The in vivo sensitivity of Plasmodium falciparum to chloroquine and to sulphadoxine-pyrimethamine combination in Ibadan,Nigeria

Fifty-eight Nigerian children with Plasmodium falciparum malaria were allocated randomly into two groups and treated with either chloroquine (25 mg/kg over three days) or Fansidar (35 mg sulphadoxine (+ $\text{1}\text{20}$ pyrimethamine) per kg single dose)). They were observed for 28 days during which blood films were examined periodically for malaria parasites.Asexual forms of P. falciparum, which were present in the blood films of all the patients in both groups before commencing treatment, disappeared rapidly from the blood so that by the fourth day after starting treatment no parasites were seen in the blood films. The blood films thereafter remained negative in both groups throughout the rest of the 28-day observation period. The rate of fever clearance was also similar in both groups. The study did not show resistance to Fansidar or to chloroquine. There is therefore, at present, no case for the indiscriminate use of Fansidar on the basis of suspected chloroquine resistance.     

In vivo assessment of the sensitivity of Plasmodium falciparum to sulphadoxine/pyrimethamine combination (Fansidar) in six localities in Tanzania where chloroquine-resistant P. falciparum has been detected

The sensitivity of Plasmodium falciparum strains to Fansidar (500 mg sulphadoxine/25 mg pyrimethamine) was tested in vivo in six localities in the United Republic of Tanzania where chloroquine-resistant P. falciparum strains have been demonstrated by both in vivo and in vitro tests. Single doses as recommended by the manufacturers achieved 100% clearance of parasitaemia in five localities with mean clearance period of between 2·2 and 2·9 days. In one locality (Gonja) the recommended dose failed to clear parasitaemia in two of the 38 cases (5·3%) within seven days. The possibility of using this drug combination for the treatment of chloroquine-resistant P. falciparum strains in the United Republic of Tanzania is discussed.     

A double-blind trial of a fixed combination of mefloquine plus sulfadoxine-pyrimethamine compared with sulfadoxine-pyrimethamine alone in symptomatic falciparum malaria

A total of 100 male Zambian patients with symptomatic falciparum malaria were treated with either two tablets of mefloquine plus sulfadoxine—pyrimethamine (Fansimef) or three tablets of sulfadoxine—pyrimethamine (Fansidar) as a single dose. The patients were kept under observation from day 0 (day of treatment) to day 28 and all were cured. An S-type of response was seen in all patients; one patient in the Fansimef group inexplicably remained positive for Plasmodium falciparum trophozoites until day 6. There were no cases of recrudescence.     

The activity of pyrimethamine and sulphadoxine against Plasmodium falciparum determined by the in vitro microtechnique

The reported increase of infections of Plasmodium falciparum which are no longer susceptible to a combination of pyrimethamine and sulphadoxine—Fansidar—emphasizes the need for an in vitro test to determine the presence and prevalence of drug-resistant parasites. Studies with the pyrimethaminesensitive FCB strain and the pyrimethamine-resistant FTA strain showed that the in vitro microtechnique can be used to determine differences in the susceptibility of these two strains to pyrimethamine and to pyrimethamine-sulphadoxine combinations. The FCB strain was almost six times more susceptible to pyrimethamine than the FTA strain. Although relatively high concentrations of sulphadoxine alone exerted no detectable antimalarial effects, the sulphonamide potentiated the activity of pyrimethamine against both strains of P. falciparum. This synergistic activity was relatively more pronounced against the pyrimethamine-resistant strain, particularly at lower concentrations of sulphadoxine. Further studies are indicated to determine to what extent findings obtained with the in vitro microtechnique can be correlated with the response of falciparum infections to treatment with pyrimethamine-sulphadoxine combinations.     

Chloroquine-resistant falciparum malaria imported into the Netherlands

A prospective study of imported chloroquine-resistant falciparum malaria in the Netherlands is described. From 1979 to 1983, 77 non-immune patients were investigated; in 41 (53%) decreased sensitivity of Plasmodium falciparum to chloroquine could be confirmed. Signs and symptoms in these patients differ from the classical picture. Resistance to sulfadoxine—pyrimethamine (Fansidar) was established in 6 patients. Parasitaemia was found twice during dapsone—pyrimethamine (Maloprim) prophylaxis. The implications for advice on treatment and prophylaxis are discussed.     

