Comprehensive case management of malaria: Operational research informing policy.

In 2013, the Odisha state Vector Borne Disease Control Programme led a five year operational research project, under programmatic conditions, in close collaboration with several partners. This Comprehensive Case Management Project covered a population of 900,000 across paired control and intervention blocks in four districts, each with different transmission intensities. Key gaps in access to malaria services were identified through household surveys and a detailed situation analysis. The interventions included ensuring adequate stocks of rapid diagnostic tests and antimalarial drugs at the village level, the capacity building of health workers and ASHAs, setting up microscopy centres at the primary health care level, and conducting mass screening and treatment in poorly accessible areas. The programme strengthened the routine health system, and improved malaria surveillance as well as the access to and quality of care. Initially, the programme led to increased case reporting due to improved detection, followed by a decline in malaria incidence. Lessons from the project were then scaled up statewide in the form of a new initiative-Durgama Anchalare Malaria Nirakaran (DAMaN).

Small-scale (Phase II) evaluation of the efficacy and residual activity of SumiShield® 50 WG (clothianidin 50%, w/w) for indoor residual spraying in comparison to deltamethrin, bendiocarb and pirimiphos-methyl for malaria vector control in Karnataka state, India.

BACKGROUND & OBJECTIVES: There is an urgent need of introducing new insecticide molecules with novel modes of action to counter the ever growing insecticide-resistance in mosquito vectors. In the present study, a new insecticide molecule, SumiShield 50 WG (clothianidin 50%, w/w) was investigated for its efficacy as an indoor residual spray along with its residual action in comparison to deltamethrin, pirimiphos-methyl and bendiocarb. METHODS: The study area included three villages in Almatti Dam catchment area in Bagalkot district, Karnataka, India. Spraying was done using Hudson sprayers with the following dosages-Clothianidin, 300 mg AI/m2; deltamethrin, 25 mg AI/m2; bendiocarb, 400 mg AI/m2; and pirimiphos-methyl, 1 g AI/m2. Cone bioassays were conducted on cement and mud plastered surfaces at fortnightly intervals to assess the bioefficacy and residual activity. Mosquito densities in the sprayed houses were recorded at regular intervals for assessment of the insecticidal efficacy. Filter paper samples collected from the sprayed houses were analyzed for insecticide content sprayed on different wall surfaces at the Walloon Agricultural Research Institute, Gembloux, Belgium. RESULTS: Chemical content analysis of filter paper samples revealed that the applied to target ratios were in the acceptable range (1 + 0.5) for all the treatment types. Duration of persistence of effectiveness of bendiocarb (≥80% mortality in cone bioassays) was 19 to 21 wk on cement plastered surfaces and 15 to 19 wk on mud plastered surfaces. Duration of persistence of effectiveness of deltamethrin was 17 to 21 wk on both mud and cement plastered surfaces and that of pirimiphos-methyl was 15 to 19 wk. For SumiShield, it was 17 to 25 wk on both types of surfaces, indicating slow action of SumiShield. The densities of Anopheles culicifacies were lower in bendiocarb sprayed houses throughout the observation period, followed by pirimiphos methyl, deltamethrin and clothianidin sprayed houses. In case of other mosquitoes also, similar trend was observed. INTERPRETATION & CONCLUSION: Considering the persistence of effectiveness of SumiShield on sprayed surfaces, effectiveness in reducing the density of mosquitoes, operational feasibility, safety and community acceptance, the formulation of clothianidin is a better option for IRS for the control of insecticide-resistant mosquito vectors.

Village-scale (Phase III) evaluation of the efficacy and residual activity of SumiShield® 50 WG (Clothianidin 50%, w/w) for indoor spraying for the control of pyrethroid-resistant Anopheles culicifacies Giles in Karnataka state, India.

