#pone.0043430.s006" target="_blank">Table S3</a></b> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043430#pone.0043430.s001" target="_blank">Fig S1</a></b> for more details on these sequence haplotypes and their country-wise distributions.</p

Sequence diversity in the N- and C-terminal non-repeat region of PfCSP.

<p>(<b>A</b>) Sequence alignment showing polymorphisms in the non-repeat N-terminal T cell epitope region (amino acid residue 84 to 104) of CSP in 161 samples. The shaded region (amino acid residue 93 to 97) is a conserved motif involved in sporozoite invasion of mosquito salivary gland as well as in binding to hepatocytes prior to invasion. (<b>B</b>) Sequence alignment showing polymorphisms in the non-repeat C-terminal T cell epitope regions (Th2R spanning from amino acid residues 311 to 327 and Th3R from amino acid residues 341 to 364) of CSP in 179 samples. The highly conserved sequences flanking the Th2R and Th3R domains are shaded grey. The eight laboratory-adapted strains are also included in this alignment. Numbers on the right indicate numbers of samples belonging to that particular haplotype. Dots represent amino acid positions identical to the 3D7 haplotype, whereas those different are indicated. C, community cohort; H, hospital cohort; T, total; WC, Wellcome.</p

Nucleotide diversity and tests of neutrality for the C-terminal epitope region (Th2R/Th3R) of the <i>P. falciparum</i> csp gene in global isolates.

<p><i>Note:</i> N, Number of isolates analyzed from each country; H, Number of haplotypes; S, Number of segregating (polymorphic) sites; Hd, Haplotype diversity; π, Observed average pairwise nucleotide diversity; dN-dS, rate of non-synonymous mutations minus rate of synonymous mutations; PNG, Papua New Guinea; SD, Standard deviation; SE, Standard error. *, <i>P</i>>0.10; **, <i>P</i>>0.05; ***, <i>P</i><0.05.</p