Therapeutic efficacy of chloroquine and sequence variation inpfcrtgene among patients withfalciparummalaria in central India

Objectives To assess the therapeutic efficacy of chloroquine (CQ) treatment against uncomplicated Plasmodium falciparum infections in a tribal population of central India (Madhya Pradesh) and to investigate the prevalence of mutant P. falciparum chloroquine-resistant transporter (pfcrt) gene in the parasite population. Methods Clinical and parasitological response was determined by in-vivo testing. For molecular testing, the parasite DNA was extracted from blood samples and used to amplify and sequence parts of the pfcrt (44–177 codons), MSP1 (block 2) and MSP2 (central repeat region) genes. Results Of 463 patients presenting fever, 137 tested positive for P. falciparum. They were treated with CQ. Of these, 58% participated in the study. Overall, treatment failure occurred in 53% of participants. Children under 5 years of age showed significantly more CQ resistance than adults. Mutant genotype S72V73M74N75T76 was prevalent among both CQ responders (61.29%) and non-responders (66.7%). Interestingly, several patients from the CQ non-responder group (33.3%, n = 39) were harbouring parasite with wild type C72V73M74N75K76 genotype of the pfcrt gene. Microsatellite sequences downstream of exon 2 varied widely among both wild type and mutant pfcrt haplotypes. Conclusion The high rate of treatment failure in the present study clearly indicates the need to reassess the use of CQ as first-line antimalarial therapy in central India. This is supported by the presence of mutant pfcrt genotype among majority of the parasite population of the CQ non-responder group of patients. However, the presence of wild type amino acid at codon 76 of the pfcrt gene among several patients with CQ non-responders requires further investigations. Objectifs: Evaluer l’efficacité thérapeutique du traitement à la chloroquine (CQ) contre les infections non compliquées àPlasmodium falciparum dans une population tribale dans le centre de l’Inde (Madhya Pradesh) et étudier la prévalence des mutants P. falciparum du pfcrt, gène porteur de résistance à la CQ dans la population du parasite. Méthodes: La réponse clinique et parasitologique a été déterminée par les tests in-vivo. Pour les tests moléculaires, l’ADN du parasite a été extrait à partir d’échantillons de sang et utilisé pour amplifier et séquencer des fragments des gènes pfcrt (44 à 177 codons), MSP1 (bloc 2) et MSP2 (région répétitive centrale). Résultats: 137 des 463 cas de fièvre testés se sont avérés positifs pour P. falciparum. Ils ont été traités avec la CQ. Parmi eux, 58% ont participéà l’étude. L’échec du traitement global a été enregistré chez 53% des participants. La résistance à la CQ chez les enfants de moins de 5 ans était beaucoup plus importante que chez les adultes. Le génotype mutant S72V73M74N75T76était répandu tant chez les répondants à la CQ (61.29%) que chez les non-répondants (66,7%). De façon intéressante, plusieurs patients non-répondants (33,3%, n = 39) portaient des parasites de génotype sauvage C72V73M74N75K76 du pfcrt. Les séquences de microsatellites en aval de l’exon 2 variaient largement autant dans les haplotypes pfcrt de type sauvage et mutant. Conclusion: Le taux élevé d’échec du traitement dans la présente étude indique clairement la nécessité de réévaluer l’utilisation de la CQ en tant que traitement de première intention de la malaria en Inde centrale. Cette hypothèse est étayée par la présence du génotype mutant pfcrt dans la majorité de la population du parasite chez les patients non répondants CQ. Toutefois, la présence d’acides aminés de type sauvage au niveau du codon 76 du gène pfcrt chez plusieurs cas de non répondants CQ nécessite des investigations supplémentaires. Objetivos: Evaluar la eficacia terapéutica del tratamiento con cloroquina (CQ) en casos de infección no complicada de Plasmodium falciparum en una población tribal de la India Central (Madhya Pradesh) e investigar la prevalencia del gen pfcrt entre la población de parásitos. Métodos: Respuesta clínica y parasitológica determinada mediante pruebas in-vivo. Para las pruebas moleculares se extrajo el ADN del parásito a partir de muestras de sangre y se utilizó para amplificar y secuenciar partes de los genes pfcrt (codones 44-177), MSP1 (bloque 2), y MSP2 (región central repetitiva). Resultados: 137 de 463 casos de fiebre dieron positivos para P. falciparum y fueron tratados con CQ. De estos, 58% participaron en el estudio. El fallo terapéutico ocurrió en un 53% de los participantes. Los niños menores de 5 años mostraron una mayor resistencia a la CQ que los adultos. El genotipo mutante S72V73M74N75T76 era prevalente tanto entre los respondedores a CQ (61.29%) como entre los no-respondedores (66.7%). Varios pacientes del grupo que no respondió a la CQ (33.3%, n=39) tenían parásitos con el genotipo salvaje C72V73M74N75K76 del gen pfcrt. Las secuencias de microsatélites corriente abajo del Exón 2 variaron ampliamente tanto entre el genotipo salvaje como en los haplotipos pfcrt mutantes. Conclusión: La alta tasa de fallo terapéutico en este estudio indica claramente que es necesario reevaluar el uso de CQ como primera línea de tratamiento antimalárico en India central. Este hecho se ve apoyado por la presencia del genotipo pfcrt mutante entre la mayoría de la población parasitaria del grupo de pacientes que no respondieron al tratamiento con CQ. Sin embargo, la presencia del amino ácido salvaje en el codón 76 del gen pfcrt entre varios casos de no-respondedores a la CQ requiere ser investigado más a fondo. Malaria is the major public health problem in Madhya Pradesh, central India (Figure 1). Dindori district (population 713 126 in 2005) contributes 12% of patients with malaria in the state of Madhya Pradesh, although its population is only 1% of the state’s population (Annual Report, State VBDCP 2004). Both Plasmodium falciparum and Plasmodium vivax are common and prevalent in all age groups. Bajag Primary Health Centre (PHC), where this study was carried out, is the most malarious PHC in the state. Transmission is perennial with preponderance of P. falciparum. This PHC contributes about 40% of patients with malaria in the district, while its population is only 12% of district population. Two efficient vectors, Anopheles culicifacies and Anopheles fluviatilis, prevail throughout the year. Both the vectors transmit both P. falciparum and P. vivax. The sporozoite rate of An. culicifacies is 1.2% and that of An. fluviatilis is 0.5%. The villages are very remote and inaccessible for 2–3 months during rainy seasons. Early detection and prompt treatment of malaria with effective drugs is the key for effective control. Map showing district Dindori, Madhya Pradesh, central India. Chloroquine (CQ) is still the drug of choice in most part of Madhya Pradesh. However, an accurate understanding of the frequency of CQ treatment failure is needed for effective implementation of treatment policy. Plasmodium falciparum resistance to CQ has contributed to increasing rates of morbidity and mortality from malaria in Madhya Pradesh (Singh et al. 2004). Numerous factors contribute to the spread and intensification of drug resistance, of which drug pressure is considered one of the most important (Bloland 2001). Tracking the spread of drug-resistant malaria is a major challenge for the global control of the disease (Mayor et al. 1998, 2001). This requires regular surveillance at field level using molecular markers such as pfcrt. Mutations in this transporter gene, located on chromosome 7, have been linked to the CQ resistance (Fidock et al. 2000). Several studies have shown the mutant pfcrt gene among field isolates whose in-vivo and in vitro CQ resistance had been established (Cox-Singh et al. 1995;Basco & Ringwald 2001;Chen et al. 2001; Djimdéet al. 2001; Mittra et al. 2006). In-vivo evaluation of the efficacy of antimalarial drugs is based on clinical and parasitological responses after treatment (WHO 1996). Recurrence of parasitaemia within 28 days of aminoquinoline treatment is considered a recrudescent infection in most malaria-endemic