OBJECTIVE: There is an urgent need to test and incorporate new molecules with promising efficacy and novel mode of action to control insecticide-resistant mosquito vectors for disease control. We tested a new compound, clothianidin (SumiShield 50 WG), for its efficacy as an indoor residual spray (IRS) for the control of pyrethroid-resistant Anopheles culicifacies (Diptera: Culicidae) in comparison with pirimiphos methyl (Actellic CS) as a positive control. METHODS: Ten villages were selected, five each for IRS with clothianidin (300 mg AI/m2 ) and pirimiphos methyl (1000 mg AI/m2 ) in Almatti Dam catchment area in Karnataka state, India. Entomological parameters were monitored in these sprayed villages using standard methods. Assessment of quality of spray was performed by analysing the insecticide content in the filter paper samples collected from sprayed houses. Perceptions of spray men and inhabitants were recorded post-spray on safety of these molecules. RESULTS: The mean applied to target ratio of content was 1.7 (n = 29) for clothianidin and 1.8 (n = 50) for pirimiphos methyl on filter paper samples analysed. Residual activity (≥80% mortality in exposed mosquitoes) after 24 h post-exposure of SumiShield WG was 5 months and increased to 6 months when the holding period was extended to 120 h and that of Actellic CS was 3 months at 24-h holding period and extended to 4 months at 120-h extended holding period. The mean densities of An. culicifacies in both arms fell drastically post-spray. In light trap collections, density of mosquitoes collected indoors was lower than outdoors in both arms indicating effectiveness of IRS. SumiShield WG was more efficacious in reducing the per-structure density than Actellic CS. The proportion of nulliparous mosquitoes was higher than that of parous mosquitoes during post-spray collections in both arms. The majority of adverse events reported were transitory and subsided without medication. CONCLUSION: Indoor residual spraying with SumiShield WG was found effective, operationally feasible and safe, and it is effective for up to 6 months.

Burden of asymptomatic malaria among a tribal population in a forested village of central India: a hidden challenge for malaria control in India.

OBJECTIVE: Chhattisgarh in India is a malaria-endemic state with seven southern districts that contributes approximately 50-60% of the reported malaria cases in the state every year. The problem is further complicated due to asymptomatic malaria cases which are largely responsible for persistent transmission. This study was undertaken in one of the forested villages of the Keshkal subdistrict in Kondagaon district to ascertain the proportion of the population harbouring subclinical malarial infections. STUDY DESIGN: Community-based cross-sectional study. METHODS: Mass blood surveys were undertaken of the entire population of the village in the post-monsoon seasons of 2013 and 2014. Fingerprick blood smears were prepared from individuals of all ages to detect malaria infections in their blood. Individuals with fever at the time of the survey were tested with rapid diagnostic tests, and parasitaemia in thick blood smears was confirmed by microscopy. Malaria-positive cases were treated with anti-malarials in accordance with the national drug policy. RESULTS: Peripheral blood smears of 134 and 159 individuals, including children, were screened for malaria infection in 2013 and 2014, respectively. Overall, the malaria slide positivity rates were 27.6% and 27.7% in 2013 and 2014, respectively, and the prevalence rates of asymptomatic malaria were 20% and 22.8%. This study showed that, for two consecutive years, the prevalence of asymptomatic malaria infection was significantly higher among children aged ≤14 years (34.4% and 34.1% for 2013 and 2014, respectively) compared with adults (15.2% and 18.2% for 2013 and 2014, respectively; P = 0.023 and 0.04, respectively). CONCLUSION: The number of asymptomatic malaria cases, especially Plasmodium falciparum, is significant, reinforcing the underlying challenge facing the malaria elimination programme in India.

Comparison of three PCR-based assays for the non-invasive diagnosis of malaria: detection of Plasmodium parasites in blood and saliva.

