Malaria is a life-threatening disease and is widespread in the tropical and subtropical regions mainly around the equator. Malaria is a protozoan disease and transmitted through infected female anopheles mosquitoes. There are five Plasmodium species which can infect human beings namely Plasmodium falciparum (Pf), Plasmodium vivax (Pv), Plasmodium malariae (Pm), Plasmodium ovale (Po) and Plasmodium knowlesi (Pk). In India, Pf and Pv are equally contributing to the malaria burden in the country, but a gradual increase in Pf cases is observed since last five years. According to NVBDCP, around 0.88 million malaria cases were reported in India and amongst them 0.46 million are Pf cases as it is more virulent (NVBDCP 2013). The highest number of malaria cases reported in India were from Odisha (25.6%) followed by Chhattisgarh (13.3%), Jharkhand (11.6%), Madhya Pradesh (8.7%), Gujarat (6.7%), Maharashtra (5.2%), other states (14.3%) and North Eastern states, which contributes 8.3% malaria cases in the country (NVBDCP-2013).
In the past, chloroquine (CQ) was effective for treating nearly all malaria cases. However, CQ resistance of Pf was first reported in Assam, India in 1973 (Sehgal et al.1973) and number of studies until 1977 indicated widespread presence of CQ resistance Pf in Assam, Arunachal Pradesh, Mizoram and Nagaland. Since then drug resistance has been reported from several other parts of the country (Dua et al; 2003, Baruah et al; 2005, Valecha et al; 2009).
Epidemiological studies have also confirmed the association of CQ resistance with a mutation in the transporter gene pfcrt. The amino acid substitution at pfcrt codon 76 (K to T) have shown a determinant association with the resistant phenotype (Lopes et al.1993, Babiker et al. 2001). The transporter for CQ resistance is located in the membrane of the food vacuoles where CQ is suggested to act by binding to hematin, a toxic by-product from the digestion of hemoglobin, thereby preventing synthesis of non-toxic hemozoin (Fitch et al. 1998, Bray et al. 1998).
To overcome the problem of CQ drug resistance, sulphadoxine-pyrimethamine (SP) combination was recommended by the National Programme in the country (National antimalarial programme, 1982). SP acts by interfering with two enzymes in the biosynthesis of folate. Sulphadoxine(SDX) is analogous to p-amino benzoic acid and competitively inhibits dihydropteroate synthase (DHPS) while pyrimethamine (PYR) is a competitive inhibitor of dihydrofolate reductase (DHFR). The inhibition of these two key enzymes affects the synthesis of tetrahydrofolate, which is needed in the production of dTTP and amino acids methionine and glycine (Sibley et al. 2001). As a result the parasites get killed because of impaired synthesis of DNA and amino acids.
Regretfully, resistance to SP developed rapidly in Southeast Asia even before the wide use of the drug (Wangsrpchanalai et al. 2002). Several factors contributed to the fast development of resistance to SP, one of which is long elimination half life of 10 and 4 days for SDX and PYR respectively.
Use of antimalarial treatment for febrile episodes and self-treatment are common in high malaria-endemic areas (Nwanyanwu et al.1996, Mahomva et al. 1996). Irrational treatment practices by the clinicians and also self treatment with antimalarials have been reported in the past (Nsimba et al. 2005). Uncontrolled and unnecessary use of antimalarials may increase drug pressure on the parasites and encourage parasite resistance. A number of important questions concerning factors related to self-treatment, full dose and adherence to self-treatment and the role of self-treatment in malaria morbidity or mortality remain challenge (Mccombie 2002, Hodel et al. 2009). The World Health Organization (WHO) protocol for monitoring antimalarial drug efficacy also does not exclude patients with a history of previous antimalarial drug use or the presence of antimalarial drugs in the urine or blood (World Health Organization 2003). Earlier studies have also investigated self’reporting drug intake (Nwanyanwu et al.1996, Mahomva et al. 1996) and presence of residual antimalarial in biological samples (Hodel et al. 2009).
Based on earlier studies, a significant trend for higher frequencies of the resistance markers with increasing CQ concentrations was observed in Pf malaria i.e. prior use of CQ in enrolled patients (Ehrhardt et al. 2005). Pre-treatment of blood CQ concentration has an inverse relation with degree of Pf resistance to CQ (Quashie et al. 2005).
High pretreatment blood CQ concentration assists in eliminating CQ resistant strains of the parasites during drug treatment (Quashie et al. 2005). However, the scope of examining the impact of pre-hospital CQ and SDX on the resolution of malaria following treatment with antimalarials such as artemisinin based combination therapy, which is the first line of drug for the management of Pf malaria, still remains open. Keeping the above points in mind the following objectives were set for my research work:
‘ To monitor the residual antimalarial levels in malaria patients in high endemic districts in the country.
