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Therapeutic efficacy of chloroquine and sequence variation in pfcrt gene among patients with falciparum malaria in central India

2009; Wiley; Linguagem: Inglês

10.1111/j.1365-3156.2009.02425.x

1365-3156

Praveen K. Bharti, Mohammad Tauqeer Alam, Robert Boxer, Man Mohan Shukla, Sant P. Gautam, Yagya D. Sharma, Neeru Singh,

Mosquito-borne diseases and control

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 whom, 153 tested positive for malaria (137 P. falciparum, 13 P. vivax, and three mixed infections of both P. falciparum and P. vivax). Of the 137 P. falciparum, 80 patients met the enrolment criteria for inclusion in the study. Their baseline characteristics are described in Table 1. The therapeutic outcome was determined for 78 of the 80 enrolled patients (97.5%). Two patients did not complete the study (one left the study area and the other declined participation at subsequent follow-up). Treatment failed in 41 of 78 patients (53%): 20 patients with ETF (26%), six patients with LCF (8%) and 15 patients with LPF (19%); the remaining 37 subjects (47%) responded clinically to CQ (Table 1). Treatment failed more frequently in children under 5 years of age 75% (12/16) than in adults, 38% (6/16) (Fisher’s exact test P < 0.053). Further, 75% (9/12) of the treatment failures in children under 5 years of age were ETFs, whereas this category only comprised 50% of treatment failures in adults (Table 2). The DNA from blood samples of all 80 enrolled patients (including two patients who did not complete the study) collected on day 0 was PCR amplified and sequenced for the pfcrt gene. Ten samples (eight from CQ responders and two from CQ non-responder group) did not show the amplification of the gene. The wild type genotype, C72V73M74N75K76, was present in 32.86% (23 of 70) of isolates in both CQ responders (32.26%, n = 31) and non-responders (33.33%, n = 39). There were two mutant pfcrt genotypes in the parasite population where S72V73M74N75T76 was highly prevalent (64.29%, n = 70); C72V73I74E75T76 was present in 2.9% (n = 70) patients. Interestingly, the mutant genotype S72V73M74N75T76 was prevalent in both CQ responders (61.29%, n = 31) and non-responders (66.67%, n = 39) (Table 3). Further, the mutant genotype C72V73I74E75T76 was recorded in isolates from CQ responders only. Together, these data suggest 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 LPF (76.92%, n = 13) and ETF (70%, n = 20), converse was true for LCF patients where wild type C72V73M74N75K76 was prevalent (66.67%, n = 6). To understand whether late treatment failures represented recrudescence or reinfection, DNA samples from patients in the LPF and LCF groups were sequenced and analysed successfully for the MSP1 and MSP2 genes. In the LCF group, isolates from all five patients analysed showed identical MSP1 and MSP2 alleles at the time of presentation and at treatment failure (Table 4). Similarly, in the LPF group, 12 of 13 isolates were found to contain the same allele in subsequent samples for both the genes MSP1 and MSP2, whereas only a single patient had isolates in which the MSP1 allele differed at the time of treatment failure (Table 4). Further, no variations were also observed in the microsatellite data to confirm the recrudescent. The data suggest that the majority of late treatment failures are likely because of recrudescent infection rather than reinfection with a new isolate. Analysis of the dinucleotide repeats (microsatellites) in the downstream of exon 2 of the pfcrt gene revealed a total of 15 alleles. AT repeats varied from 8 to 24 among these isolates. These isolates lack 13 or 17 AT repeats. Twenty percentage of isolates (n = 70) contained 24 AT repeats. In the mutant (S72V73M74N75T76) haplotype, the microsatellite variations were limited and ranged from 20 to 24 AT repeats except in one isolate which had nine AT repeats and another isolate with 15 AT repeats. However, isolates with wild type haplotype C72V73M74N75K76 contained the variation widespread in this region, where the number of AT repeats varied from 8 to 25. Clinical assessment of the efficacy of CQ and other commonly used antimalarial drugs provide useful data to guide the national drug policy and malaria control programme. However, molecular surveillance can give an advanced indication that a particular drug may lose its efficacy in the near future. In-vivo