High-throughput analysis of antimalarial susceptibility data by the WorldWide Antimalarial Resistance Network (WWARN) in vitro analysis and reporting tool.

Assessment of in vitro susceptibility is a fundamental component of antimalarial surveillance studies, but wide variations in the measurement of parasite growth and the calculation of inhibitory constants make comparisons of data from different laboratories difficult. Here we describe a Web-based, high-throughput in vitro analysis and reporting tool (IVART) generating inhibitory constants for large data sets. Fourteen primary data sets examining laboratory-determined susceptibility to artemisinin derivatives and artemisinin combination therapy partner drugs were collated from 11 laboratories. Drug concentrations associated with half-maximal inhibition of growth (IC50s) were determined by a modified sigmoid Emax model-fitting algorithm, allowing standardized analysis of 7,350 concentration-inhibition assays involving 1,592 isolates. Examination of concentration-inhibition data revealed evidence of apparent paradoxical growth at high concentrations of nonartemisinin drugs, supporting amendment of the method for calculating the maximal drug effect in each assay. Criteria for defining more-reliable IC50s based on estimated confidence intervals and growth ratios improved correlation coefficients for the drug pairs mefloquine-quinine and chloroquine-desethylamodiaquine in 9 of 11 and 8 of 8 data sets, respectively. Further analysis showed that maximal drug inhibition was higher for artemisinins than for other drugs, particularly in ELISA (enzyme-linked immunosorbent assay)-based assays, a finding consistent with the earlier onset of action of these drugs in the parasite life cycle. This is the first high-throughput analytical approach to apply consistent constraints and reliability criteria to large, diverse antimalarial susceptibility data sets. The data also illustrate the distinct biological properties of artemisinins and underline the need to apply more sensitive approaches to assessing in vitro susceptibility to these drugs.

Long term persistence of clonal malaria parasite Plasmodium falciparum lineages in the Colombian Pacific region.

BACKGROUND: Resistance to chloroquine and antifolate drugs has evolved independently in South America, suggesting that genotype - phenotype studies aimed at understanding the genetic basis of resistance to these and other drugs should be conducted in this continent. This research was conducted to better understand the population structure of Colombian Plasmodium falciparum in preparation for such studies. RESULTS: A set of 384 SNPs were genotyped in blood spot DNA samples from 447 P. falciparum infected subjects collected over a ten year period from four provinces of the Colombian Pacific coast to evaluate clonality, population structure and linkage disequilibrium (LD). Most infections (81%) contained a single predominant clone. These clustered into 136 multilocus genotypes (MLGs), with 32% of MLGs recovered from multiple (2 - 28) independent subjects. We observed extremely low genotypic richness (R = 0.42) and long persistence of MLGs through time (median = 537 days, range = 1 - 2,997 days). There was a high probability (>5%) of sampling parasites from the same MLG in different subjects within 28 days, suggesting caution is needed when using genotyping methods to assess treatment success in clinical drug trials. Panmixia was rejected as four well differentiated subpopulations (FST = 0.084 - 0.279) were identified. These occurred sympatrically but varied in frequency within the four provinces. Linkage disequilibrium (LD) decayed more rapidly (r2 = 0.17 for markers <10 kb apart) than observed previously in South American samples. CONCLUSIONS: We conclude that Colombian populations have several advantages for association studies, because multiple clone infections are uncommon and LD decays over the scale of one or a few genes. However, the extensive population structure and low genotype richness will need to be accounted for when designing and analyzing association studies.

Sentinel network for monitoring in vitro susceptibility of Plasmodium falciparum to antimalarial drugs in Colombia: a proof of concept.

Drug resistance is one of the principal obstacles blocking worldwide malaria control. In Colombia, malaria remains a major public health concern and drug-resistant parasites have been reported. In vitro drug susceptibility assays are a useful tool for monitoring the emergence and spread of drug-resistant Plasmodium falciparum. The present study was conducted as a proof of concept for an antimalarial drug resistance surveillance network based on in vitro susceptibility testing in Colombia. Sentinel laboratories were set up in three malaria endemic areas. The enzyme linked immunosorbent assay-histidine rich protein 2 and schizont maturation methods were used to assess the susceptibility of fresh P. falciparum isolates to six antimalarial drugs. This study demonstrates that an antimalarial drug resistance surveillance network based on in vitro methods is feasible in the field with the participation of a research institute, local health institutions and universities. It could also serve as a model for a regional surveillance network. Preliminary susceptibility results showed widespread chloroquine resistance, which was consistent with previous reports for the Pacific region. However, high susceptibility to dihydroartemisinin and lumefantrine compounds, currently used for treatment in the country, was also reported. The implementation process identified critical points and opportunities for the improvement of network sustainability strategies.