Comparison of in vitro pyrimethamine assays and in vivo response to sulphadoxine-pyrimethamine in Plasmodium falciparum from Papua New Guinea

The in vitro pyrimethamine sensitivity of 20 Plasmodium falciparum isolates from Papua New Guinea children was determined. The children were treated with sulphadoxine-pyrimethamine and six were found to have clinically resistant malaria. The P. falciparum isolated from these subjects were more resistant to pyrimethamine in vitro than 13 of the isolates from sensitive cases. These results suggest that pyrimethamine sensitivity alone may be a good indicator of in vitro response to sulphadoxine-pyrimethamine.     

A new in vitro test for pyrimethamine/sulfadoxine susceptibility of Plasmodium falciparum and its correlation with in vivo resistance in Kenya

A useful in vitro method for field evaluation of Plasmodium falciparum sensitivity to pyrimethamine/sulfadoxine is described. Thirty-five Kenyan schoolchildren infected with P. falciparum were treated with this drug combination and followed up for 5 weeks. In vitro tests for sensitivity to these drugs and to chloroquine were performed before starting treatment. All infections cleared within 7 days of treatment, but 5 children had recurrent parasitaemia within 35 days. The original isolates from 4 of these 5 children had an in vitro response to pyrimethamine/sulfadoxine similar to a known strain that was resistant to these drugs; only 4 of the remaining 30 isolates from patients in whom recurrent parasitaemia did not occur had a resistant in vitro response (P = 0.006). In the patient with recurrent parasitaemia whose initial isolate appeared sensitive to pyrimethamine/sulfadoxine, the recurrent isolate had a resistant pattern in vitro, suggesting either reinfection or selection of a resistant subpopulation following treatment. The in vitro response to this drug combination was correlated with the in vitro response to either drug alone and with the in vitro response to chloroquine. Two of the 5 infections with recurrent parasitaemia after initial pyrimethamine/sulfadoxine treatment were resistant to chloroquine in vivo. The in vitro test for pyrimethamine/sulfadoxine should be useful for mapping the spread of multidrug-resistant P. falciparum.     

Chloroquine-Resistant Haplotype Plasmodium falciparum Parasites, Haiti

Plasmodium falciparum parasites have been endemic to Haiti for >40 years without evidence of chloroquine (CQ) resistance. In 2006 and 2007, we obtained blood smears for rapid diagnostic tests (RDTs) and filter paper blots of blood from 821 persons by passive and active case detection. P. falciparum infections diagnosed for 79 persons by blood smear or RDT were confirmed by PCR for the small subunit rRNA gene of P. falciparum. Amplification of the P. falciparum CQ resistance transporter (pfcrt) gene yielded 10 samples with amplicons resistant to cleavage by ApoI. A total of 5 of 9 samples had threonine at position 76 of pfcrt, which is consistent with CQ resistance (haplotypes at positions 72–76 were CVIET [n = 4] and CVMNT [n = 1]); 4 had only the wild-type haplotype associated with CQ susceptibility (CVMNK). These results indicate that CQ-resistant haplotype P. falciparum malaria parasites are present in Haiti.     

DNA sequence polymorphisms of the pfmdr1 gene and association of mutations with the pfcrt gene in Indian Plasmodium falciparum isolates

Mutations in the Plasmodium falciparum multidrug resistance (pfmdr1) gene are known to provide compensatory fitness benefits to the chloroquine (CQ)-resistant malaria parasites and are often associated with specific mutations in the P. falciparum CQ resistant transporter (pfcrt) gene. Prevalence of the specific mutations in these two genes across different malaria endemic regions was mostly studies. However, reports on mutations in the pfmdr1 gene and their genetic associations with mutations in the pfcrt gene in Indian P. falciparum field isolates are scarce. We have sequenced a 560 bp region of pfmdr1 coding sequence in 64 P. falciparum isolates collected from different malaria endemic populations in India. Twenty out of these 64 isolates were laboratory cultured with known in vitro CQ sensitiveness (10 sensitive and 10 resistant). Three low frequency mutations (two non-synonymous and one synonymous) in the pfmdr1 gene were segregating in Indian isolates in addition to the predominant Y₈₆ and Y₁₈₄ ones, with high haplotype and nucleotide diversity in the field isolates in comparison to the cultured ones. No statistically significant genetic association between the mutations in the pfmdr1 and pfcrt gene could be detected; almost all observed associations were intragenic in nature. The results on the genetic diversity of the pfmdr1 gene were discussed in term of evolutionary perspectives in Indian P. falciparum, with possible future potential of gaining further insights on this gene in view of evolving malaria parasites resistant to artemisinin partner drugs.     