response to drug treatment is partly dependent on host factors, including the level of acquired immunity and pharmacokinetic variations. Clinical judgment of therapeutic failure does not necessarily indicate the presence of drug-resistant parasites because the persistence or recrudescence of parasitaemia may be attributable to causes independent of the parasites, such as poor compliance, unreported vomiting, inadequate absorption or biotransformation into biologically active metabolites and reinfection (Bloland 2001). In this study, clinical, parasitological and molecular techniques were used simultaneously to characterize treatment response to CQ in P. falciparum-infected patients from central India. To date, CQ continues to be the first-line drug for the treatment of malaria in most part of India, despite falling sensitivity reported in patients with P. falciparum. Clinical assessment and parasitological monitoring of the patients demonstrated high rates of CQ treatment failure in P. falciparum-infected patients, which confirmed the continuous decline in the sensitivity of P. falciparum infection to CQ (Singh & Shukla 1990;Rastogi et al. 1991; Satpathy et al. 1997; Biswas et al. 2003; Wijeyaratne et al. 2005; Mittra et al. 2006). Overall, 53% of patients failed to respond adequately to standard CQ treatment, of whom, 26% had ETF. Incomplete parasitological cure may lead to anaemia or the return of clinical illness which can progress to severe disease (Wijeyaratne et al. 2005). The molecular data suggest that the majority of late treatment failures resulted from LPF (15 of 21). Parasitaemia in the absence of clinical symptoms, as in LPF, is also important from a transmission point of view as such patients are unlikely to seek treatment and may contribute significantly to the maintenance of the parasite reservoir (Payne 1987). The CQ efficacy is on the decline in nearby districts: Singh and Shukla (1990) reported only 5.2% CQ resistance in 1990, which had increased to 53% in 2005. High CQ treatment failure rate have been reported from all over India: 18.3% in Orrisa (Satpathy et al.1997), 21% at the India–Nepal border (Wijeyaratne et al. 2005), 23.15% (Biswas et al. 2003) and 30.43% (Dua et al. 2003) in Assam. The high rate of CQ treatment failure may reflect increased prevalence of CQ-resistant strains in the settings where CQ has been used as first-line therapy. In such settings, the molecular markers involved in the CQ resistance need to be determined. More importantly, for public health, the key Lys76Thr amino acid substitution was suggested to be a genetic marker associated with in-vivo resistance to CQ in a clinical study conducted in Mali, where malaria transmission is seasonal (Djimdéet al. 2001). Prevalence of parasite population with K76T mutation also correlates well with the prevalence of in-vivo CQ non-responders in the present study.While it is true that K76T mutation is associated with CQ resistance, this mutation is not absolute, because a large number of CQ responders were also found to harbour this mutation, and it is highly prevalent among Indian isolates (Vinayak et al. 2003; Vathsala et al. 2004). This finding highlights the importance of other factors to therapeutic response, including the status of the host immune system which may be able to clear the parasite irrespective of its being CQ-resistant or not (Djimdéet al. 2003). Similarly, differences in the drug absorption and metabolic rate of individuals could affect the outcome of CQ treatment. We observed several parasite isolates from the CQ non-responders (33.33%) with wild type pfcrt at 72–76 codons. Earlier studies have also reported wild type amino acid at K76 position of pfcrt among in vitro-tested CQ-resistant parasite (Vinayak et al. 2003). This is intriguing and requires further investigations to find out whether other mutations in pfcrt or mutation in additional genes is involved to give rise to CQ resistance (Mu et al. 2003). Overall, the in-vivo clinical resistance data is in consistence with the prevalence of drug-resistant mutations in the pfcrt gene. We are grateful to the former Director General Dr N.K. Ganguly, Indian Council of Medical Research and Prof. A.P. Dash for constant encouragement and support. Thanks are also extended to the patients who consented to participate in this study. The work was funded by the Indian Council of Medical Research (ICMR) New Delhi.