Increased prevalence of the pfdhfr/phdhps quintuple mutant and rapid emergence of pfdhps resistance mutations at codons 581 and 613 in Kisumu, Kenya

<p>Abstract</p> <p>Background</p> <p>Anti-malarial drug resistance in Kenya prompted two drug policy changes within a decade: sulphadoxine-pyrimethamine (SP) replaced chloroquine (CQ) as the first-line anti-malarial in 1998 and artemether-lumefantrine (AL) replaced SP in 2004. Two cross-sectional studies were conducted to monitor changes in the prevalence of molecular markers of drug resistance over the period in which SP was used as the first-line anti-malarial. The baseline study was carried out from 1999-2000, shortly after implementation of SP, and the follow-up study occurred from 2003-2005, during the transition to AL.</p> <p>Materials and methods</p> <p>Blood was collected from malaria smear-positive, symptomatic patients presenting to outpatient centers in Kisumu, Kenya, during the baseline and follow-up studies. Isolates were genotyped at codons associated with SP and CQ resistance. <it>In vitro </it>IC₅₀values for antifolates and quinolones were determined for isolates from the follow-up study.</p> <p>Results</p> <p>The prevalence of isolates containing the <it>pfdhfr </it>N51I/C59R/S108N/<it>pfdhps </it>A437G/K540E quintuple mutant associated with SP-resistance rose from 21% in the baseline study to 53% in the follow-up study (p < 0.001). Isolates containing the <it>pfdhfr </it>I164L mutation were absent from both studies. The <it>pfdhps </it>mutations A581G and A613S/T were absent from the baseline study but were present in 85% and 61%, respectively, of isolates from the follow-up study. At follow-up, parasites with mutations at five <it>pfdhps </it>codons, 436, 437, 540, 581, and 613, accounted for 39% of isolates. The CQ resistance-associated mutations <it>pfcrt </it>K76T and <it>pfmdr1 </it>N86Y rose from 82% to 97% (p = 0.001) and 44% to 76% (p < 0.001), respectively, from baseline to follow-up.</p> <p>Conclusions</p> <p>During the period in which SP was the first-line anti-malarial in Kenya, highly SP-resistant parasites emerged, including isolates harboring <it>pfdhps </it>mutations not previously observed there. SP continues to be widely used in Kenya; however, given the highly resistant genotypes observed in this study, its use as a first-line anti-malarial should be discouraged, particularly for populations without acquired immunity to malaria. The increase in the <it>pfcrt </it>K76T prevalence, despite efforts to reduce CQ use, suggests that either these efforts are not adequate to alleviate CQ pressure in Kisumu, or that drug pressure is derived from another source, such as the second-line anti-malarial amodiaquine.</p

falciparum dihydrofolate reductase and

Trends in drug resistance codons in Plasmodiu

Low Complexity of Infection Is Associated With Molecular Persistence of Plasmodium falciparum in Kenya and Tanzania

Background Plasmodium falciparum resistance to artemisinin-based combination therapies (ACTs) is a threat to malaria elimination. ACT-resistance in Asia raises concerns for emergence of resistance in Africa. While most data show high efficacy of ACT regimens in Africa, there have been reports describing declining efficacy, as measured by both clinical failure and prolonged parasite clearance times. Methods Three hundred children aged 2–10 years with uncomplicated P. falciparum infection were enrolled in Kenya and Tanzania after receiving treatment with artemether-lumefantrine. Blood samples were taken at 0, 24, 48, and 72 h, and weekly thereafter until 28 days post-treatment. Parasite and host genetics were assessed, as well as clinical, behavioral, and environmental characteristics, and host anti-malarial serologic response. Results While there was a broad range of clearance rates at both sites, 85% and 96% of Kenyan and Tanzanian samples, respectively, were qPCR-positive but microscopy-negative at 72 h post-treatment. A greater complexity of infection (COI) was negatively associated with qPCR-detectable parasitemia at 72 h (OR: 0.70, 95% CI: 0.53–0.94), and a greater baseline parasitemia was marginally associated with qPCR-detectable parasitemia (1,000 parasites/uL change, OR: 1.02, 95% CI: 1.01–1.03). Demographic, serological, and host genotyping characteristics showed no association with qPCR-detectable parasitemia at 72 h. Parasite haplotype-specific clearance slopes were grouped around the mean with no association detected between specific haplotypes and slower clearance rates. Conclusions Identifying risk factors for slow clearing P. falciparum infections, such as COI, are essential for ongoing surveillance of ACT treatment failure in Kenya, Tanzania, and more broadly in sub-Saharan Africa

Antiplasmodial activity of flavan derivatives from rootbark of Cassia abbreviata Oliv.

The root bark of Cassia abbreviata has been traditionally used by the native population of the coastal region of Kenya to treat malaria. As part of our ongoing investigations into compounds with activity against malaria parasites, we tested the in vitro antiplasmodial activity against Plasmodium falciparum strain namely; chloroquine-resistant W2 and chloroquine-sensitive D6. The methanolic root extract of the plant was active against the chloroquine-sensitive (IC50 = 20.56 μg/ml) and the chloroquine-resistant (IC50 = 13.31 μg/ml) strains of P. falciparum. Two flavans 1 and 2 were purified, identified and further shown to be antiplasmodial. Compound 2 was more active than compound 1 against both strains of P. falciparum with IC50 values of 8.12 μg/ml (D6); 8.89 μg/ml (W2) and 26.02 μg/ml (D6); 25.97 μg/ml (W2), respectively. This study partly provides evidence to support the use of C. abbreviata as a malaria remedy, as used by the native populations

Anthraquinones of the Roots of Pentas micrantha

molecule

The Genotypic and Phenotypic Stability of Plasmodium falciparum Field Isolates in Continuous In Vitro Culture.

