Drug-Resistant Malaria in Bangui, Central African Republic: An in Vitro Assessment

Ménard, Didier; Djallé, Djibrine; Manirakiza, Alexandre; Yapou, Ferdinand; Siadoua, Valerie; S, Sana; Matsika-Claquin, Marcelle Diane; Madji, Nestor; Talarmin, Antoine

doi:10.4269/ajtmh.2005.73.239

Cited by 21 publications

(26 citation statements)

References 20 publications

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“…However, study inclusion criteria in this range could potentially lead to the exclusion of a considerable proportion of malaria-positive cases in most endemic regions and could introduce a major selection bias. 12 The results obtained by Bacon and others, who used a mean starting parasitemia of 0.75% for their comparison of the SYBR Green I and the HRP2 assay, confirm the good performance of the SYBR Green I assay at higher parasite densities. 8 Particularly under field conditions, where parasite densities tend to be low, a lack in sensitivity can preclude the testing of samples that may otherwise provide valuable information.…”

Section: Discussionmentioning

confidence: 55%

The SYBR Green I Malaria Drug Sensitivity Assay: Performance in Low Parasitemia Samples

Vossen¹,

Pferschy²,

Chiba³

et al. 2010

The American Society of Tropical Medicine and Hygiene

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INTRODUCTIONFast and reliable in vitro growth assessment of malaria parasites is the key to the assessment of in vitro drug susceptibility of malaria parasites, and to high throughput screening of newly developed drugs and drug combinations. Although there are several highly sensitive assays available today, the time and effort necessary to test a single microtiter plate remains substantial. Our goal was to assess the value of the SYBR Green I dye for drug susceptibility testing in Plasmodium falciparum cultures, both, under controlled, simulated field conditions and under optimal laboratory conditions.Recently, several new in vitro flourescene assays have been reported. 1 -4 The assays detect the presence of malaria DNA in infected erythrocytes as a measure of parasite growth and propagation and parasite growth inhibition by antimalarial drugs .Although fast and relatively inexpensive, growth assessment using nucleic acid stains has inherent limitations because these stains are not specific for malaria DNA. The SYBR Green I binds to any double-stranded DNA, including the DNA inherently present in whole blood samples, thereby resulting in high background readings.SYBR Green I has been used in molecular biology as a substitute for ethidium bromide for several years. It is an asymmetrical cyanine dye, binding to double stranded DNA, preferring G and C base pairs.2 When intercalated into DNA, it is highly fluorescent, absorbing light at a wavelength between 390 and 505 nm, with a peak at 497 nm and a secondary peak near 254 nm. It emits light at 505 to 615 nm, with a peak at 520 nm. MATERIALS AND METHODSA series of experiments were conducted to evaluate the usefulness of SYBR Green I for in vitro drug susceptibility testing of malaria parasites, not only at the previously tested higher, 4 but also at lower parasite densities. For all tests 20 µL of a 10× concentration of SYBR Green I plus 80 µL either undiluted or, if postincubation parasitemia exceeded ~3.5% infected red blood cells (IRBC), diluted (1:2) sample was used. The dye was obtained as 10,000X stock in DMSO (Sigma-Aldrich GesmbH, Vienna, Austria) and diluted in sterilized distilled water. The enzymelinked immunosorbent assay (ELISA) assays were done after dilution of the samples in distilled water as previously described. 6 Background fluorescence of the medium was analyzed by comparing regular RPMI 1640 medium containing HEPES, gentamycin, and phenol red to RPMI medium with HEPES and gentamycine, but without phenol red. The latter medium was then measured with and without addition of gentamycine.Following initial testing the phenol red-free medium containing HEPES and gentamycine was used as standard medium. To assess the influence of uninfected erythrocytes on the measurements, these were tested against the standard medium at different hematocrit levels. In addition, whole blood collected in heparinized tubes and fresh erythrocyte concentrate, both washed three times in RPMI medium, were analyzed. Two-fold serial dilutions of infected blood, with and w...

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Section: Discussionmentioning

confidence: 55%

The SYBR Green I Malaria Drug Sensitivity Assay: Performance in Low Parasitemia Samples

Vossen¹,

Pferschy²,

Chiba³

et al. 2010

The American Society of Tropical Medicine and Hygiene

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“…Our results suggest that, using an HRP2-based assay, the cutoff for in vitro resistance to CQ might be lowered. AQ often retains efficacy against CQ-resistant parasites, probably due to improved potency over CQ, but in vitro resistance to AQ and MDAQ has been described in Africa (32,55). Our study showed that parasites with IC 50 s above the accepted cutoff for resistance to MDAQ are common in Kampala (58% of tested samples had IC 50 s of Ͼ60 nM).…”

Section: Discussionmentioning

confidence: 70%

In Vitro Sensitivities of Plasmodium falciparum to Different Antimalarial Drugs in Uganda