Origin and dissemination across the Colombian Andes mountain range of sulfadoxine-pyrimethamine resistance in Plasmodium falciparum.

The therapeutic efficacy of sulfadoxine-pyrimethamine (SP) in treating uncomplicated Plasmodium falciparum malaria is unevenly distributed in Colombia. The Andes mountain range separates regions in the west where malaria is endemic from those in the east and constitutes a barrier against gene flow and the dispersal of parasite populations. The distribution of dhfr and dhps genotypes of 146 P. falciparum samples from the eastern Amazon and Orinoco basins and Northwest and Southwest Pacific regions of Colombia was consistent with the documented levels of therapeutic efficacy of SP. The diversity of four dhfr- and dhps-linked microsatellites indicated that double- and triple-mutant alleles for both resistance loci have a single origin. Likewise, multilocus association genotypes, including two unlinked microsatellite loci, suggested that genetic exchanges between the eastern Orinoco and Northwest Pacific populations has taken place across the Andes, most probably via migration of infected people.

Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests.

BACKGROUND: Accurate diagnosis is essential for prompt and appropriate treatment of malaria. While rapid diagnostic tests (RDTs) offer great potential to improve malaria diagnosis, the sensitivity of RDTs has been reported to be highly variable. One possible factor contributing to variable test performance is the diversity of parasite antigens. This is of particular concern for Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-detecting RDTs since PfHRP2 has been reported to be highly variable in isolates of the Asia-Pacific region. METHODS: The pfhrp2 exon 2 fragment from 458 isolates of P. falciparum collected from 38 countries was amplified and sequenced. For a subset of 80 isolates, the exon 2 fragment of histidine-rich protein 3 (pfhrp3) was also amplified and sequenced. DNA sequence and statistical analysis of the variation observed in these genes was conducted. The potential impact of the pfhrp2 variation on RDT detection rates was examined by analysing the relationship between sequence characteristics of this gene and the results of the WHO product testing of malaria RDTs: Round 1 (2008), for 34 PfHRP2-detecting RDTs. RESULTS: Sequence analysis revealed extensive variations in the number and arrangement of various repeats encoded by the genes in parasite populations world-wide. However, no statistically robust correlation between gene structure and RDT detection rate for P. falciparum parasites at 200 parasites per microlitre was identified. CONCLUSIONS: The results suggest that despite extreme sequence variation, diversity of PfHRP2 does not appear to be a major cause of RDT sensitivity variation.

Evidence of Self-Medication with Chloroquine before Consultation for Malaria in the Southern Pacific Coast Region of Colombia.

Self-medication with antimalarial drugs is a major factor in the development of drug resistance, exerting subtherapeutic drug pressure on circulating parasite populations. Data on self-medication with antimalarials from the Southern Pacific coast region of Colombia, where 4-aminoquinolines resistance and political instability prevail, are vital to elimination strategies. We present results of an exploratory study of 254 individuals having malaria symptoms who sought malaria diagnosis in two hospitals in Tumaco, Department of Nariño, Colombia. Thirty-two percent (82/254) of participants had positive Saker-Solomons urine tests, indicating self-medication with chloroquine (CQ) before consultation for diagnosis. Notably, among 30 pregnant women participating in the study, 43% were Saker--Solomons positive. Molecular analysis of the K76T position encoded by the pfcrt gene revealed the mutant allele in all four samples that were both positive for Plasmodium falciparum and positive for the Saker-Solomons test, suggesting persistent CQ pressure. The high frequency of self-medication, particularly among pregnant women merits attention by public health authorities and comprehensive investigation.

In vitro susceptibility of P. falciparum populations from Colombia and Tanzania to a new synthetic peroxide (OZ277).