areas (WHO 1973). However, infections recurring between day 14 and 28 in areas of high transmission could be because of newly acquired infections. Therefore, in vivo clinical efficacy studies of antimalarial drugs should not only include clinical and parasitological responses, but also use genetic polymorphism analysis to distinguish between recrudescent infections and reinfections and thereby aid in the identification of true treatment failures. We assessed the epidemiology of CQ treatment failure in patients with P. falciparum infection in a remote community to investigate pfcrt mutations in the parasite population. This study was conducted in four villages of the Bajag PHC of Dindori district, Madhya Pradesh, India, from July 2005 through November 2005, where almost all of the inhabitants belong to the Baiga and Gond tribe. Patients aged 1–59 presenting with fever and symptoms of P. falciparum malaria were screened for malaria parasites after obtaining written consent. Fever history was obtained from the patient or a guardian in the case of children. The patients were examined by a physician and their axillary temperature was recorded. Thick and thin blood films were prepared from finger prick blood for Plasmodium species identification. Only P. falciparum-infected patients who were willing to participate and who fit the enrolment criteria were included in this study as per WHO protocol (WHO 1996). The enrolment criteria were (i) fever or a history of fever in the preceding 24–48 h; (ii) parasitaemia of 1000–50 000 asexual forms per μl of blood; (iii) no history of antimalarial drug ingestion in the preceding 2 weeks of presentation. Patients who could not be included in the study were also given a full course of treatment. Each patient was treated with CQ orally (25 mg/kg of body weight/day) for 3 days. Clinical observations were recorded daily for the first 8 days (0–7 days) and during follow-up on days 14, 21 and 28. Thick and thin blood films were prepared from each patient on days 0, 2, 3, 7, 14, 21 and 28, stained with Jaswant Singh & Bhattacharya stain (Singh & Bhattacharyaji 1944) and examined by light microscopy to monitor the parasitological response to the CQ treatment. Parasitaemia in thick films was estimated by counting the number of asexual forms of P. falciparum corresponding to 200 leucocytes. The parasite density was calculated by assuming a leucocyte count of 8000/μl in blood (WHO 1991). Treatment outcomes i.e. early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF) and adequate clinical and parasitological response were determined according to WHO protocol (WHO 1996). All patients who failed to respond to CQ, as defined below, were treated with sulfadoxine-pyrimethamine (SP), and patients failed to SP were given mefloquine. Two to three drops of finger prick blood samples were also collected on 3-mm filter paper (Whatman International Ltd., Maidstone, UK) at each of the same time points (i.e. days 0.2, 3, 7, 14, 21 and 28) for extraction of parasite DNA. The parasite DNA was extracted from blood samples collected on filter paper using the Tris–EDTA buffer-based method as described previously (Bereczky et al. 2005). The extracted DNA was used for PCR amplification of the region spanning codons 44–177 of pfcrt. A primary PCR was set up for amplification of a 1.6-kb fragment of pfcrt using the primers PFCF (forward): 5′-CCGTTAATAATAAATACAGGCAG-3′ and PFCR (reverse): 5′-CTTTTAAAAATGGAAGGGTGTATAC-3′. The primary PCR product was diluted 1:10 and was used for nested PCR amplification of a 582-bp secondary product. The primers used for the nested PCR were Pf72 5′-TGTGCTCATGTGTTTAAACTTAT-3′ and Pr72 5′-AAAATAGTATACTTACCTATATCT-3′. The primary PCR was performed in a volume of 20 μl with 0.2 U of Taq DNA polymerase, 0.2 mm each dNTP, 1 μm each primer and 1.5 mm MgCl2. The reaction was allowed to proceed for 40 cycles after an initial denaturation at 94 °C for 30 s, annealing at 56 °C for 1 min and extension at 60 °C for 90 s. Final extension was at 60 °C for 3 min. The nested PCR for codons 44–177 was performed