Use of the in vitro microtechnique for the assessment of drug sensitivity of Plasmodium falciparum in Sennar, Sudan

In 1978, studies on the chloroquine sensitivity of Plasmodium falciparum were carried out in the district of Sennar, Sudan. The results of the in vivo tests showed parasites resistant at the RI level only, but the mean clearance time of trophozoites from the blood was higher than for strains found in many other areas of tropical Africa. The in vitro tests, using the microtechnique, indicated a lower sensitivity to chloroquine in the local P. falciparum isolates than in those of most other African countries. However, similar results have been reported from Ethiopia. The chloroquine sensitivity of P. falciparum from Sennar is close to the critical level of resistance. The in vitro microtechnique was also used to test for the sensitivity to Dabequin, 4-aminobenzo-quinoline, and was generally found to be a suitable and reproducible method, with a greater potential than the standard macro method. At parasite densities of over 100 000 asexual parasites per microlitre of blood the effect of a given concentration of chloroquine was related to the parasite density owing to the selective uptake of the compound by the parasitized cells.     

Chloroquine sensitivity of Plasmodium falciparum in Ibadan,Nigeria

Thirty-five children with Plasmodium falciparum malaria were treated with 25 mg/kg chloroquine over three days and observed for seven days during which blood films were examined daily for malaria parasites.Asexual forms of P. falciparum which were present in the blood films of all the patients before commencing treatment disappeared rapidly from the blood so that by the third day no parasites were seen in the blood film. The blood films remained negative for the rest of the seven-day observation period.Plasma chloroquine determination in eight of the patients showed high blood levels during the first three days.The results do not confirm the suspicion of chloroquine-resistant P. falciparum in the area studied although RI level of resistance by WHO criteria was not excluded.     

Chloroquine sensitivity of Plasmodium falciparum in Ibadan,Nigeria: II. Correlation of in vitrowith in vivo sensitivity

Plasmodium falciparum malaria was treated in 82 children with 25 mg/kg chloroquine orally over three days. They were observed for 28 days during which blood films were examined periodically for malaria parasites. Asexual forms of P. falciparum, present in the blood films of all the patients before commencing treatment, disappeared rapidly and by the third day no parasites were seen in blood films from any of them. Among the patients observed for more than three days, blood films remained negative throughout the observation period. In vitro tests of sensitivity of blood samples from 10 patients showed chloroquine concentrations of 0·5 to 0·8 nmol/ml to inhibit completely maturation from ring forms to schizonts.This suggests that P. falciparum in the Ibadan area is probably still fully sensitive to chloroquine.     

Resistance of ten Thai isolates of Plasmodium falciparum to chloroquine and pyrimethamine by in vitro tests

In vitro drug resistance tests of ten isolates of Plasmodium falciparum from three different collection points in Central Thailand have been carried out, and the results compared with those of similar tests with a drug-sensitive West African isolate. Judged by concentration of drug tolerated, the Thai isolates appeared to be about 10 times as resistant to chloroquine, and usually about 10⁵ times as resistant to pyrimethamine, as the African isolate. A little variation amonst the Thai isolates was detected.     

Reduced heterozygosity at intragenic and flanking microsatellites of pfcrt gene establishes natural selection based molecular evolution of chloroquine-resistant Plasmodium falciparum in India

