Naman K. Shah

Pfmdr1 copy number and arteminisin derivatives combination therapy failure in falciparum malaria in Cambodia

BackgroundThe combination of artesunate and mefloquine was introduced as the national first-line treatment for Plasmodium falciparum malaria in Cambodia in 2000. However, recent clinical trials performed at the Thai-Cambodian border have pointed to the declining efficacy of both artesunate-mefloquine and artemether-lumefantrine. Since pfmdr1 modulates susceptibility to mefloquine and artemisinin derivatives, the aim of this study was to assess the link between pfmdr1 copy number, in vitro susceptibility to individual drugs and treatment failure to combination therapy.MethodsBlood samples were collected from P. falciparum-infected patients enrolled in two in vivo efficacy studies in north-western Cambodia: 135 patients were treated with artemether-lumefantrine (AL group) in Sampovloun in 2002 and 2003, and 140 patients with artesunate-mefloquine (AM group) in Sampovloun and Veal Veng in 2003 and 2004. At enrollment, the in vitro IC50 was tested and the strains were genotyped for pfmdr1 copy number by real-time PCR.ResultsThe pfmdr1 copy number was analysed for 115 isolates in the AM group, and for 109 isolates in the AL group. Parasites with increased pfmdr1 copy number had significantly reduced in vitro susceptibility to mefloquine, lumefantrine and artesunate. There was no association between pfmdr1 polymorphisms and in vitro susceptibilities. In the patients treated with AM, the mean pfmdr1 copy number was lower in subjects with adequate clinical and parasitological response compared to those who experienced late treatment failure (n = 112, p < 0.001). This was not observed in the patients treated with AL (n = 96, p = 0.364). The presence of three or more copies of pfmdr1 were associated with recrudescence in artesunate-mefloquine treated patients (hazard ratio (HR) = 7.80 [95%CI: 2.09–29.10], N = 115), p = 0.002) but not with recrudescence in artemether-lumefantrine treated patients (HR = 1.03 [95%CI: 0.24–4.44], N = 109, p = 0.969).ConclusionThis study shows that pfmdr1 copy number is a molecular marker of AM treatment failure in falciparum malaria on the Thai-Cambodian border. However, while it is associated with increased IC50 for lumefantrine, pfmdr1 copy number is not associated with AL treatment failure in the area, suggesting involvement of other molecular mechanisms in AL treatment failures in Cambodia.

Origin and evolution of sulfadoxine resistant Plasmodium falciparum.

The Thailand-Cambodia border is the epicenter for drug-resistant falciparum malaria. Previous studies have shown that chloroquine (CQ) and pyrimethamine resistance originated in this region and eventually spread to other Asian countries and Africa. However, there is a dearth in understanding the origin and evolution of dhps alleles associated with sulfadoxine resistance. The present study was designed to reveal the origin(s) of sulfadoxine resistance in Cambodia and its evolutionary relationship to African and South American dhps alleles. We sequenced 234 Cambodian Plasmodium falciparum isolates for the dhps codons S436A/F, A437G, K540E, A581G and A613S/T implicated in sulfadoxine resistance. We also genotyped 10 microsatellite loci around dhps to determine the genetic backgrounds of various alleles and compared them with the backgrounds of alleles prevalent in Africa and South America. In addition to previously known highly-resistant triple mutant dhps alleles SGEGA and AGEAA (codons 436, 437, 540, 581, 613 are sequentially indicated), a large proportion of the isolates (19.3%) contained a 540N mutation in association with 437G/581G yielding a previously unreported triple mutant allele, SGNGA. Microsatellite data strongly suggest the strength of selection was greater on triple mutant dhps alleles followed by the double and single mutants. We provide evidence for at least three independent origins for the double mutants, one each for the SGKGA, AGKAA and SGEAA alleles. Our data suggest that the triple mutant allele SGEGA and the novel allele SGNGA have common origin on the SGKGA background, whereas the AGEAA triple mutant was derived from AGKAA on multiple, albeit limited, genetic backgrounds. The SGEAA did not share haplotypes with any of the triple mutants. Comparative analysis of the microsatellite haplotypes flanking dhps alleles from Cambodia, Kenya, Cameroon and Venezuela revealed an independent origin of sulfadoxine resistant alleles in each of these regions.