The conventional molecular diagnosis of malaria uses 18S rRNA-based PCR assay employing blood samples. This assay presents limitation in terms of long turnaround time and increased chances of false-positive results. Here, we evaluated one-step singleplex or multiplex PCR assay based on high copy species-specific consensus repeat sequences (CRS) along with standard 18S rRNA nested PCR (18S n-PCR) assay to detect P. falciparum and P. vivax infection using blood and saliva samples from Indian febrile patients. Out of 327 patients, 187 were found to be positive for malaria parasites by microscopic examination of peripheral blood smears. Among these 130 were P. vivax and 57 were P. falciparum cases. The 18S n-PCR assay and CRS PCR assay identified 186 out of 187 cases (99.4 %). Multiplex CRS PCR assay detected Plasmodium in 176 out of 187 cases (94.1 %). Both singleplex and multiplex CRS PCR assay identified 6 mixed infection cases, while 18S n-PCR assay detected 10 mixed infection cases of P. vivax and P. falciparum, which were not recognized by microscopy. Non-invasive Plasmodium detection rate with DNA derived from saliva samples was highest for 18S n-PCR (87.36 %), followed by singleplex CRS (81 %) and multiplex CRS PCR assay (70.5 %). Specificity for P. vivax and P. falciparum detection for all assays was 98.48 % and 100 % respectively. Detection rate for P. vivax in saliva correlated with parasite density for CRS target-based assays. The species-specific CRS PCR, either as a singleplex or multiplex assay, can have an impact on diagnosis and epidemiological studies in malaria.

Genetic diversity of Plasmodium falciparum field isolates from south western Nigeria.

BACKGROUND: Plasmodium falciparum the main causative agent of malaria is an important public health vector. With the use of PCR, its genetic diversity has been extensively studied with dearth information from Nigeria. METHODS: In this study, 100 P. falciparum strains merozoite surface protein 1(msp-1), merozoite surface protein 2 (msp-2) and Glutamate rich protein (Glurp) from Ogun State General Hospitals were characterized. The genetic diversity of P. falciparum isolates was analyzed by restriction fragment length polymorphism following gel electrophoresis of DNA products from nested polymerase chain reactions (PCR) of their respective allelic families KI, MAD 20, RO33 (MSP-1);FC27, 3D7 (MSP-2) and Glutamate rich protein respectively. RESULTS: Majority of the patients showed monoclonal infections while multiplicity of the infection for msp-1 and msp-2 were 1.1 and 1.2 respectively. The estimated number of genotypes was 8 msp-1 (4 KI; 3 MAD; 1 RO33) and 6 msp-2 (3 FC27; 3 3D7). 80% of the isolates coded for Glurp with allelic size ranged between 700 and 900 bp. CONCLUSION: The allelic distributions however were similar to those previously reported in other endemic malaria countries. Future studies will be designed to include other malaria endemic regions of Nigeria such as the oil exploration regions.

Low efficacy of chloroquine: time to switchover to artemisinin-based combination therapy for falciparum malaria in India.

Drug resistance in Plasmodium falciparum poses a major threat to malaria control globally; including India. Chloroquine is still the most widely used drug in the country because of its safety and cost effectiveness. Although chloroquine resistance was first reported in 1973 in North Eastern India, the extent of the problem was realized only after the more intensive 28-day drug efficacy studies were used to monitor drug resistance. In the present study, efficacy of chloroquine in treatment of uncomplicated falciparum malaria was investigated using standard World Health Organization (WHO) procedures in three distinct epidemiological settings. The prevalence of molecular markers of drug resistance, Pfcrt K76T, Pfmdr1 N86Y, was also studied. A total of 374 children and adults with uncomplicated P. falciparum malaria were enrolled at six sites in four states, treated with chloroquine and follow-up was done for 28 days. The cumulative incidence of success of chloroquine at Day 28 by the Kaplan Meier analysis in the state of Orissa (District Sundargarh, CHC Bisra and Kuarmunda) was 57 (95% CI 43-68) and 54 (95% CI 40-66); in the state of Jharkhand (District Ranchi, PHC Angara and District Simdega, PHC Jaldega) it was 72 (95% CI 59-81) and 65 (95% CI 50-76); in the state of Goa (District North-Goa, Panaji Town), it was 20 (95% CI 10-2) and in the state of Rajasthan (District Udaipur, PHC Rishabdev), it was 96 (95% CI 85-99). Treatment failure was related to Pfcrt mutations but not Pfmdr mutations. Early treatment failure was observed only in 15.8% out of total failures, probably due to the semi-immune nature of the population. This type of response may give false perception about efficacy of the failing drug to patients, clinicians and National Authorities. In a large country like India it is not feasible to conduct in vivo studies in all districts and lack of direct correlation between molecular markers, in vitro studies and treatment outcome makes it difficult to predict the areas requiring change of policy. In this scenario, it is a challenge for National Programmes to make evidence-based revisions in the drug policy. However, considering the global, especially Southeast Asian, scenario and interpretation of available in vivo data, trends of mutations, availability of effective drugs and support of international donors, India should consider changing the first line treatment, at least for all diagnosed P. falciparum cases.