Sensitivity of Plasmodium falciparum populations from Colombia (N = 38) and Tanzania (N = 45) to the newly developed, fully synthetic peroxide OZ277 was investigated using a standard isotopic microtest. OZ277 showed excellent activity against chloroquine-resistant isolates in Colombia with median IC(50) [range] values of 2.5 ng/mL [0.34-8] (4.4 nM [0.6-14]) and Tanzania with 1.5 ng/mL [0.22-10] (2.65 nM [0.4-17.7]). The potency of OZ277 was similar to artesunate, showing median IC(50) values of 1.5 ng/mL [0.42-8.6] (3.8 nM [1.1-22.3]) and 1.8 ng/mL [0.2-10] (4.7 nM [0.5-26.04]) in Colombia and Tanzania, respectively. These results support the development of this new antimalarial compound.

Short report: polymorphisms in the pfcrt and pfmdr1 genes of Plasmodium falciparum and in vitro susceptibility to amodiaquine and desethylamodiaquine.

The potential role of polymorphisms in the pfcrt and pfmdr1 genes and in vitro susceptibility to amodiaquine and desethylamodiaquine were explored in 15 chloroquine-resistant Colombian Plasmodium falciparum isolates. Single nucleotide polymorphisms in the pfcrt gene, including a newly reported mutation (S334N), were seen in isolates with decreased susceptibility to amodiaquine and desethylamodiaquine. The lowest susceptibility found to amodiaquine was observed in an isolate carrying a pfcrt and pfmdr1 Dd2-like haplotype, whereas a pfcrt haplotype related to the 7G8 Brazilian strain was found in a Colombian isolate with the lowest susceptibility to desethylamodiaquine. This exploratory study suggests that polymorphisms in the pfcrt and pfmdr1 genes play a role in amodiaquine and desethylamodiaquine resistance and warrants further study.

Polymorphisms in cg2 and pfcrt genes and resistance to chloroquine and other antimalarials in vitro in Plasmodium falciparum isolates from Colombia.

Polymorphisms in Plasmodium falciparum cg2 and pfcrt genes and their association with chloroquine resistance in vitro in Colombian parasites were evaluated in this study. Association of chloroquine resistance with resistance to other antimalarial drugs in vitro was also examined. Polymerase chain reactions (PCR) for kappa and omega cg2 regions and nested PCR and digestion with ApoI enzyme for K-76T pfcrt point mutation defined corresponding polymorphisms in 83 samples collected between 1995 and 1999. The isotopic microtest was used to evaluate sensitivity in vitro in a subgroup of 18 isolates. The predominant cg2 pattern observed was 13K/14omega repeats (46/83 [55.4%]) and all samples presented the K-76T mutant allele. Seventy-eight percent of samples were resistant to chloroquine in vitro, 35.3% to amodiaquine, 16.7% to mefloquine, and 5.6% to quinine. Significant correlations (P < 0.05) were observed between the IC50s of chloroquine and arteether, and among IC50s of arteether, mefloquine, and quinine. These results suggest the development of multiple and cross-resistance of Colombian P. falciparum isolates to second- and third-line antimalarials and new alternative drugs.

Single nucleotide polymorphisms in Plasmodium falciparum V type H(+) pyrophosphatase gene (pfvp2) and their associations with pfcrt and pfmdr1 polymorphisms.

BACKGROUND: Chloroquine resistance in Plasmodium falciparum malaria has been associated with pfcrt 76T (chloroquine resistance transporter gene) and pfmdr1 86Y (multidrug resistance gene 1) alleles. Pfcrt 76T enables transport of protonated chloroquine out of the parasites digestive vacuole resulting in a loss of hydrogen ions (H(+)). V type H(+) pyrophosphatase (PfVP2) is thought to pump H(+) into the digestive vacuole. This study aimed to describe the geographic distribution of single nucleotide polymorphisms in pfvp2 and their possible associations with pfcrt and pfmdr1 polymorphisms. METHODS: Blood samples from 384 patients collected (1981-2009) in Honduras (n=35), Colombia (n=50), Liberia (n=50), Guinea Bissau (n=50), Tanzania (n=50), Iran (n=50), Thailand (n=49) and Vanuatu (n=50) were analysed. The pfcrt 72-76 haplotype, pfmdr1 copy numbers, pfmdr1 N86Y and pfvp2 V405I, K582R and P711S alleles were identified using PCR based methods. RESULTS: Pfvp2 was amplified in 344 samples. The pfvp2 allele proportions were V405 (97%), 405I (3%), K582 (99%), 582R (1%), P711 (97%) and 711S (3%). The number of patients with any of pfvp2 405I, 582R and/or 711S were as follows: Honduras (2/30), Colombia (0/46), Liberia (7/48), Guinea-Bissau (4/50), Tanzania (3/48), Iran (3/50), Thailand (1/49) and Vanuatu (0/31). The alleles were most common in Liberia (P=0.01) and Liberia+Guinea-Bissau (P=0.01). The VKP haplotype was found in 189/194 (97%) and 131/145 (90%) samples harbouring pfcrt 76T and pfcrt K76 respectively (P=0.007). CONCLUSIONS: The VKP haplotype was dominant. Most pfvp2 405I, 582R and 711S SNPs were seen where CQ resistance was not highly prevalent at the time of blood sampling possibly due to greater genetic variation prior to the bottle neck event of spreading CQ resistance. The association between the pfvp2 VKP haplotype and pfcrt 76T, which may indicate that pfvp2 is involved in CQ resistance, should therefore be interpreted with caution.