‘ To correlate the residual antimalarial levels with molecular marker of drug resistance for Chloroquine, Sulphadoxine and Pyrimethamine.
‘ To establish links between presence of residual antimalarials and therapeutic outcome, if any.
In this chapter deal in the aspect the existing knowledge about malarial life cycles, vectors, diagnosis s, treatment , distribution, mode of action antimalarials, antimalarial drug resistance and factor affecting to drug resistance with special emphasis on irrational use of antimalarial drug and its effect in the community have been review.
Malaria is a life-threatening disease and is widespread in the tropical and subtropical regions mainly around the equator. Malaria is a protozoan disease and transmitted through infected female anopheles mosquitoes. Malaria parasite is require two hosts to complete their life cycle; one is definitive host (Sexual cycle) in Anopheles mosquito and second intermediate host (Asexual cycle) in human. Haploid parasite adopts three different cellular strategies in the distinct phases of the complex life cycle. In the human, schizogony (Asexual reproduction) occurs and this schizogony is found as two types, one erythrocytic schizogony ‘ found in erythrocytes and second exo-erythrocytic schizogony ‘ found in other tissues (Liver).
There are 430 species of Anopheles mosquitoes, and out of these, 58 species are identifying in India. Seven of these have been known as the main malaria vectors in India, namely An. culicifacies, An. dirus, An. fluviatilis, An. minimus, An. Sundaicus, An. Stephensi and An. Philippinesis..
Epidemiology of malaria
Globally, an estimated 3.4 billion people are at risk of malaria. WHO estimates that 207 million cases of malaria occurred globally in 2012 and 627 000 deaths. Most cases deaths reported in Africa and under 5 years children were in most deaths (77%) (WHO malaria report 2013). In South East Asia (SEA) contributed only 3.9% malaria burden in globally but India is contributing alone more 50% reported malaria cases from SEA followed by Myanmar (24%) and Indonesia (22%).
Prompt and accurate diagnosis is critical to the effective management of malaria. Malaria diagnosis involves identifying malaria parasites or antigens/products in patient blood. Various diagnostic methods for malaria parasite identification includes clinical diagnosis, microscopy, QBC method, rapid diagnostic test kits, serological test, molecular methods like PCR, flow cytometry, LAMP, microarray, mass spectrometry.
AS+SP, is recommended for all uncomplicated Pf case in the county except North eastern (NE) states. In NE states, the combination of Artemether-lumefantrine (AL) is recommended).
According to WHO, resistance to antimalarial drugs can be defined as ‘the ability of a parasite strain to survive and/or to multiply despite the administration and absorption of a drug given in doses equal to, or higher than, those usually recommended, but within the limits of tolerance of the subject’ (WHO).
CQ resistance is associated with a decrease accumulation of CQ concentration in the food vacuole of parasites, which is the site of action for C (Fidock et al., 2000.). The substitution of lysine to threonine (K76T) at codon 76 of the pfcrt gene is associated with in vivo and in vitro CQ resistance in Africa, South America, and Southeast Asia (Anvikar et al., 2012; Garcia et al., 2004; Ojurongbe. et al., 2007).
SDX and PYR is inhibiting to the DHPS and DHFR enzymes, respectively, specific point mutations in Pfdhps & Pfdhfr gene encoding these enzymes lead to a lower binding affinity for sulphadoxine-pyrimethamine drugs. High frequency of mutations in codon S108N followed by codon C59R and double mutant (S108N+C59R) genotypes are prevalent in India (Mishra et al. 2012). Apart from this, mutations at codon 51 and 164 are also responsible for increasing the tolerance of parasite towards the drug (Lumb et al. 2009). Pf showed variable levels of resistant to sulphdoxine with sequence variation in dhps gene at codon S436 to A436, A437 to G437, K540 to E540, A581 to G581 and A613 to S/T613. High frequency of mutation in pfdhps gene was observed in Cor-Nicobar Island (Lumb et al. 2009). In Northeastern region of Indian, where witness of treatment failure in AS+ SP regimen, with high prevalence of mutations in dhps gene at codon 436, 437 and 540 (Mishra et al.,2014).