The positive selection of a nucleotide substitution in exon 2 of Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene (mutation responsible for chloroquine resistance) causes a reduction in variation of neutral loci close to the gene. This reduction in allelic diversity around flanking regions of pfcrt gene was reported in worldwide chloroquine resistant isolates and referred as selective sweep. In Plasmodium falciparum isolates of India, the selective sweep in flanking loci of pfcrt gene is well established, however, high allelic diversity observed in intragenic microsatellites of pfcrt gene implied an ongoing genetic recombination. To understand, if molecular evolution of chloroquine-resistant P. falciparum isolates in India follow a selective sweep model, we analyzed genetic diversity at both seven intragenic and seven flanking microsatellites of pfcrt (−24 to +106 kb) gene in chloroquine sensitive and resistant parasites originating from high and low transmission areas. We observed low expected heterozygosity at all loci of resistant pfcrt-haplotypes (H_e = 0–0.77) compared to the wild-type (H_e = 0.38–0.96). Resistant SVMNT from high transmission areas showed significantly higher mean H_e (P = 0.03, t-test) at both intragenic and pfcrt-flanking loci (−24 to +22 kb) in comparison to low transmission areas. Our observation of reduction in variation at both intragenic and flanking loci of mutant pfcrt gene confirmed the selective sweep model of natural selection in chloroquine resistant P. falciparum isolates in India.     

Evaluation of an in vitro test method for the assessment of sensitivity of Plasmodium falciparum to pyrimethamine and sulfadoxine

Twenty three Plasmodium falciparum isolates collected from two highly pyrimethamine/sulfadoxine-resistant areas of Thailand were evaluated for their in vitro responses to pyrimethamine, sulfadoxine and combinations of these two drugs in various conditions. The test procedure was based on inhibition of parasite multiplication and of schizont formation, using the recommended modified RPMI medium 1640 with PABA 0.5 μg per litre and folic acid 10 μg per litre (LPLF). The optimum blood/medium ratios and inoculum sizes for parasite multiplication and for schizont formation were 1:19, 100 μl/well and 1:9, 50 μl/well, respectively. The appropriate incubation period was 48 hours. It was found that inhibition of either parasite multiplication or schizont formation could be used as the endpoint for evaluating the antiplasmodial action of pyrimethamine and combined pyrimethamine/sulfadoxine in vitro for field investigations; however, inhibition of only parasite multiplication should be used for determination of sulfadoxine activity. The actions of pyrimethamine in the combination pyrimethamine/sulfadoxine in ratios of 1:80 and 1:200 were similar. In vitro testing using combined pyrimethamine/sulfadoxine should be more precise than pyrimethamine alone for monitoring parasite susceptibility to the combined drug (Fansidar).     

Studies on chloroquine-resistance of Plasmodium falciparum in Upper Volta and Liberia, West Africa

The emergence of strains of Plasmodium falciparum resistant to normal dosages of chloroquine or other 4-aminoquinolines has caused concern in a number of malarious areas. Well-documented reports have confirmed this resistance in wide areas of South-East Asia and in some areas of South America. Occasional reports have been received from parts of West Africa, and the present investigation sought to clarify the situation in two areas, Ouagadougou, Upper Volta, and Kpain, Liberia, from which such reports had emanated. Studies were carried out to determine the sensitivity of local strains of P. falciparum to chloroquine. The results, based on the rapidity of parasite clearance, indicated a normal sensitivity in the cases treated. Although it is concluded that strains of P. falciparum with significant resistance to chloroquine are not at present a problem in the areas concerned, a watch should be kept for the possible future emergence of this problem.     

In Vitro Confirmation of Artemisinin Resistance in Plasmodium falciparum from Patient Isolates,Southern Rwanda, 2019

Artemisinin resistance in Plasmodium falciparum is conferred by mutations in the kelch 13 (K13) gene. In Rwanda, K13 mutations have increased over the past decade, including mutations associated with delayed parasite clearance. We document artemisinin resistance in P. falciparum patient isolates from Rwanda carrying K13 R561H, A675V, and C469F mutations.     

DNA Sequence Polymorphism of the Lactate Dehydrogenase Genefrom Iranian Plasmodium vivax and Plasmodium falciparum Isolates

Background:

Parasite lactate dehydrogenase (pLDH) is extensively employed as malaria rapid diagnostic tests (RDTs). Moreover, it is a well-known drug target candidate. However, the genetic diversity of this gene might influence performance of RDT kits and its drug target candidacy. This study aimed to determine polymorphism of pLDH gene from Iranian isolates of P. vivax and P. falciparum.

Methods:

Genomic DNA was extracted from whole blood of microscopically confirmed P. vivax and P. falciparum infected patients. pLDH gene of P. falciparum and P. vivax was amplified using conventional PCR from 43 symptomatic malaria patients from Sistan and Baluchistan Province, Southeast Iran from 2012 to 2013.