Nsobya

Kiggundu

Nanyunja

et al. 2010

Antimicrob Agents Chemother

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The control of malaria is challenged by resistance of Plasmodium falciparum to multiple drugs. New combination regimens are now advocated for the treatment of uncomplicated falciparum malaria, but the extent of resistance to newer agents is incompletely understood. We measured the in vitro sensitivity of P. falciparum parasites cultured from children enrolled in a drug efficacy trial in Kampala, Uganda, from 2006 to 2008. Sensitivities were measured by comparing levels of histidine-rich protein-2 in parasites incubated with different concentrations of drugs with those in untreated controls. The cultured parasites exhibited a wide range of sensitivities to chloroquine (CQ); monodesethylamodiaquine (MDAQ), the major active metabolite of amodiaquine; and quinine (QN). Mean 50% inhibitory concentration (IC 50 ) results were above standard cutoffs for resistance for CQ and MDAQ. Parasites were generally sensitive to dihydroartemisinin (DHA), lumefantrine (LM), and piperaquine (PQ). For CQ, MDAQ, and QN but not the other drugs, activities against individual strains were highly correlated. We also assessed known resistance-mediating polymorphisms in two putative transporters, pfcrt and pfmdr1. When parasites that were least and most sensitive to each drug were compared, the pfmdr1 86Y mutation was significantly more common in parasites that were most resistant to CQ and MDAQ, and the pfmdr1 D1246Y mutation was significantly more common in parasites that were most resistant to MDAQ and QN. In summary, we demonstrated in parasites from Kampala a range of sensitivities to older drugs; correlation of sensitivities to CQ, MDAQ, and QN; and good activity against nearly all strains for DHA, LM, and PQ.Resistance of Plasmodium falciparum to available drugs remains a major challenge to the control of malaria. Older drugs, including the aminoquinolines chloroquine (CQ) and amodiaquine (AQ) and the antifolate sulfadoxine/pyrimethamine (SP), are already seriously compromised, with unacceptable levels of treatment failure in most of Africa (61). In the setting of increasing drug resistance, the WHO has recommended artemisininbased combination therapy (ACT) for the treatment of uncomplicated falciparum malaria (42). The commonly used ACTs in Africa are artemether/lumefantrine (AM/LM), artesunate/ amodiaquine (AS/AQ), and dihydroartemisinin/piperaquine (DHA/PQ), each containing an artemisinin combined with a longer-acting drug. These ACTs have shown excellent efficacy for the treatment of malaria in Africa. However, there is concern that heavy use of ACTs will offer strong selective pressure for parasites with diminished sensitivity to the drugs. This development may seriously jeopardize the efficacy of ACTs.Multiple recent studies in Africa have demonstrated excellent efficacy of AM/LM, AS/AQ, and DHA/PQ for the treatment of falciparum malaria (8,18,29,30,34,46,63,64). Clinical trials provide our primary means of assessing antimalarial drug efficacy, but they offer only an indirect measure of the sensitivity of parasites to drugs be...

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“…Of 19,597 isolates tested, just 130 (0.7%) were positive for I164L [3]. The mutation occurs at low level in multiple locations; 3% prevalence in western Kenya [4, 5]; 4% in Blantyre, Malawi [6]; 0.6% in the Central African Republic [7]; 1.1% in Comoros [8]; 1.0% in southern Madagascar [8] and 2% in Angola [9]. So far, it only occurs in significant numbers in Uganda [10, 11] and Rwanda.…”

Section: Introductionmentioning

confidence: 99%

Travel and the emergence of high-level drug resistance in Plasmodium falciparum in southwest Uganda: results from a population-based study

Lynch

Pearce

Pota

et al. 2017

Malar J

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BackgroundThe I164L mutation on the dhfr gene confers high level resistance to sulfadoxine–pyrimethamine (SP) but it is rare in Africa except in a cluster of reports where prevalence >10% in highland areas of southwest Uganda and eastern Rwanda. The occurrence of the dhfr I164L mutation was investigated in community surveys in this area and examined the relationship to migration.MethodsA cross-sectional prevalence survey was undertaken in among villages within the catchment areas of two health facilities in a highland site (Kabale) and a highland fringe site (Rukungiri) in 2007. Sociodemographic details, including recent migration, were collected for each person included in the study. A total of 206 Plasmodium falciparum positive subjects were detected by rapid diagnostic test; 203 in Rukungiri and 3 in Kabale. Bloodspot samples were taken and were screened for dhfr I164L.ResultsSequence analysis confirmed the presence of the I164L mutations in twelve P. falciparum positive samples giving an estimated prevalence of 8.6% in Rukungiri. Of the three parasite positive samples in Kabale, none had I164L mutations. Among the twelve I164L positives three were male, ages ranged from 5 to 90 years of age. None of those with the I164L mutation had travelled in the 8 weeks prior to the survey, although three were from households from which at least one household member had travelled during that period. Haplotypes were determined in non-mixed infections and showed the dhfr I164L mutation occurs in both as a N51I + S108N + I164L haplotype (n = 2) and N51I + C59R + S108N + I164L haplotype (n = 5). Genotyping of flanking microsatellite markers showed that the I164L occurred independently on the triple mutant (N51I, C59R + S108N) and double mutant (N51I + S108N) background.ConclusionsThere is sustained local transmission of parasites with the dhfr I164L mutation in Rukungiri and no evidence to indicate its occurrence is associated with recent travel to highly resistant neighbouring areas. The emergence of a regional cluster of I164L in SW Uganda and Rwanda indicates that transmission of I164L is facilitated by strong drug pressure in low transmission areas potentially catalysed in those areas by travel and the importation of parasites from relatively higher transmission settings.

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Drug-Resistant Malaria in Bangui, Central African Republic: An in Vitro Assessment

Cited by 21 publications

References 20 publications

The SYBR Green I Malaria Drug Sensitivity Assay: Performance in Low Parasitemia Samples

The SYBR Green I Malaria Drug Sensitivity Assay: Performance in Low Parasitemia Samples

In Vitro Sensitivities of Plasmodium falciparum to Different Antimalarial Drugs in Uganda

Travel and the emergence of high-level drug resistance in Plasmodium falciparum in southwest Uganda: results from a population-based study

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