Antimalarial drug resistance of Plasmodium falciparum in India: changes over time and space

After the launch of the National Malaria Control Programme in 1953, the number of malaria cases reported in India fell to an all-time low of 0·1 million in 1965. However, the initial success could not be maintained and a resurgence of malaria began in the late 1960s. Resistance of Plasmodium falciparum to chloroquine was first reported in 1973 and increases in antimalarial resistance, along with rapid urbanisation and labour migration, complicated the challenge that Indias large geographical area and population size already pose for malaria control. Although several institutions have done drug-resistance monitoring in India, a complete analysis of countrywide data across institutions does not exist. We did a systematic review of P falciparum malaria drug-efficacy studies in India to summarise drug-resistance data and describe changes over the past 30 years to inform future policy. Continued use of chloroquine for treatment of P falciparum malaria in India will likely be ineffective. Resistance to sulfa-pyrimethamine should be closely monitored to protect the effectiveness of treatment with artesunate plus sulfadoxine-pyrimethamine, which is the new first-line treatment for P falciparum malaria. Strategies to reduce the emergence and spread of future drug resistance need to be proactive and supported by intensive monitoring.

Multiple Genetic Backgrounds of the Amplified Plasmodium falciparum Multidrug Resistance (pfmdr1) Gene and Selective Sweep of 184F Mutation in Cambodia

BACKGROUND The emergence of artesunate-mefloquine (AS+MQ)-resistant Plasmodium falciparum in the Thailand-Cambodia region is a major concern for malaria control. Studies indicate that copy number increase and key alleles in the pfmdr1 gene are associated with AS+MQ resistance. In the present study, we investigated evidence for a selective sweep around pfmdr1 because of the spread of adaptive mutation and/or multiple copies of this gene in the P. falciparum population in Cambodia. METHODS We characterized 13 microsatellite loci flanking (+/-99 kb) pfmdr1 in 93 single-clone P. falciparum infections, of which 31 had multiple copies and 62 had a single copy of the pfmdr1 gene. RESULTS Genetic analysis revealed no difference in the mean (+/- standard deviation) expected heterozygosity (H(e)) at loci around single (0.75+/-0.03) and multiple (0.76+/-0.04) copies of pfmdr1. Evidence of genetic hitchhiking with the selective sweep of certain haplotypes was seen around mutant (184F) pfmdr1 allele, irrespective of the copy number. There was an overall reduction of 28% in mean H(e) (+/-SD) around mutant allele (0.56+/-0.05), compared with wild-type allele (0.84+/-0.02). Significant linkage disequilibrium was also observed between the loci flanking mutant pfmdr1 allele. CONCLUSION The 184F mutant allele is under selection, whereas amplification of pfmdr1 gene in this population occurs on multiple genetic backgrounds.

Molecular surveillance for multidrug-resistant Plasmodium falciparum, Cambodia

We conducted surveillance for multidrug-resistant Plasmodium falciparum in Cambodia during 2004–2006 by assessing molecular changes in pfmdr1. The high prevalence of isolates with multiple pfmdr1 copies found in western Cambodia near the Thai border, where artesunate–mefloquine therapy failures occur, contrasts with isolates from eastern Cambodia, where this combination therapy remains highly effective.