Results:

Sequence analysis of 15 P. vivax LDH showed fourteen had 100% identity with P. vivax Sal-1 and Belem strains. Two nucleotide substitutions were detected with only one resulted in amino acid change. Analysis of P. falciparum LDH sequences showed six of the seven sequences had 100% homology with P. falciparum 3D7 and Mzr-1. Moreover, PfLDH displayed three nucleotide changes that resulted in changing only one amino acid. PvLDH and PfLDH showed 75%–76% nucleotide and 90.4%–90.76% amino acid homology.

Conclusion:

pLDH gene from Iranian P. falciparum and P. vivax isolates displayed 98.8–100% homology with 1–3 nucleotide substitutions. This indicated this gene was relatively conserved. Additional studies can be done weather this genetic variation can influence the performance of pLDH based RDTs or not.     

Analyses of genetic variations at microsatellite loci present in-and-around the Pfcrt gene in Indian Plasmodium falciparum

Evolution and spread of chloroquine resistant (CQR) malaria parasite Plasmodium falciparum have posed great threat in malaria intervention across the globe. The occurrence of K76T mutation in the P. falciparum chloroquine resistance transporter (pfcrt) gene has been widely attributed to CQR with four neighboring mutations providing compensatory fitness benefit to the parasite survival. Understanding evolutionary patterns of the pfcrt gene is of great relevance not only for devising new malaria control measures but also could serve as a model to understand evolution and spread of other human drug-resistant pathogens. Several studies, mainly based on differential patterns of diversities of the microsatellite loci placed in-and-around the pfcrt gene have indicated the role of positive natural selection under the ‘hitchhiking’ model of molecular evolution. However, the studies were restricted to limited number of microsatellite loci present inside the pfcrt gene. Moreover, comparatively higher level of diversities in microsatellite loci present inside the pfcrt gene than the loci flanking the pfcrt gene are hallmarks of Indian P. falciparum, presenting contrasting evolutionary models to global isolates. With a view to infer evolutionary patterns of the pfcrt gene in Indian P. falciparum, we have adopted a unique sampling scheme of two types of populations (cultured and field collected) and utilized 20 polymorphic microsatellite loci (16 located inside the pfcrt gene and four in the two flanking regions) to disentangle between genetic drift (inbred cultured isolates) and natural selection (field isolates). Data analyses employing different population genetic tests could not straightforwardly explain either the model invoking ‘genetic hitchhiking’ or ‘genetic drift’. However, complex evolutionary models influenced by both demography and natural selection or an alternative model of natural selection (e.g. diversifying/balancing selection) might better explain the observed microsatellite variation in-and-around the pfcrt gene in Indian P. falciparum.     

Molecular Detection of Malaria in South Punjab with Higher Proportion of Mixed Infections

Background

Malaria is well known for its fatalities worldwide, Plasmodium vivax and the Plasmodium falciparum are the two important species of malaria reported from Pakistan and creating lots of morbidities across the country.

Method

Study was conducted to determine the Surveillance of malaria in South Punjab by microscopy and Polymerase chain reaction (PCR).

Result

samples out of 100 patients were found positive for malarial parasites. One patient was found with mixed infection, whereas P. falciparum and P. vivax infections were detected in 17 and 22 patients, respectively. In nested PCR, genus-specific primers for Plasmodium species. in round 1 and species-specific primers for P. falciparum and P. vivax in round 2 were used. By the application of PCR 41% were found to be infected by Plasmodium spp. Among Plasmodium positive patients: mixed, P. falciparum and P. vivax infection were detected in 10, 15 and 16 patients, respectively. Thirty nine microscopically positive patients confirmed to have Plasmodium spp. One negative by PCR, 2 microscopically negative patients had shown Plasmodium spp. infection (P. falciparum and P. vivax) by PCR. In total samples, P. falciparum, P. vivax and mixed infection accounted for 36.6%, 39.0% and 24.3%, respectively.

Conclusion

Microscopy was found deficient for interpretation of mixed infections, low parasitaemia, and species specific diagnosis. The sensitivity, specificity and efficacy of nested PCR was calculated 95%, 98% and 97%, respectively, showing PCR as a more effective and efficient diagnostic tool for malaria.