The MSPDBL2 codon 591 polymorphism is associated with lumefantrine in vitro drug responses in Plasmodium falciparum isolates from Kilifi, Kenya.

The mechanisms of drug resistance development in the Plasmodium falciparum parasite to lumefantrine (LUM), commonly used in combination with artemisinin, are still unclear. We assessed the polymorphisms of Pfmspdbl2 for associations with LUM activity in a Kenyan population. MSPDBL2 codon 591S was associated with reduced susceptibility to LUM (P = 0.04). The high frequency of Pfmspdbl2 codon 591S in Kenya may be driven by the widespread use of lumefantrine in artemisinin combination therapy (Coartem).

A genome wide association study of Plasmodium falciparum susceptibility to 22 antimalarial drugs in Kenya.

BACKGROUND: Drug resistance remains a chief concern for malaria control. In order to determine the genetic markers of drug resistant parasites, we tested the genome-wide associations (GWA) of sequence-based genotypes from 35 Kenyan P. falciparum parasites with the activities of 22 antimalarial drugs. METHODS AND PRINCIPAL FINDINGS: Parasites isolated from children with acute febrile malaria were adapted to culture, and sensitivity was determined by in vitro growth in the presence of anti-malarial drugs. Parasites were genotyped using whole genome sequencing techniques. Associations between 6250 single nucleotide polymorphisms (SNPs) and resistance to individual anti-malarial agents were determined, with false discovery rate adjustment for multiple hypothesis testing. We identified expected associations in the pfcrt region with chloroquine (CQ) activity, and other novel loci associated with amodiaquine, quinazoline, and quinine activities. Signals for CQ and primaquine (PQ) overlap in and around pfcrt, and interestingly the phenotypes are inversely related for these two drugs. We catalog the variation in dhfr, dhps, mdr1, nhe, and crt, including novel SNPs, and confirm the presence of a dhfr-164L quadruple mutant in coastal Kenya. Mutations implicated in sulfadoxine-pyrimethamine resistance are at or near fixation in this sample set. CONCLUSIONS/SIGNIFICANCE: Sequence-based GWA studies are powerful tools for phenotypic association tests. Using this approach on falciparum parasites from coastal Kenya we identified known and previously unreported genes associated with phenotypic resistance to anti-malarial drugs, and observe in high-resolution haplotype visualizations a possible signature of an inverse selective relationship between CQ and PQ.

Repeat polymorphisms in the low-complexity regions of Plasmodium falciparum ABC transporters and associations with in vitro antimalarial responses.

The Plasmodium falciparum genome is rich in regions of low amino acid complexity which evolve with few constraints on size. To explore the extent of diversity in these loci, we sequenced repeat regions in pfmdr1, pfmdr5, pfmdr6, pfmrp2, and the antigenic locus pfmsp8 in laboratory and cultured-adapted clinical isolates. We further assessed associations between the repeats and parasite in vitro responses to 7 antimalarials to determine possible adaptive roles of these repeats in drug tolerance. Our results show extensive repeat variations in the reference and clinical isolates in all loci. We also observed a modest increase in dihydroartemisinin activity in parasites harboring the pfmdr1 sequence profile 7-2-10 (reflecting the number of asparagine repeats, number of aspartate repeats, and number of asparagine repeats in the final series of the gene product) (P = 0.0321) and reduced sensitivity to chloroquine, mefloquine, quinine, and dihydroartemisinin in those with the 7-2-11 profile (P = 0.0051, 0.0068, 0.0011, and 0.0052, respectively). Interestingly, we noted an inverse association between two drugs whereby isolates with 6 asparagine repeats encoded by pfmdr6 were significantly more susceptible to piperaquine than those with 8 (P = 0.0057). Against lumefantrine, those with 8 repeats were, however, more sensitive (P = 0.0144). In pfmrp2, the 7-DNNNTS/NNNNTS (number of DNNNTS or NNNNTS motifs; underlining indicates dimorphism) repeat group was significantly associated with a higher lumefantrine 50% inhibitory concentration (IC50) (P = 0.008) than in those without. No associations were observed with pfmsp8. These results hint at the probable utility of some repeat conformations as markers of in vitro antimalarial response; hence, biochemical functional studies to ascertain their role in P. falciparum are required.

