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 188.8.131.52).
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.
CRF information & residual antimalarial
A total of 295 patients were analysed for the previous drug intakes using case record form (CRF) information; 21.1% patients did not took drug, 75.4% patients were not aware about previous intake and 3.5% of the patients were in category where information was not recorded. However, blood samples were analysed on day 0 by HPLC, 24.2 % patients had residual antimalarial.
Rarasite density with residual CQ:
Parasitemia was compared between patients had residual antimalarials (n=27) on day 0; 1056 to 78240 asexual parasites/”l (mean ”SD: 20394”19735 asexual parasites/”l) and with patients not having residual CQ on day 0 (n=262), that is, 616 to 99290 asexual parasites/”l (mean” SD: 23687”25835 asexual parasites/”l). Low level of residual CQ in those samples which had higher parasite density per microliter of blood and vice versa.
Parasite density with residual SDX:
Parasitemia was compared between patients had residual SDX in (n=49) on day 0; 1154 to 80238 asexual parasites/”l (mean ”SD: 16733”18077asexual parasites/”l, P<0.05**) and those with no SDX on day 0 (n=240), that is, 616 to 99290 asexual parasites/”l (mean” SD: 25023”26665asexual parasites/”l, P<0.05**). Low level of residual SDX in those samples which had higher parasite density per microliter of blood and vice versa
Molecular markers of drug resistance
Pfdhfr: To monitor the PYR drug resistance in collected 295 samples, out of these samples, 288 samples could be amplified for dhfr and dhps both gene. In dhfr gene, double mutation was observed highest frequency (71.9%) followed by single (9%) and triples (2.4%) mutation and 16.7% samples had wild type genotype.
Frequency of double mutation (59+108) was higher in Madhya Pradesh (97.5%) followed by Chhattisgarh (75.4%), Jharkhand (64.8%) and Odisha (46.5%) of the samples. Single mutation was seen 18.3%, 12.7%, 3% and 2.5% in Odisha, Jharkhand, Chhattisgarh and Madhya Pradesh samples, respectively. Triple mutation was observed in 9.1% isolate in Chhattisgarh and only 1.4% isolates from Jharkhand. While wild genotype was observed in highest frequency (35.2%) in Odisha followed by Jharkhand (21.1%) and Chhattisgarh isolates (12.1%).
Pfdhps:- To monitor the SDX drug resistance pattern in amplified of 288 samples, wild type dhps genotype was prevalent with highest frequency (51%) followed by dhps triple (26%), double (11.8%), & single (11.1%) mutation.
Frequency of wild type genotype was higher in Odisha (76.1%), followed by Chhattisgarh (74.2%), Madhya Pradesh (31.3%) and Jharkhand (26.8%). Triple mutation (436+437+540) in dhps gene was observed with frequency of 52.1%, 31.3%, 16.9% and 1.5% in the isolates collected from Jharkhand, Madhya Pradesh, Odisha and Chhattisgarh, respectively. However, double mutation was highly prevalent in Madhya Pradesh i.e. 30.0% as compared to the frequency of 7.6%, 4.2% and 2.8%in Chhattisgarh, Odisha and Jharkhand of isolates, respectively. Single mutation in pfdhps was found in the18.3%, 16.7%, 7.5% and 2.8%of the samples from Jharkhand, Chhattisgarh Madhya Pradesh and Odisha, respectively.
Mutation pattern in dhps gene in relation with residual SDX:
Mutation was analyzed in dhps gene in two groups; residual levels SDX (+ve) (n=45) and SDX (-ve) (n=235) on day 0. Mutation rate in dhps gene was higher in those patients had residual SDX (57.8%) as compared to without residual SDX (48.5%) concentration on day on day 0. Triple mutation in dhps was also higher (33.3%) in SDX (+) as compared to SDX (-) patients on day 0. Mutation in codon K540E was higher (57.8% )with presence of residual levels of SDX as compared to those patients not having residual SDX on day 0 (p value<0.05).