Therapeutic responses of Plasmodium vivax and P. falciparum to chloroquine, in an area of western India where P. vivax predominates.

In 2003-2005, following an increase in the local incidence of human malaria, the therapeutic efficacy of chloroquine (CQ) in the treatment of Plasmodium vivax and P. falciparum malaria was evaluated in the Anand district of Gujarat state, in western India. After oral administration of CQ, clinical and parasitological responses were measured over a follow-up period of 28 days, following the standard protocol of the World Health Organization. Most of the recurrent infections were checked, by genotyping, to see whether they were the result of treatment failure or re-infection during the follow-up. At the primary health centre (PHC) in Deva, all 57 P. vivax cases included in the study responded to CQ within 3 days. At the Pansora PHC, however, only 59 [90.8%, with a 95% confidence interval (CI) of 83.7%-97.8%] of the 65 P. vivax cases appeared to respond completely, recurrent infections being observed in the other six cases (9.2%; CI=2.2%-16.3%). Of the four recurrent infections checked by genotyping, however, only two appeared to be the result of true treatment failure. Twenty-seven (81.8%; CI=67.2%-94.4%) of the 33 P. falciparum cases who were enrolled in the study, all from Pansora PHC also showed apparent treatment failure, with one early failure, 17 late clinical failures and nine late parasitological failures. All 23 P. falciparum cases that showed apparent treatment failure and were investigated by genotyping appeared to be true cases of failure, none showing any evidence of re-infection during follow-up. The mean parasite-clearance times for those infected with P. falciparum, both those considered CQ-sensitive and the treatment failures, exceeded 2 days. These results indicate the presence of CQ-resistant P. vivax and P. falciparum in Anand district. The high frequency of CQ failure against P. falciparum observed in this study led to a change in the drug policy at the Pansora PHC, with artemisinin-based combination therapy now being used for the first-line treatment of P. falciparum malaria. Chloroquine remains the recommended first-line treatment for P. vivax infections in the area but the treatment failure seen in at least two P. vivax cases indicates a need for further monitoring of the therapeutic efficacy of CQ against such infections, in central Gujarat and elsewhere.

Allelic polymorphism in the Plasmodium vivax dihydrofolate reductase gene among Indian field isolates.

In total, 129 Plasmodium vivax isolates from different geographical areas in India were analysed for point mutations in the P. vivax dihydrofolate reductase gene that were associated with pyrimethamine resistance. A gradual increase in the frequency of mutant genotypes was observed from north to south (p <0.0001). In the northern region (Delhi, Panna and Nadiad), the wild-type genotype was most prevalent, while the mutant genotype predominated in the coastal regions of southern India (Navi Mumbai, Goa and Chennai). Isolates from the Car-Nicobar islands showed only mutant genotypes. The differential geographical pattern of mutations may be associated with the transmission pattern.

Therapeutic efficacy of antimalarial drugs along the eastern Indo-Nepal border: a cross-border collaborative study.

This collaborative cross-border study was performed to determine the therapeutic efficacy of antimalarial drugs used by the National Programmes for falciparum malaria along the eastern Indo-Nepal border where there is unregulated population movement across the border. The study was conducted at sites in Jhapa District, Nepal and Darjeeling District, India. The study was conducted from August 2003 to February 2004, following the WHO 28 day treatment protocol. The efficacy of chloroquine was tested in India among 91 subjects and of sulfadoxine-pyrimethamine in Nepal among 107 subjects with laboratory-confirmed Plasmodium falciparum malaria. Of the 102 subjects who completed the study in Nepal, there were 21 (20.6%) treatment failures comprising 7 (6.9%) early treatment failures (ETF) and 14 (14.7%) late treatment failures (LTF) (5 late clinical failures [LCF] and 9 late parasitological failures [LPF]). Of the 89 subjects who completed the study in India, there were 46 (51.7%) treatment failures comprising 7 (7.9%) ETFs and 39 (43.8%) LTFs (13 LCFs and 26 LPFs). Based on WHO guidelines both countries need to review their drug policy urgently and make appropriate changes, taking into account aspects of cross-border collaboration in the control of drug-resistant malaria.

