In vitro sensitivity pattern of chloroquine and artemisinin in Plasmodium falciparum.

Artemisinin (ART) and its derivatives form the mainstay of antimalarial therapy. Emergence of resistance to them poses a potential threat to future malaria control and elimination on a global level. It is important to know the mechanism of action of drug and development of drug resistance. We put forwards probable correlation between the mode of action of chloroquine (CQ) and ART. Modified trophozoite maturation inhibition assay, WHO Mark III assay and molecular marker study for CQ resistance at K76T codon in Plasmodium falciparum CQ-resistant transporter gene were carried out on cultured P. falciparum. On comparing trophozoite and schizont growth for both CQ-sensitive (MRC-2) and CQ-resistant (RKL-9) culture isolates, it was observed that the clearance of trophozoites and schizonts was similar with both drugs. The experiment supports that CQ interferes with heme detoxification pathway in food vacuoles of parasite, and this may be correlated as one of the plausible mechanisms of ART.

In vitro assessment of drug resistance in Plasmodium falciparum in five States of India.

Background & objectives: In vitro assays are an important tool to assess baseline sensitivity and monitor the drug response of Plasmodium falciparum over time and place and, therefore, can provide background information for the development and evaluation of drug policies. This study was aimed at determining the in vitro sensitivity of P. falciparum isolates to antimalarials. Methods: The in vitro activity of 108 P. falciparum isolates obtained from five States of India was evaluated using WHO microtest (Mark III) to chloroquine, monodesethylamodiaquine, dihydroartesunate and mefloquine. Samples were collected from the States of Orissa, Jharkhand, Karnataka, Goa and Chhattisgarh from September 2007 to August 2009. In addition, representative samples from different States of India cryopreserved and culture adapted in the Malaria Parasite Bank of National Institute of Malaria Research, New Delhi, were also evaluated. Results: The proportion of isolates resistant to chloroquine and monodesethylamodiaquine was 44.4 and 25 per cent, respectively. Of the 27 isolates resistant to monodesethylamodiaquine, 16 (59.3%) were cross-resistant to chloroquine. No isolate showed resistance to dihydroartesunate and mefloquine. Isolates from Orissa showed the highest degree of resistance to chloroquine and amodiaquine followed by Jharkhand. Forty two isolates were genotyped for pfcrt T76K chloroquine resistant mutation; mutations were seen in 38 (90.47%) isolates. Interpretation & conclusions: The Indian P. falciparum isolates showed a high degree of resistance to chloroquine followed by monodesethylamodiaquine. No resistance was recorded to mefloquine and dihydroartesunate.

Investigation of malaria outbreak in Bahraich district, Uttar Pradesh.

BACKGROUND & OBJECTIVES: Based on the reports of 139 fever related deaths in Jarwal primary health centre (PHC) of Bahraich district, Uttar Pradesh (UP) during April to September 1999, a study was undertaken to explore the possibility of outbreak of Plasmodium falciparum malaria in the area and reasons of outbreak. METHODS: The study was undertaken during September-October 1999 in Bahraich district, UP. The study included a parasitological and an entomological survey. Blood slides from fever cases were collected and examined following standard procedures for detection of species and stage of parasite. The resting adult mosquitoes were collected from human dwellings and cattle sheds from selected villages. Susceptibility status of Anopheles culicifacies to 4 per cent DDT and 0.05 per cent deltamethrin was determined under laboratory conditions following the WHO procedure. In vitro drug sensitivity of P. falciparum to chloroquine was also estimated. RESULTS: Overall slide positivity rate (SPR) was found to be 33.8 with a preponderance of P. falciparum (88.4%). There was an outbreak of Pf malaria in Jarwal and surrounding areas as well. Foci of P. falciparum malaria were found in Jarwal, Fakharpur and Hazoorpur PHCs around Kaisarganj PHC. In addition, P. falciparum cases, were also reported from Motipur and Tejwapur. INTERPRETATION & CONCLUSION: Poor surveillance of affected areas resulting in low annual parasite incidence (API), lack of insecticidal spray in the currently affected PHCs as the API was less than 2 and development of resistance in P. falciparum to chloroquine were found as the possible reasons for the outbreak. It is recommended that surveillance be strengthened in all PHCs of Bahraich district to contain further extension of malaria in northeastern UP.