The Plasmodium falciparum in vitro culture system is critical for genotypic and phenotypic analyses of the parasites. For genotypic analysis, the genomic DNA can be obtained directly from the patient blood sample or from culture adapted parasites whereas for phenotypic analysis, immediate ex vivo or in vitro culture adapted parasites are used. However, parasite biology studies have not investigated whether culture adaptation process affects genotypic and/or phenotypic characteristics of the parasites in short- or long-term cultures. Here, we set out to study the dynamics and stability of parasite genetic and phenotypic profiles as field isolate parasites were adapted in continuous cultures. Parasites collected from three different patients presenting with uncomplicated malaria were adapted and maintained in drug-free continuous cultures. Aliquots from the continuous cultures were collected every 24-48 hours for analyses. Each aliquot was treated as a separate parasite sample. For genetic analysis, microsatellite (MS) typing and single nucleotide polymorphism (SNP) analyses of 23 drug resistance markers were done. The 50% inhibitory concentrations (IC50) for some of the samples were also established for four antimalarial drugs. Samples from each patient (parasite-line) were compared as they were passed through the continuous culture. Data revealed genotypic and phenotypic profiles for the three parasite-lines fluctuated from one generation to the next with no specific pattern or periodicity. With few exceptions, multilocus analysis revealed samples from each parasite-line had high genetic diversity with unique haplotypes. Interestingly, changes in MS and SNP profiles occurred simultaneously. The difference in the IC50s of samples in each parasite-line reached statistical significance. However, phenotypic changes did not correspond or correlate to genotypic changes. Our study revealed parasite genetic and phenotypic characteristics fluctuates in short- and long-term cultures, which indicates parasite genetic information obtained even in short cultures is likely to be different from the natural infection parasites

Busseihydroquinones A–D from the Roots of <i>Pentas bussei</i>

Four new naphthohydroquinones, named busseihydroquinones A–D (<b>1</b>–<b>4</b>), along with a known homoprenylated dihydronaphthoquinone (<b>5</b>), were isolated from the CH₂Cl₂/MeOH (1:1) extract of the roots of <i>Pentas bussei</i>. Although the genus <i>Pentas</i> is frequently used by traditional healers for the treatment of malaria, only marginal activities against the chloroquine-sensitive (D6) and the chloroquine-resistant (W2) strains of <i>Plasmodium falciparum</i> were observed for the crude root extract and the isolated constituents of this plant

Four Prenylflavone Derivatives with Antiplasmodial Activities from the Stem of Tephrosia purpurea subsp. leptostachya

Four new flavones with modified prenyl groups, namely (E)-5-hydroxytephrostachin (1), purleptone (2), (E)-5-hydroxyanhydrotephrostachin (3), and terpurlepflavone (4), along with seven known compounds (5–11), were isolated from the CH2Cl2/MeOH (1:1) extract of the stem of Tephrosia purpurea subsp. leptostachya, a widely used medicinal plant. Their structures were elucidated on the basis of NMR spectroscopic and mass spectrometric evidence. Some of the isolated compounds showed antiplasmodial activity against the chloroquine-sensitive D6 strains of Plasmodium falciparum, with (E)-5-hydroxytephrostachin (1) being the most active, IC50 1.7 ± 0.1 μM, with relatively low cytotoxicity, IC50 &gt; 21 μM, against four cell-lines

Distinctive Kaposi Sarcoma-Associated Herpesvirus Serological Profile during Acute <i>Plasmodium falciparum</i> Malaria Episodes

The seroprevalence of Kaposi sarcoma-associated herpesvirus (KSHV) and the incidence of endemic Kaposi sarcoma (KS) overlap with regions of malaria endemicity in sub-Saharan Africa. Multiple studies have shown an increased risk of KSHV seroconversion in children from high malaria compared to low malaria regions; however, the impact of acute episodes of Plasmodium falciparum (P. falciparum) malaria on KSHV’s biphasic life cycle and lytic reactivation has not been determined. Here, we examined KSHV serological profiles and viral loads in 134 children with acute malaria and 221 healthy children from high malaria regions in Kisumu, as well as 77 healthy children from low malaria regions in Nandi. We assayed KSHV, Epstein–Barr virus (EBV), and P. falciparum malaria antibody responses in these three by multiplexed Luminex assay. We confirmed that KSHV seroprevalence was significantly associated with malaria endemicity (OR = 1.95, 1.18–3.24 95% CI, p = 0.01) with 71–77% seropositivity in high-malaria (Kisumu) compared to 28% in low-malaria (Nandi) regions. Furthermore, KSHV serological profiles during acute malaria episodes were distinct from age-matched non-malaria-infected children from the same region. Paired IgG levels also varied after malaria treatment, with significantly higher anti-ORF59 at day 0 but elevated ORF38, ORF73, and K8.1 at day 3. Acute malaria episodes is characterized by perturbation of KSHV latency in seropositive children, providing further evidence that malaria endemicity contributes to the observed increase in endemic KS incidence in sub-Saharan Africa