Anti-malarial activity of some xanthones isolated from the roots of Andrographis paniculata.

Four xanthones were isolated from the roots of Andrographis paniculata using a combination of column and thin-layer chromatographic methods. They were characterized as (i) 1,8-di-hydroxy-3,7-dimethoxy-xanthone, (ii) 4,8-dihydroxy-2,7-dimethoxy-xanthone, (iii) 1,2-dihydroxy-6,8-dimethoxy-xanthone and (iv) 3,7,8-trimethoxy-1-hydroxy xanthone by IR, MS and NMR spectroscopic methods. In vitro study revealed that compound 1,2-dihydroxy-6,8-dimethoxy-xanthone possessed substantial anti-plasmodial activity against Plasmodium falciparum with its IC(50) value of 4 microg ml(-1). Xanthones bearing hydroxyl group at 2 position demonstrated most potent activity while xanthones with hydroxyl group at 1,4 or 8 position possessed very low activity. In vivo anti-malarial sensitivity test of this compound on Swiss Albino mice with Plasmodium berghei infection using Peters' 4-day test gave substantial reduction (62%) in parasitaemia after treating the mice with 30 mg kg(-1) dose. In vitro cytotoxicity against mammalian cells revealed that 1,2-dihydroxy-6,8-dimethoxy-xanthone is non-cytotoxic with its IC(50) > 32 microg ml(-1).

The hospital- and field-based performances of the OptiMAL test, for malaria diagnosis and treatment monitoring in central India.

The performance of the OptiMAL test, to detect and differentiate Plasmodium falciparum and P. vivax, was evaluated in central India. The subjects were either symptomatic patients, who presented at a referral hospital in urban Jabalpur, or the inhabitants of remote, tribal, forested villages where malaria is a major public-health problem. In each setting, the results of conventional microscopy were used as the 'gold standard'. Under hospital conditions, the test had excellent sensitivity (100%), good specificity (97%), a high positive predictive value (98%) and a high negative predictive value (100%). The corresponding values in the field-based study in the tribal villages (100%, 67%, 84% and 100%, respectively) were almost as good. The results of OptiMAL testing reveal the decline in parasitaemias (of P. falciparum or P. vivax) after drug administration. For monitoring the effectiveness of treatment, the test could therefore be a useful alternative to microscopy, particularly (1) in places where the facilities for microscopy are poor or non-existent and (2) among hospitalized patients with severe, complicated malaria (in whom parasitaemia and drug response need to be followed very carefully). Follow-up (within 28 days of diagnosis) of the 58 malaria cases detected in the field revealed that the OptiMAL test can be used to detect re-infection with a different Plasmodium sp. (sensitivity = 100%; specificity = 100%; J-index = 1) or recrudescence/re-infection with the same Plasmodium sp. (sensitivity = 83%; specificity = 100%; J-index = 0.83) accurately. The ability to use the test to distinguish P. falciparum from P. vivax, and to identify mixed infections of these two species, is of great significance in areas where the preferred and effective therapy for P. falciparum malaria differs from that for P. vivax.

Isolation and antimalarial activity of peroxydisulfate oxidation products of primaquine.

Five compounds formed by peroxydisulfate oxidation of primaquine were isolated using chromatographic methods and evaluated for antimalarial activity in vitro. One compound 6-methoxy-5,8 bis(4'-amino-1'-methylbutylamino)quinoline [P(1)] was found to have good gametocytocidal activity against Plasmodium yoelli infected mice at 10mg kg(-1) dose in vivo.

Ahaptoglobinemia (HpO) and malaria in India.

Haptoglobin (Hp) polymorphism analysed among P. vivax and P. falciparum patients and malaria negative subjects from areas with different epidemiological situations had shown high incidence of ahaptoglobinemia (HpO) among malaria patients. A definite association of HpO with P. vivax as well as P. falciparum malaria in Indian subjects had been observed. However, low sensitivity and reliability of HpO index indicates that it can not be a good indicator for determination of malaria endemicity. About 75 per cent of HpO subjects with P. vivax infection when treated with chloroquine showed typable Hp polymorphs by 8-9 days of post-treatment.