with a final MgCl2 concentration of 1 mm and annealing at 52 °C for 25 cycles. Other nested PCR conditions were the same as those described for the primary PCR. The PCR products were resolved on a 2% agarose gel. The primary PCR was set up for the amplification of block 2 region by using the primers MSP1A (forward): 5′-CACAATGTGTAACACATGAAAG-3′ and MSP1B (reverse): 5′-AGTACGTCTAATTCATTTGCAC-3′. The 646-bp primary PCR product was diluted 1:10 and was used for the nested PCR. A nested PCR of a 555-bp product was amplified by using the primers MSP1C (forward): 5′-TAGAAGCTTTAGAAGATGCAG-3′ and MSP1 D (reverse): 5′-GACAATAATCATTAGCACATAC-3′ and sequenced. The primary PCR was performed in a volume of 20 μl with 0.175 U of Taq DNA polymerase, 0.2 mm each dNTP, 0.4 μm each primer and 1 mm MgCl2. The reaction was allowed to proceed for 35 cycles after an initial denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 1 min. Final extension was at 72 °C for 10 min. The nested PCR was performed with annealing at 53 °C for 25 cycles. Other nested PCR conditions were the same as those described for the primary PCR. The PCR products were resolved on a 2% agarose gel. The primary PCR was set up for the amplification of the central repeat region of MSP2 by using the primers MSP2A (forward): 5′-ATGAAGGTAATTAAAACATTGTC-3′ and MSP2B (reverse): 5′-TTATTGAAGCAATATTACTAGAG-3′. The 760-bp primary PCR product was diluted 1:10 and was used for the nested PCR. A nested PCR of a 634-bp product was amplified by using primers MSP2C (forward): 5′-AGCAACACATTCATAAACAATG-3′ and MSP2 D (reverse): 5′-CACAGTTTTCTTTGTTACCATC-3′ and sequenced. The primary PCR was performed in a volume of 20 μl with 0.175 U of Taq DNA polymerase, 0.2 mm each dNTP, 0.4 μm each primer and 1 mm MgCl2. The reaction was allowed to proceed for 35 cycles after an initial denaturation at 94 °C for 30 s, annealing at 53 °C for 45 s and extension at 72 °C for 1 min. Final extension was at 72 °C for 15 min. The nested PCR was performed with annealing at 54 °C for 25 cycles. Other nested PCR conditions were the same as those described for the primary PCR. The PCR products were resolved on a 2% agarose gel. The PCR products were purified from the agarose gel by using HyYeld™ gel/PCR DNA extraction kit (Real Biotech Corp., Teipei Country, Taiwan), as per the manufacturer’s recommended protocol. From 200 to 250 ng of the gel-purified product was used with the ABI Big Dye Terminator Ready Reaction kit Version 3.1 (PE Applied Biosystems, Foster City, CA, USA) for the sequencing PCR. The sequencing PCR of Pfcrt, MSP1 and MSP2 were performed in a volume of 20 μl with 1 μl of Terminator Ready Reaction Mix (TRR), 3.2 pmol of gene specific primers Pf72 (44–177 codon), MSP1C (555 bp of block 2 region) and MSP2C (634 bp of central repeat region) and 0.5× sequencing buffer. Cycling conditions for the sequencing PCR included 25 cycles of denaturation at 96 °C for 10 s, annealing at 50 °C for 5 s and extension at 60 °C for 4 min. Templates were purified and sequenced on an ABI Prism 310 Genetic Analyzer (PE Applied Biosystems). Sequence obtained was translated using the Edit Sequence tool (DNASTAR). The translated sequences were then aligned using the MEGALIGN program (DNASTAR, Inc., Madison, WI). The study was approved by the Scientific Advisory Committee, Ethical Committee of Regional Medical Research Centre for Tribals Jabalpur, MP, India, and informed consent and human subjects guidelines were followed. We screened 463 patients presenting with fever, of tested positive for malaria P. P. and three infections of both P. falciparum and P. Of the 137 P. patients the enrolment criteria for in the study. are described in The therapeutic was determined for of the patients Two patients not the study the study and the at Treatment failed in of patients 20 patients with patients with and 15 patients with the subjects to CQ 1). Treatment failed more in under 5 years of age than in of the treatment in under 5 years of age were this only of treatment in The DNA from blood samples of all patients patients who not the collected