The microbiologic safety of umbilical cord blood transfusion for children with severe anemia in Mombasa, Kenya.

BACKGROUND: Severe anemia requiring blood transfusion is common in hospitalized young children in sub-Saharan Africa but blood is often in short supply. Umbilical cord blood may be a useful source of blood if microbiologic safety concerns can be addressed. STUDY DESIGN AND METHODS: Cord blood, donated on the labor ward at the provincial hospital in Mombasa, was cultured soon after collection (screening culture) and after a period of storage (poststorage culture). Conventional blood transfused to children at the hospital was cultured only at the time of issue (poststorage culture). Maternal sera (cord blood) and conventional blood donations were also screened for transfusion-transmitted infection. RESULTS: At poststorage culture, the overall contamination rate of cord blood was one-third that of conventional blood (13/449 vs. 38/434; odds ratio [OR], 0.31; 95% confidence interval [CI], 0.15-0.61) and for bacteria of high pathogenic potential it was half that of conventional blood (4/449 vs. 7/434; OR, 0.55; 95% CI, 0.12-2.18). Screening cultures were positive in 50% (2/4) of cord blood packs where an organism of high pathogenic potential was isolated at poststorage culture. Cord blood donors had a lower seroreactivity than conventional donors for human immunodeficiency virus (OR, 0.63; 95% CI, 0.29-1.18), hepatitis B virus (OR, 0.32; 95% CI, 0.16-0.59), and hepatitis C virus (OR, 0.20; 95% CI, 0.24-0.76). For syphilis, initial seroreactivity in cord blood donors was 3.8% compared to 1.8% in conventional blood donors (OR, 2.10; 95% CI, 1.15-3.60) but was 0.5% after retesting. CONCLUSION: With respect to bacterial contamination and seroreactivity for transfusion-transmitted infection, the safety of cord blood in Mombasa compares favorably with conventional blood. Clinical trials of cord blood transfusion are justified.

Baseline in vitro activities of the antimalarials pyronaridine and methylene blue against Plasmodium falciparum isolates from Kenya.

We have analyzed the in vitro activities of pyronaridine and methylene blue against 59 Plasmodium falciparum isolates from Kenya in association with polymorphisms in Pfcrt (codon 76), Pfmdr1 (codon 86), and Pfnhe (full sequence). The median inhibitory concentrations that kill 50% of parasites were 13.5 and 3.3 nM for pyronaridine and methylene blue, respectively. Their activities were not associated with polymorphisms in these genes. The drugs' high in vitro activities indicate that they would be efficacious against Kenyan isolates in vivo.

In vitro activities of quinine and other antimalarials and pfnhe polymorphisms in Plasmodium isolates from Kenya.

Resistance to the amino alcohol quinine has been associated with polymorphisms in pfnhe, a sodium hydrogen exchanger. We investigated the role of this gene in quinine resistance in vitro in isolates from Kenya. We analyzed pfnhe whole-gene polymorphisms, using capillary sequencing, and pfcrt at codon 76 (pfcrt-76) and pfmdr1 at codon 86 (pfmdr1-86), using PCR-enzyme restriction methodology, in 29 isolates from Kilifi, Kenya, for association with the in vitro activities of quinine and 2 amino alcohols, mefloquine and halofantrine. In vitro activity was assessed as the drug concentration that inhibits 50% of parasite growth (IC50). The median IC50s of quinine, halofantrine, and mefloquine were 92, 22, and 18 nM, respectively. The presence of 2 DNNND repeats in microsatellite ms4760 of pfnhe was associated with reduced susceptibility to quinine (60 versus 227 nM for 1 and 2 repeats, respectively; P<0.05), while 3 repeats were associated with restoration of susceptibility. The decrease in susceptibility conferred by the 2 DNNND repeats was more pronounced in parasites harboring the pfmdr1-86 mutation. No association was found between susceptibility to quinine and the pfcrt-76 mutation or between susceptibility to mefloquine or halofantrine and the pfnhe gene and the pfcrt-76 and pfmdr1-86 mutations. Using previously published data on the in vitro activities of chloroquine, lumefantrine, piperaquine, and dihydroartemisinin, we investigated the association of their activities with pfnhe polymorphism. With the exception of a modulation of the activity of lumefantrine by a mutation at position 1437, pfnhe did not modulate their activities. Two DNNND repeats combined with the pfmdr1-86 mutation could be used as an indicator of reduced susceptibility to quinine.