A multicentric study with arteether in patients of uncomplicated falciparum malaria.

Two hundred and sixty seven patients of uncomplicated P. falciparum malaria completed study in a multicentric phase III clinical trial of Arteether. Arteether was given intramuscularly in a dose of 150 mg daily for three consecutive days. Each patient was followed upto 28 days of alpha, beta arteether therapy. The cure rate was 97% with fever clearance time between 1-7 days (24-168 hours) and parasite clearance time between 1-3 days (24-72 hours). Parasite reappearance rate was found to be 3% and reported at only three of the centres. Following the treatment no adverse effect was observed on haematological, biochemical and vital clinical parameters.

Antimalarial t-butylperoxyamines.

Twelve t-butylperoxyamines (6-17) were synthesized as targeted antimalarials and evaluated for antimalarial activity in vivo against Plasmodium berghei in mice and in vitro against both chloroquine sensitive and chloroquine resistant strains of Plasmodium falciparum. Compound 8 was found to have highest potency with activity at 80 and 160mg/kg dose in vivo and compound 11 exhibited highest efficacy in vitro.

Plasmodium vivax polymorphism in a clinical drug trial.

Data from a double-blind randomized clinical drug trial were analyzed to find the comparative responses of two antirelapse drugs, bulaquine and primaquine, against different relapsing forms of Plasmodium vivax infection. A 1-year follow-up study strongly suggests that the duration of preerythrocytic development of P. vivax is a polymorphic characteristic, exhibited by two strains of hypnozoites responsible for early and late manifestations after primary infection. Short-term relapses were significantly higher in the first half year than long-term relapses, and the reverse was true in the second half year. Clinical drug response data showed that the hypnozoites characterized for short-term relapse were not susceptible to either of the antirelapse drugs in the currently administered dose, whereas hypnozoites characterized for long incubation were significantly susceptible.

Field evaluation of the ICT malaria P.f/P.v immunochromatographic test for diagnosis of Plasmodium falciparum and P.vivax infection in forest villages of Chhindwara, central India.

A rapid new immunochromatographic test (ICT malaria P.f/P.v) for diagnosis of Plasmodium falciparum and P.vivax was evaluated against thick blood smears in forest villages of Chhindwara, Madhya Pradesh, where both Plasmodium falciparum and P.vivax are prevalent. 344 symptomatic patients (Gond ethnic tribe) in five villages were screened by field staff of the Malaria Research Centre in October 1999. For P.falciparum, the ICT was 97.5% sensitive and 88% specific, with a positive predictive value (PPV) of 87.6% and a negative predictive value (NPV) of 97.6%. For P.vivax the sensitivity was only 72%, the specificity 99%, with a PPV of 92% and an NPV of 96%. Although a negative test result was inadequate to exclude parasitaemia < or = 300/microl for P.falciparum and < or = 1500/microl for P.vivax, the test is potentially useful in remote areas.

Evaluation of a rapid diagnostic test, 'Determine malaria pf', in epidemic-prone, forest villages of central India (Madhya Pradesh).

A rapid, immunochromatographic test for malaria diagnosis, 'Determine malaria pf', was evaluated by a field team in the epidemic-affected, forest setting of Chhindwara district, in Madhya Pradesh, central India. In all, 526 fever cases were screened for Plasmodium falciparum in October or November, 1999. Those found to be infected were treated with sulfadoxine-pyrimethamine and primaquine. Using microscopy as the gold standard, the new test had a sensitivity of 98% and a specificity of 87%. The positive and negative predictive values were 88% and 98%, respectively. Although follow-up of 64 subjects on day 7 post-treatment revealed that 20% of those who then appeared smear-negative were still antigenaemic, 34% of the subjects were still smear-positive, for asexual parasites, at that time. The Determine test was found to be very easy to perform and the results could be read reliably by field workers, without any supervision. The ease of use of the test indicates that it could be useful in the management of malaria, particularly in remote and inaccessible areas, provided that its accuracy can be assured and that it can be made affordable.