Large variation in detection of histidine-rich protein 2 in Plasmodium falciparum isolates from Colombia.

Most rapid diagnostic tests (RDTs) available use histidine-rich protein 2 (HRP2) as a target. However, it has been reported that sequence variations of this protein affects its sensitivity. Currently, there is insufficient evidence for HRP2 variability in Plasmodium falciparum isolates from Colombia and its relationship with RDT performance. To determine possible geographic differences and their effects on the performance of RDTs, 22 blood samples from patients with P. falciparum malaria from Tumaco and Buenaventura, Colombia were assessed by measurement of HRP2 concentration by an HRP2 enzyme-linked immunosorbent assay, RDTs, and thick blood smear. Statistical analysis showed an association between RDT performance and HRP2 concentrations. No significant difference was found between locations. A large variation of antigen concentration in samples was found at same parasitemia. In contrast to previously reports, there was no correlation between initial parasitemia and HRP2 concentration. Our results indicate that antigen quantity should be studied more carefully because the sensitivity of the RDT is affected more by antigen concentration than by parasitemia.

Susceptibility of Colombian Plasmodium falciparum isolates to 4-aminoquinolines and the definition of amodiaquine resistance in vitro.

There are wide variations in the threshold used to define in vitro resistance of Plasmodium falciparum to amodiaquine (AQ), probably due to differences in methodology and interpretation. In vitro susceptibility data of Colombian P. falciparum strains to AQ and N-desethylamodiaquine is used to illustrate the need to standardized methodologies and compare inhibitory concentrations, instead of resistant/susceptible phenotypes, when studying the mechanisms of resistance to AQ and monitoring drug susceptibility trends in the field.

Sentinel network for monitoring in vitro susceptibility of Plasmodium falciparum to antimalarial drugs in Colombia: a proof of concept

Drug resistance is one of the principal obstacles blocking worldwide malaria control. In Colombia, malaria remains a major public health concern and drug-resistant parasites have been reported. In vitro drug susceptibility assays are a useful tool for monitoring the emergence and spread of drug-resistant Plasmodium falciparum. The present study was conducted as a proof of concept for an antimalarial drug resistance surveillance network based on in vitro susceptibility testing in Colombia. Sentinel laboratories were set up in three malaria endemic areas. The enzyme linked immunosorbent assay-histidine rich protein 2 and schizont maturation methods were used to assess the susceptibility of fresh P. falciparum isolates to six antimalarial drugs. This study demonstrates that an antimalarial drug resistance surveillance network based on in vitro methods is feasible in the field with the participation of a research institute, local health institutions and universities. It could also serve as a model for a regional surveillance network. Preliminary susceptibility results showed widespread chloroquine resistance, which was consistent with previous reports for the Pacific region. However, high susceptibility to dihydroartemisinin and lumefantrine compounds, currently used for treatment in the country, was also reported. The implementation process identified critical points and opportunities for the improvement of network sustainability strategies.

Susceptibility of Colombian Plasmodium falciparum isolates to 4-aminoquinolines and the definition of amodiaquine resistance in vitro

There are wide variations in the threshold used to define in vitro resistance of Plasmodium falciparum to amodiaquine (AQ), probably due to differences in methodology and interpretation. In vitro susceptibility data of Colombian P. falciparum strains to AQ and N-desethylamodiaquine is used to illustrate the need to standardized methodologies and compare inhibitory concentrations, instead of resistant/susceptible phenotypes, when studying the mechanisms of resistance to AQ and monitoring drug susceptibility trends in the field.