Frequency of pfcrt mutation in different geographical region
To monitor the CQ drug resistance pattern in 295 samples, out of these samples, 276 samples was amplified for pfcrt gene. Two hundred and ten (76.1%) samples had 76T mutation while 50(18.1%) samples showed wild genotype and only 16 (5.8%) samples showed mix type (K76 + 76T) pattern. Mutation in codon 76T was more prevalent in in samples from Madhya Pradesh (98.7%) followed by Jharkhand (84.1), Chhattisgarh (81.3%) and Odisha (36.4%).
Correlation between residual CQ and Pfcrt mutation
The mutation in the pfcrt gene in codon76 (Lysine to Tyrosine) was correlated with the patients who had or did not have residual CQ level. The frequency of 76T mutation was higher in those having residual CQ (96%) as compared to those not having residual CQ (74.6%).The samples which had residual levels of CQ more than minimum inhibitor concentration (MIC) showed 100% mutant genotype in pfcrt gene at codon K76T.
Follow-up and therapeutic outcome
Out of 295 enrolled patients, 271 patients completed follow-up till 28 or 42 days. All follow- up completed patients showed adequate clinical parasitological response (100% cure rate). Withdraw
Residual antimalarial on day 0 and therapeutic outcome
The effect of residual CQ on parasites persistence after patients treated with AS+ SP was analysed. It was observed that in parasitemia was clear within 48 hrs with residual antimalarial, while parasitemia were persisting ’48 hour in patients not residuals antimalarial on day 0.
To our knowledge this is the first study to investigate the presence of five antimalarials (CQ, SDX, PYR, QN and MQ) in blood of patients from Chhattisgarh, Madhya Pradesh, Jharkhand and Odisha with Pf malaria before the onset of treatment. Out of these five antimalarial drugs, CQ is currently recommended as first line antimalarial for uncomplicated P. vivax (Pv) malaria cases in the country, while SP is being recommended as partner drug in the recommended ACT for the treatment of uncomplicated Pf malaria throughout the country except in the north-eastern India. QN is recommended as rescue for case where treatment failure cases to ACT is observed while MQ is advised for long term chemoprophylaxis. The measurement of these five antimalarials allowed a comprehensive assessment of the circulating drugs in the community under study.
Malaria is a major public health problem in India and its dynamics vary from place to place. The forest and tribal areas of Madhya Pradesh, Chhattisgarh, Jharkhand & Odisha where malaria outbreaks are frequently recorded.
Both Pf and Pv are prevalent species in these states with a preponderance of Pf. Drug resistance is a major problem for control and eradication of malaria. Besides the genetic factors, drug pressure in the community also plays a major role in emergence of drug resistant parasites (Hodel et al. 2009). To assess the drug pressure, residual level of antimalarials was monitored by HPLC in the patients enrolled at selected study sites under therapeutic efficacy studies.
We found that out of 295 patients enrolled in our study, 70 (24.2%) carried blood residual antimalarials above the lower limit of detection (50 ng/ml) at inclusion (Hodel et al. 2010) This could be a worrying factor and a possible indication of tolerance to residues which could act as a precursor for the development precipitation of resistance. This is a matter of concern and the issue needs to be carefully monitored at state as well as national level.
In our study, we found residual level of SDX in blood of 49 (17%) patients on day 0 at a mean concentration of 10765.3ng/ml. This finding corroborates with the results obtained from the study in which residual plasma concentration of antimalarials was detected prior to the treatment (Hodel et al. 2009). PYR is usually given at a lower dose of 1.25 mg/kg in combination with SDX and its half-life is approximately 4 days; shorter than that of SDX which has a half-life of 7.2 days (Hodel et al. 2009; Weidekamm et al.1982 ). In present study, the mean concentration obtained for SDX was comparatively higher than the reported study; but there was no conspicuous difference in the concentration range of the residual antimalarials (Hodel et al. 2009). There are various factors which might be responsible for such differences the in mean concentration of residual antimalarials: (1) Differences in the Samples (dried blood spots) extraction procedure (2) Instrumentation technique used i.e. they have used liquid chromatography-tandem mass spectrometry which is highly sensitive as compared to HPLC used in the present study.