In vitro schizontocidal activity of standard antimalarial drugs on chloroquine-sensitive and chloroquine-resistant isolates of Plasmodium falciparum.

The expanding foci of multiple drug resistant malaria and emergence of different strains requires the reassessment of antimalarial activity with various drugs. In vitro response of a chloroquine sensitive and a chloroquine resistant isolate of P. falciparum to a group of 6 quinine derived and 3 artemisinin derived standard drugs has been screened, to evaluate schizontocidal activity of the drugs. In a conventional test system the IC50s were derived from the log dose response curves and evaluated by a rigorous statistical interpretation. Analysis by Tukey's test was significant for the quinine related drugs (Q < or = 0.01) and excludes the statistical significance of artemisinin related drugs in these isolates. The dose-responses of these two isolates vary with quinine derivatives, with some overlap at lower doses for the sensitive isolate than for the resistant one which manifests at higher doses.

Role of macrophages in experimental malaria: VII--studies on adoptive transfer of macrophages.

Adoptive transfer of purified macrophages harvested from normal, Plasmodium berghei infected and latent/cured mice and also macrophages exposed to parasites in vitro were carried out to see the role of macrophages in transferring immunity against P. berghei infection. Macrophages obtained from mice having high parasitaemia at a dose of one million cells/animal showed significant increase in survival period (SP) and K values, compared to controls. Macrophages exposed to low parasite density conferred significant K values only. There was a decrease in prepatent period (PP) in the animal which received macrophages from animals cured 7-11 months compared to controls. The adoptive transfer studies with macrophages conditioned in vitro to parasite contributed towards increased protection of host against P. berghei as expressed by K values only. These studies showed that the macrophages harvested from infected mice were capable of acting as immunogen against P. berghei infection.

Role of macrophages in experimental malaria: VI--Effect of Freund's complete adjuvant in Plasmodium berghei infected mice.

Freund's complete adjuvant (FCA) treated group of mice when challenged with lethal Plasmodium berghei showed increased survival value; survival period (SP) and median survival day (MSD) compared to their respective control groups. K values were affected and mean parasitaemia during infection period was lower than that of control. In general survival rate after 35 days of infection was 10.5% in FCA recipients. The survival rate in a particular group of animals which received 0.2 ml FCA 3 days before challenge was 22.7%. FCA was found to contribute to increased survival of the host against P. berghei infection. The study indicates that adjuvants, like FCA induce protective immunity and future studies should include non-specific immunization against human malaria.

Role of macrophages in experimental malaria: V--Effect of ethyl palmitate on macrophages in Plasmodium berghei infected mice.

Ethyl palmitate (EP) was used as a macrophage cytotoxin. The response of P. berghei after exposing the macrophage to EP was opposite to what was seen with other agents like Silica, Antimacrophage serum and Freund's complete adjuvant. EP at dose of 5 mg and above decreased the survival period (SP), median survival day (MSD) and parasite density 24 hrs. before death (K values). Prepatent period (PP) was lower at doses 10 mg and 20 mg per day for 5 days before challenge compared to their corresponding controls. EP at a dose of 5 mg and above was found to be toxic to host, mice. EP in dosage of 3 mg per mouse administered 48 hrs. before challenge resulted in an increase in the mean survival period, survival rate (30%) and decrease in the mean parasitaemia per day when compared with the corresponding control. The interfering agents affected differently both the host and/or parasite. A proper modulation of the macrophage during the course of infection may help the host in surviving this lethal infection.

Role of macrophages in experimental malaria: IV--Bioassay of silica in immunity against Plasmodium berghei infection.

Silica treated mice when challenged with Plasmodium berghei showed increase in duration of prepatent(PP) and survival period (SP) and median survival day(MSD) as compared with controls. Daily parasite density curve during the course of infection was similar to control. Response to the parasite challenge, however, was dependent on the dose of silica. No increase in SP at 0.7 mg and in PP at 35 mg (cumulative doses) dose was observed. A dose upto 5 mg per mouse before challenge resulted in protection of the animal. No mortality was recorded in mice which received silica alone (35 mg; 5 mg/day x 7 days). Death due to lethal P.berghei infection could be delayed or prevented by altering/reducing the functional activities of macrophages during the course of infection.