on day was PCR amplified and sequenced for the pfcrt gene. samples from CQ responders and from CQ non-responder not the amplification of the gene. The wild type was present in of of isolates in both CQ responders n = and non-responders n = were mutant pfcrt in the parasite population where S72V73M74N75T76 was prevalent n = was present in = patients. Interestingly, the mutant genotype S72V73M74N75T76 was prevalent in both CQ responders n = and non-responders n = 39) the mutant genotype was recorded in isolates from CQ responders that the mutant genotype S72V73M74N75T76 of the pfcrt gene is widely spread in P. falciparum isolates in central India. However, the S72V73M74N75T76 genotype was more prevalent in n = and n = was true for patients where wild type C72V73M74N75K76 was prevalent n = To late treatment or DNA samples from patients in the and were sequenced and for the MSP1 and MSP2 genes. the isolates from all patients showed MSP1 and MSP2 at the time of and at treatment failure in the of isolates were to the same in samples for both the genes MSP1 and only a patient had isolates in which the MSP1 at the time of treatment failure no were also in the to the The that the majority of late treatment are because of recrudescent infection than with a of the in the downstream of exon 2 of the pfcrt gene a of 15 varied from 8 to among isolates or of isolates = the mutant the were and from 20 to in one which had and with 15 However, isolates with wild type C72V73M74N75K76 the in this where the number of varied from 8 to Clinical of the efficacy of CQ and used antimalarial drugs to the drug and malaria control However, molecular surveillance an that a drug its efficacy in the In-vivo response to drug treatment is on the level of acquired and Clinical of therapeutic failure not the presence of drug-resistant parasites because the or of parasitaemia be to of the such as or and (Bloland 2001). this parasitological and molecular were used to treatment response to CQ in P. falciparum-infected patients from central India. To CQ to be the first-line drug for the treatment of malaria in most part of India, in patients with P. falciparum. Clinical and parasitological of the patients high rates of CQ treatment failure in P. falciparum-infected which the in the of P. falciparum infection to CQ (Singh & et al. et al. et al. et al. Mittra et al. 2006). Overall, 53% of patients failed to respond to CQ of had parasitological to or the of clinical which to disease et al. 2005). The molecular that the majority of late treatment from of Parasitaemia in the of clinical as in is also important from a transmission of as such patients are to treatment and contribute significantly to the of the parasite The CQ efficacy is on the in Singh and only CQ resistance in which had to 53% in CQ treatment failure rate have been from all in et at the et al. et al. and et al. in The high rate of CQ treatment failure prevalence of in the where CQ has been used as first-line such the molecular markers in the CQ resistance need to be for public the key amino acid was to be a genetic with in-vivo resistance to CQ in a clinical study conducted in where malaria transmission is al. 2001). of parasite population with also with the prevalence of in-vivo CQ non-responders in the present is true that is with CQ resistance, this is not because a number of CQ responders were also to this and is prevalent among isolates et al. et al. 2004). This the of factors to therapeutic the of the which be to the parasite of its or not al. in the drug and rate of could the of CQ treatment. We several parasite isolates from the CQ non-responders with wild type pfcrt at studies have also wild type amino acid at of pfcrt among in parasite et al. This is and requires further investigations to mutations in pfcrt or in genes is to to CQ resistance et al. Overall, the in-vivo clinical resistance is in with the prevalence of drug-resistant mutations in the pfcrt gene. We are to the of Medical Research and for and are also to the patients who to participate in this study. The was by the of Medical Research