Based on the day 0 and day 7 SDX concentration (C0 and C7), and assuming that the patients with residual SDX in their blood had taken a single dose of SDX according to their body weight, we can infer by using back estimation method that these patients must have taken SP approximately within one month (29 mean days) prior to inclusion in our study. Furthermore, it is also possible that patients might have taken a sub-therapeutic dose or counterfeit form of SP more recently (Basco et al. 2004).
Although, CQ has been withdrawn to be treatment for Pv infection (Valecha et al; 2009 ) still first line treatment for the Pv case across the country; it was detected in the blood of 27 (9.3%) patients in our study at a mean concentration of 147 ng/ml. Hodel et al., 2010 also reported the residual plasma concentration of CQ, MQ and QN before initiating the antimalarials treatment in patients (Hodel et al., 2010). This might be due to easy availability of CQ in the private clinics and pharmacy shops or due to its wide use in the treatment for a previous episode of Pv malaria. Previous study from our laboratory has reported that CQ is the most common antimalarial sold across the counter with high frequency of prescription in public as well as private health facilities as compared to other antimalarials (Mishra et at. 2011)
As we employed dried blood spot method for sample collection, it was not possible to detect any artemisinin compounds. Also, short half-life of artemsinin requires sophisticated techniques as well as plasma sample collection. However, the presence of residual artemisinin cannot be ruled out as high prescription rate of this antimalarial has been previously observed in the country (Mishra et at., 2011).
Both MQ and QN are not recommended for uncomplicated malaria, but their residual levels were still found to be present in the patients, although in very few samples and that too at low concentrations.
By detecting the presence of residual anti-malarial levels on day 0, it can concluded that this be due to; (1) Self-medication due to non-availability of reachable drug to the dispensing facility or the practitioner leading to inadequate dosing and there by inability to control infection(Hodel et al. 2009; Hodel et al. 2010; Jombo et al. 2011; Nsimaba et al. 2005; Souares et al. 2009), (2) Irrational treatment practices by the physician at the study sites with high transmission intensity of malaria parasites, where all febrile patients were treated with a variety of available antimalarial drugs(Aborah et al. 2013 and Khan et al. 2012), (3) Unawareness regarding the suitable antimalarial drug to be used for treating malaria (4) Treatment of previous episode of infection with antimalarial drug after then re-infection with a new parasite resulting of previously consumed episode of drug as residue in blood (Quashie et al. 2005; Stepniewska and White 2008 ). These factors contribute to increased drug pressure on the parasite thus encouraging resistance in Plasmodium species (Jamison et al. 2006). With these observations, it can be deduced that the entry criteria based on self reporting of previous drug intake or information collected in case record forms are not reliable at least in this population.
Assuming that the patients with residual SDX in their whole blood had taken a single dose of SP according to body weight, most patients must have taken the drug, 1 month (median 29 days) prior blood withdrawal. Furthermore, it is also possible that patients might have taken a sub-therapeutic dose of SP more recently. Detection of a drug with very trace and long duration does not allow to determination of whether a patient had monotherapy or as part of an ACT or complete dose had been taken.
The influence of age and sex on the probability of residual antimalarials at entry showed no significant relationship in our study indicating uniform antimalarial prescription or intake behaviour in the population. These estimates are approximate, as indicated by the wide confidence interval explained by the fair degree of inter-individual variability and residual error.
Lower parasite density/”l was observed in patients having residual CQ and SDX as compared to those patients no residual CQ and SDX intakes. This clearly showed that the residual (CQ, SDX) levels of drug in blood were not enough to control parasite replication and to overcome clinical symptoms in the patients, although the parasitemia levels were little lower in the patients with residual CO/SDX than without because. antimalarials may screened out sensitive parasites and only resistant parasite population may survived with sub-therapeutic levels of SDX due to this; patients having residual levels of SDX showed lower parasite density as compared to without residual levels. Here it is difficult to comment whether the residual drug levels were due to the result of full or incomplete treatment and the parasites causing the current episode of infection were from the same or a new infection.