Changes in Childhood Diarrhea Incidence in Nicaragua Following 3 Years of Universal Infant Rotavirus Immunization

Background: Although the pentavalent rotavirus vaccine was highly efficacious against rotavirus diarrhea in clinical trials, the effectiveness of vaccine under field conditions in the developing world is unclear. In October 2006, Nicaragua became the first developing nation to implement universal infant immunization with the pentavalent rotavirus vaccine. To assess the effect of the immunization program, we examined the incidence of diarrhea episodes between 2003 and 2009 among children in the state of León, Nicaragua. Methods: We extracted data on diarrhea episodes from health ministry records. We used scaled Poisson regression models to estimate diarrhea incidence rate ratios for the period following the programs implementation to the period before implementation. Results: Following implementation of the immunization program, diarrhea episodes among infants were reduced (incidence rate ratios: 0.85, 95% confidence interval: 0.71–1.02) during the rotavirus season, but appear to have increased during other months. Conclusions: Although the immunization program appears effective in reducing diarrhea episodes during the rotavirus season, a large burden of diarrhea still persists during the remainder of the year.

Plasmodium falciparum Gametocyte Carriage Is Associated with Subsequent Plasmodium vivax Relapse after Treatment

Mixed P. falciparum/P. vivax infections are common in southeast Asia. When patients with P. falciparum malaria are treated and followed for several weeks, a significant proportion will develop P. vivax malaria. In a combined analysis of 243 patients recruited to two malaria treatment trials in western Cambodia, 20/43 (47%) of those with P. falciparum gametocytes on admission developed P. vivax malaria by Day 28 of follow-up. The presence of Pf gametocytes on an initial blood smear was associated with a 3.5-fold greater rate of vivax parasitemia post-treatment (IRR = 3.5, 95% CI 2.0–6.0, p<0.001). The increased rate of post-treatment P. vivax infection persisted when correlates of exposure and immunity such as a history of malaria, male gender, and age were controlled for (IRR = 3.0, 95% CI 1.9–4.7, p<0.001). Polymerase chain reaction (PCR) confirmed that only a low proportion of subjects (5/55 or 9.1%) who developed vivax during follow-up had detectable Pv parasites in the peripheral blood at baseline. Molecular detection of falciparum gametocytes by reverse transcriptase PCR in a subset of patients strengthened the observed association, while PCR detection of Pv parasitemia at follow-up was similar to microscopy results. These findings suggest that the majority of vivax infections arising after treatment of falciparum malaria originate from relapsing liver-stage parasites. In settings such as western Cambodia, the presence of both sexual and asexual forms of P. falciparum on blood smear at presentation with acute falciparum malaria serves as a marker for possible occult P. vivax coinfection and subsequent relapse. These patients may benefit from empiric treatment with an 8-aminoquinolone such as primaquine.

Risk factors for malaria deaths in Jalpaiguri district, West Bengal, India: evidence for further action

BackgroundIn 2006, a cluster of malaria deaths in the highly endemic Jalpaiguri district, West Bengal, India, led to assignment of additional resources. Malaria deaths decreased, but continued to occur. A study was conducted to identify the risk factors for residual malaria deaths.MethodsMalaria death was defined as a death from fever with microscopically confirmed Plasmodium falciparum among residents of Jalpaiguri during 2007–2008. For each case, three age-, sex- and locality-matched controls were recruited among microscopically confirmed falciparum malaria patients cured during the same period. Clinical and treatment information was abstracted from records. Information about knowledge about malaria, presence of bed nets and DDT spraying was collected through interviews of the close relatives of study subjects. Odds ratio (OR) were calculated using multivariate methods.Results51 malaria deaths were matched with 153 controls, which did not differ by age (median: 35 versus 36 years) and proportion of males (63% versus 63%). On multiple logistic regression analysis, compared with survivors, malaria deaths were more likely to have been admitted with already existing complications [OR = 4.1, 95% confidence interval (CI) = 1.6–10)], treated at a private facility (OR = 3.7, 95% CI = 1.2–12), received treatment after 48 hours of fever onset (OR = 14, 95% CI = 2.9–64), received chloroquine (OR = 13.3, 95% CI = 3.7–47). Households of the deceased were also more likely to miss bed nets (OR = 6.3, 95% CI = 1.9–24) and DDT spraying (OR = 9.2, 95% CI = 2.8–31).ConclusionElimination of malaria deaths will require education of providers for prompt referral before complications, engagement of the private sector, community awareness for early treatment as well as scaled-up use of bed nets use and DDT. Use of newer generation anti-malarials must to be generalized.