The quality of stored umbilical cord and adult-donated whole blood in Mombasa, Kenya.

BACKGROUND: In sub-Saharan Africa umbilical cord blood may be a useful source of blood for transfusion. Before clinical trials, evidence is needed that cord blood donations, which vary greatly in volume, can be collected and stored into a fixed volume of anticoagulant-preservative solution obviating the need for prestorage processing. STUDY DESIGN AND METHODS: Twenty-four umbilical cord whole blood (UC-WB) donations were collected into 21 mL of CPDA-1 and refrigerated for 35 days. The Kenya Blood Transfusion Service provided 12 adult-donated whole blood (AD-WB) controls. Supernatant hemoglobin (Hb) and potassium were assayed at 7-day intervals. RESULTS: UC-WB red blood cell hemolysis and potassium loss increased throughout storage but did not differ significantly with cord blood volume. Hemolysis rates did not differ significantly between UC-WB and AD-WB but UC-WB potassium loss was slightly but significantly greater than AD-WB on Days 2, 7, and 14 (p < 0.05). In the AD-WB controls, eight were low volume (<405 mL), two had total Hb of less than 45 g, and two showed hemolysis greater than 0.8% by Day 28. CONCLUSION: Variable volumes of UC-WB can be stored for 35 days without prestorage processing and further work into its suitability for transfusion to children is justified. The quality of conventional AD-WB is a concern and needs further evaluation.

In vitro activities of piperaquine, lumefantrine, and dihydroartemisinin in Kenyan Plasmodium falciparum isolates and polymorphisms in pfcrt and pfmdr1.

We have analyzed the in vitro chemosensitivity profiles of 115 Kenyan isolates for chloroquine (CQ), piperaquine, lumefantrine (LM), and dihydroartemisinin in association with polymorphisms in pfcrt at codon 76 and pfmdr1 at codon 86, as well as with variations of the copy number of pfmdr1. The median drug concentrations that inhibit 50% of parasite growth (IC(50)s) were 41 nM (interquartile range [IQR], 18 to 73 nM), 50 nM (IQR, 29 to 96 nM), 32 nM (IQR, 17 to 46 nM), and 2 nM (IQR, 1 to 3 nM) for CQ, LM, piperaquine, and dihydroartemisinin, respectively. The activity of CQ correlated inversely with that of LM (r(2) = -0.26; P = 0.02). Interestingly, parasites for which LM IC(50)s were higher were wild type for pfcrt-76 and pfmdr1-86. All isolates had one pfmdr1 copy. Thus, the decrease in LM activity is associated with the selection of wild-type pfcrt-76 and pfmdr1-86 parasites, a feature that accounts for the inverse relationship between CQ and LM. Therefore, the use of LM-artemether is likely to lead to the selection of more CQ-susceptible parasites.

Bacterial contamination of pediatric whole blood transfusions in a Kenyan hospital.

BACKGROUND: Hospitalized children in sub-Saharan Africa frequently receive whole blood transfusions for severe anemia. The risk from bacterial contamination of blood for transfusion in sub-Saharan Africa is not known. This study assessed the frequency of bacterial contamination of pediatric whole blood transfusions at a referral hospital in Kenya. STUDY DESIGN AND METHODS: This was an observational study. Over the course of 1 year, bacteriologic cultures were performed on 434 of the 799 blood packs issued to children by the blood bank of Coast Provincial General Hospital, Mombasa. Clinical outcome was not assessed. RESULTS: Forty-four bacterial contaminants were isolated from 38 blood packs-an overall contamination frequency of 8.8% (95% confidence interval, 6.1%-11.4%). Sixty-four percent of the bacteria isolated were Gram-negative. Many of the isolates are usually found in the environment and the most likely source of contamination was considered to be the hospital blood bank. CONCLUSION: Bacterial contamination of whole blood may be a significant but unrecognized hazard of blood transfusion for children in sub-Saharan Africa. Further work is needed to clarify the extent of the problem and its clinical consequences. Increased awareness and adherence to basic principles of asepsis in the hospital blood bank may be important immediate interventions.