These regions are far apart from each other and show a diverse level of drug resistance and malaria transmission intensity viz. a worse level of drug resistance and malaria transmission (Hastings & Watkins, 2005).We were studied Pf dominate area, Odisha alone contribute 47.8 % of total Pf case reported in the country followed by 14.8% in Chhattisgarh, 6.3% in Jharkhand and Madhya Pradesh is contributing 6.0% of Pf cases (NVBDCP 2013).
Our data showed the higher frequency (71.9%) of double mutation at codon C59R and S108N in pfdhfr gene in study sites. Double mutation in the different geographical region and transmission zones are showed the divers frequency i.e Madhya Pradesh (97.5%) followed by Chhattisgarh (75.4%), Jharkhand (64.8%) and Odisha (46.5%).This data suggest that double mutations at codon 59R+108N is indicating that lower level of PYR drug resistance is existing in the Indian pollution. Triple mutation in the combination of N51I+C59R+S108N or C59R+S108N+I164L is indictor of high levels of PYR, our data showed that only 2.4% triple mutation population in the studies samples ( Basco at el.,1998&2000). The high transmission rate will lead to higher genetic variation. This would also lead to the emergence of more and more drug resistance genotypes and, and faster spread of PYR resistance in these states.
Resistance in SDX in P. falciparum is connected with mutations in pfdhps at codons; 436A/F, 437G, 540E, 581G, and 613S [Ahmad et ai.,2006]. The pfdhps mutation at codon A436 reduce the affinity in of SDX binding followed by sequential mutations at G437, E540, G581, and T/S613 which may cause increase in SDX resistance [Lumb et al.,2011], In dhps mutation we saw six variants, over wild type. In contrast, most of the P. falciparum isolates (41.2%) exposed a single haplotype (540E) for pfdhps gene followed by 437 and 436 and no mutation detected at codons 581 and 613.
Recently studies in Jalpaiguri, India has shown 9.5% failure rate in AS’WHO, and was due to SP failure because of mutations in pfdhfr(I51+R59+N108), with pfdhps(G437+E540)(Saha et al., 2012). Subsequently, high treatment failures rate( more than cutoff levels) in AS+SP regimen was observed in Northeastern states (Tripura, Mizoram, Arunachal Pradesh) of India because higher mutation frequency in dhfr and dhps genes, in 2013, artemether lumifentrine (AL) was used first line treatment of uncomplicated Pf case in this region (Mishra et al,2014, NVBDCP-2013).
Quintuple mutations in combination of dhfr-dhps gene including pfdhfr three codons (108 + 51 + 59) and two pfdhps codons (437G+ 540E) was a strong indicator of SP treatment failure (Ngoma at el.,2011), its combination was not seen our studies, it mean partner drug (SP) efficacy in this region working fine in the combination of AS+SP. Pre-indication of treatment failure was not there in this region.
The correlation study revealed higher mutation frequency (96%) in pfcrt at codon K76T in patients with residual levels of CQ on day 0 as compared to those patients not having residual levels of CQ before enrollment (74.6%). The samples with residual levels of CQ more than minimum inhibitory concentration (MIC) showed 100% mutant genotype in pfcrt gene at codon K76T. Wild genotype was not observed in this group because sub-therapeutic levels of CQ may screen out sensitive parasites and only mutant parasites population can survive. Due to mutation in pfcrt gene at codon K76T, CQ accumulation is decreased in food vacuoles of parasites, levels of CQ is not sufficient to inhibit hemozoin formation. In case of sensitive parasites, there is increased accumulation of CQ in food vacuoles, normal recommended dose is sufficient for interference of heme detoxification pathway resulting in free heme thereby produces free radicals, which damages food vacuoles membrane and other process in parasites and ultimate kill them. Continuous increase drug pressure of CQ on parasite may promote the emergence of drug resistant parasite population.
A total of 295 patients recruited in the present study, 273 patients were completed the 28 or 42days follow-up after treatment with AS+SP, 100% patients showed adequate clinical parasite response (ACPR). Prior drug intake may have an effect on the current treatment in many ways; it may lead to increased drug exposure from cumulative levels resulting either in better efficacy.
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