Research Options for Controlling Zoonotic Disease in India, 2010–2015

Background Zoonotic infections pose a significant public health challenge for low- and middle-income countries and have traditionally been a neglected area of research. The Roadmap to Combat Zoonoses in India (RCZI) initiative conducted an exercise to systematically identify and prioritize research options needed to control zoonoses in India. Methods and Findings Priority setting methods developed by the Child Health and Nutrition Research Initiative were adapted for the diversity of sectors, disciplines, diseases and populations relevant for zoonoses in India. A multidisciplinary group of experts identified priority zoonotic diseases and knowledge gaps and proposed research options to address key knowledge gaps within the next five years. Each option was scored using predefined criteria by another group of experts. The scores were weighted using relative ranks among the criteria based upon the feedback of a larger reference group. We categorized each research option by type of research, disease targeted, factorials, and level of collaboration required. We analysed the research options by tabulating them along these categories. Seventeen experts generated four universal research themes and 103 specific research options, the majority of which required a high to medium level of collaboration across sectors. Research options designated as pertaining to ‘social, political and economic’ factorials predominated and scored higher than options focussing on ecological, genetic and biological, or environmental factors. Research options related to ‘health policy and systems’ scored highest while those related to ‘research for development of new interventions’ scored the lowest. Conclusions We methodically identified research themes and specific research options incorporating perspectives of a diverse group of stakeholders. These outputs reflect the diverse nature of challenges posed by zoonoses and should be acceptable across diseases, disciplines, and sectors. The identified research options capture the need for ‘actionable research’ for advancing the prevention and control of zoonoses in India.

Nonrandomized Controlled Trial of Artesunate plus Sulfadoxine-Pyrimethamine with or without Primaquine for Preventing Posttreatment Circulation of Plasmodium falciparum Gametocytes

ABSTRACT Artemisinin combination therapies eliminate immature Plasmodium falciparum gametocytes but not mature gametocytes, which may persist for up to 1 month posttreatment. A single dose of primaquine, which is inexpensive and effective against mature gametocytes, could be added to further reduce the potential for posttreatment parasite transmission. Currently, we have few data regarding the effectiveness or safety of doing so. We collected data from 21 therapeutic efficacy trials of the National Antimalarial Drug Resistance Monitoring System of India conducted during 2009 to 2010, wherein 9 sites used single-dose primaquine (0.75 mg/kg of body weight) administered on day 2 along with artesunate plus sulfadoxine-pyrimethamine (AS+SP) while 12 did not. We estimated the effect of primaquine on posttreatment gametocyte clearance and the total number of gametocyte-weeks as determined by microscopy. We compared the median area under the curve for gametocyte density and reported adverse events. One thousand three hundred thirty-five patients completed the antimalarial drug treatment. Adjusting for region, primaquine increased the rate of gametocyte clearance (hazard ratio, 1.9; 95% confidence interval [CI], 1.1 to 3.3), prevented 45% (95% CI, 19 to 62) of posttreatment gametocyte-weeks, and decreased the area under the gametocyte density curve over the 28-day follow-up compared to AS+SP alone (P value = 0.01). The results were robust to other adjustment sets, and the estimated effect of primaquine increased during sensitivity analysis on the measurement of exposure time. No serious adverse events were detected. In conclusion, the addition of primaquine to AS+SP was effective in reducing the posttreatment presence of P. falciparum gametocytes. Primaquine was well tolerated and could be administered along with an artemisinin combination therapy as the first-line therapy.