Factors influencing emergence and spread resistant parasite
Spread of drug resistance rapidly in area of high transmission intensity of malaria incidence because clonal multiplicity increased the level of sexual recombination. If more than one gene is required to encode drug resistance, then recombination slows the evolution of resistance by breaking apart the resistant gene combinations (Hastings & D’Alessandro, 2000). In malaria endemic areas therapeutic responses vary with age as young children have little or no immunity compared with older children and adults. Immunity is play important role to decreases the chance of an infection becoming patients and also better therapeutic responses for any level of resistance [Hastings & Watkins, 2005]. The mutation rate in parasites have influence of the frequency of emerging drug resistance, higher mutation rates facilitate a faster emergence of resistance. An increased mutation rate is advantageous for the adaptation to quickly changing environments [Hastings & Watkins, 2005]. Mutation was associated with drug resistance often impart a fitness cost, the selective advantage to biological cost arising from the altered function of the mutated protein.
Irrational use of antimalarials
In Pakisthan, 35.5% of the patients had negative slide for parasite but treated with antimalarial drugs, its irrational prescription of antimalarial drugs, without laboratory confirmation of malarial disease increase drug pressure in the community (Khan et al., 2012). Self-treatment was extremely common in Kenya, 60% patients treated at home with herbal remedies or medicines (Ruebush et al., 1999). Use of antimalarial treatment for febrile episodes and self-treatment are common in high malaria-endemic areas (Nwanyanwu et al.1996, Mahomva et al. 1996). Several factors were involved in the increase drug pressure in the community such Self intake of the drugs by patients (Jombo et al., 2011; Nsimba et al., 2005; Souares et al., 2009, Hodel et al.,2009& 2010), irrational treatment practices by the physician (Aborah et al., 2013; Khan et al., 2012), unawareness regarding the suitable antimalarial drug to be used for treating malaria, long acting antimalarials post treatment prophylaxis, Mass drug administration (Stepniewska &White, 2008). Increase drug pressure on the parasites as screening resistant parasite population and spread.
Materials and methods
Population screening and sample collection
This study was carried-out during the year 2011-2013 at Bilaspur district in Chhattisgarh (n=70), Betul district in Madhya Pradesh (n=80), Simdega district in Jharkhand (n=73) and Sundergarh district in Odisha (n=72). The patients with Pf mono-infection, fulfilling inclusion criteria (WHO criteria, 2009) were enrolled in the study. Written and oral consent was obtained from each enrolled participant. Finger prick blood samples were used for identification and counting parasite density on day 0. Before the initiating the antimalarial drug treatment, three hanging blood drops on 3Chr Whatman filter paper and 100”l blood on 31ET filter paper for analyzing dhps gene mutation and estimating residual antimalarial or SDX level on day 7 respectively, was collected from each enrolled patient. The collected dried blood spot (DBS) papers were placed in zipper pouch and kept in desiccator till analysis. The patients were treated with ACT (AS+SP) according to National Drug Policy on Malaria after blood collection. The study was approved from Institutional Ethics Committee, National Institute of Malaria Research (NIMR), New Delhi.
Baseline blood samples (day 0) collected from patients reporting no antimalarial intake prior to the study were screened for the presence of five antimalarial drugs such as CQ, SDX, PYR, QN and MQ using a modified HPLC method(Blessborn et al. 2010) . The level of partner drug (SDX) of AS+SP was also determined on day 7. Extraction of the standard drugs (CQ, QN, SDX, PYR and MQ; Sigma Aldrich, USA), blank whole blood spot (control sample) and each of the collected samples were carried out according to the protocol of Blessborn et al., 2010, with slight modification. This involved the use of multi-mode solid phase extraction column (M-M SPE, Biotage, USA) and elution of the samples by methanol:triethylamine (97:3 v/v) mixture. Eluates were dried under a gentle stream of air at 70”C and were then dissolved in 100 ”l of methanol:HCl (0.01 M) 10:90 v/v. Twenty microliter (20 ”l) of each of these standards and samples were injected into the HPLC system. HPLC was performed on a Hitachi gradient system equipped with binary pump (Model L-2100/2130) and multi wavelength UV detector (Model L-2420 UV-VIS). Analytes extracted from the M-M SPE column were analyzed using two different mobile phases (A) acetonitrile:ammonium formate (20 mM in 1% formic acid) (5:95 v/v) and (B) acetonitrile:ammonium formate (10 mM in 1% formic acid) (80:20 v/v) and were run according to previously described gradient program(Blessborn et al. 2010). The compounds were analyzed on a Tosoh ” 5 ”m C18 (150 mm ” 2 mm) column protected by a precolumn security guard C8 (8mm x2 mm) (Tosoh Bioscience, PA). The UV detector was monitored at 280nm. Data acquisition and quantification were performed using HystarTM and Data AnalysisTM (Bruker, Bremen, Germany).
Estimation of dose intake time for SDX
To estimate the probable timing of drug intake, we compared the whole blood concentrations of SDX at baseline (C0) and on Day 7 (C7) after a complete treatment with AS+SP for the same patients. Assuming a terminal elimination half-life (t”) of 7.2 days for SDX, an inter-individual variability of 30% and a similar dosage on pre-study exposure and during the study, a back-calculation was done to estimate the intake time of the drug before baseline sampling:
Intake time =ln(C7/C0).t1/2/ln(2)+7[days]
The variability on t” was used to estimate a 90% confidence interval around this intake time, considering plausible inter-individual variations in elimination rate (White et al. 1999). Similar calculation was not attempted for PYR because of its short half-life period (Hodel et al. 2009).
Isolation of genomic DNA
Parasites genomic DNA was extracted from clinical samples by using QIAamp DNA min kit (Qiagen, valencia, CA) according to the manufacturer’s protocol with slight modification. Pfdhfr and pfdhps gene products were amplified using earlier reported methods (Duarai singh et al., 1998) and then digested using restriction enzymes for analysing point mutations in Pfdhfr (codon 51, 59, 108 and 164), Pfdhps gene(436, 437, 540, 581 and 613) and pfcrt mutation analysis was done according to Vathsala et al 2004. Applied Biosystem thermocycler was used for all PCR amplification reactions. Digested PCR product (5-8 microlitre) was analysed on 1.5 % agarose gel containing ethidium bromide (0.5”g/ml) and 0.5X TBE running buffer (pH 8.0). Digested PCR products were visualized under UV transilluminator and digitally captured with the help of gel documentation system (Alpha Imager EP, USA). Molecular sizes of PCR fragments were calculated using gene tool (Alpha Inotech, version 22.214.171.124).
The study was approved by Scientific Advisory Committee and Research Advisory Committee of National Institute of Malaria Research (NIMR) and ethical clearance of by institution ethical committee.
All statistical analyses were done using the SPSS software version 14. Geometric mean of parasite densities at 95% confidence interval (CI) was calculated. Frequencies were compared using the X2 test. The differences were considered statistically significant at an error probability less than 0.05 (p<0.05).
Baseline demographic data
Baseline data such as Gender (Male/Female), Age (Geometric mean”SD), Parasitaemia/”l (mean”SD & range) and previously drug intakes information was captured in Case record form (CRF).
Out of 295 samples, 289 samples were processed for monitoring residual antimalarials levels. Out of 289 patient samples, 70 (24.2%) had residual antimalarials levels on day 0. Out of these patients, 25(31.6%) patients from Madhya Pradesh had highest residual antimalarials followed by 18(25.4%) patients from Jharkhand, 17(25%) patients from Chhattisgarh and 10(14.1%) patients from Odisha.
Levels of residual antimalarials
Out of 289 patients, the presence of antimalarial drug was detected in 70 (24.2%) patients: 49(17.0%) had mean concentration SDX of 10765.3ng/ml (100-54100ng/ml), 27(9.3%) of them showed mean concentration of CQ is 147.0 ng/ml(49-263ng/ml), 5(1.7%) had mean concentration of PYR is 980ng/ml(100-1600ng/ml), 4(1.4%) had mean concentration of QN is 184.5 ng/ml(100-279 ng/ml), while MQ was present in only 2 (0.7%) patients at mean concentration of 317ng/ml (267-367ng/ml).
Therapeutic level of SDX on day 7
The level of SDX was monitored on day 7, patients enrolled and treated with AS+SP, from Chhattisgarh (n=50), Madhya Pradesh (n=48), Jharkhand (n=58) and Odisha (n=63) found SDX concentration (range between 43.7 to 48.8 ”g/ml) is; 48.8”13.3, 45.8”18.2, and 43.7”18.8 and 45.5”12.9 ”g/ml blood, respectively.
Probable time of previous SDX intake
SDX concentration on day 0 and on day 7, the back estimation method indicated a means of 29 days prior to enrolment and drug administration in the study(range 5-69 days; 90% CI), the most likely time for previous SDX intake. Majority of the patients i.e. 23 (46.9%) showed previous SDX intake estimated time of more than 28 days.
Residual antimalarial in different age groups
The patient samples were divided in three groups on the basis of age viz. ‘ 6 month to <5yrs, ‘5 yrs to <15yrs and ’15yrs to observe the effect of irrational practices in different age groups. Since, 70 patients had residual antimalarials on day 0, the age wise residual antimalarial drugs were 8.6%, 31.4% and 60.0% respectively. indicating the maximum intake in adult patients.
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