Effects of transmission reduction by insecticide-treated bed nets (ITNs) on parasite genetics population structure: I. The genetic diversity of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> parasites by microsatellite markers in western Kenya

Background

Insecticide-treated bed nets (ITNs) reduce malaria transmission and are an important prevention tool. However, there are still information gaps on how the reduction in malaria transmission by ITNs affects parasite genetics population structure. This study examined the relationship between transmission reduction from ITN use and the population genetic diversity of Plasmodium falciparum in an area of high ITN coverage in western Kenya.

Methods

Parasite genetic diversity was assessed by scoring eight single copy neutral multilocus microsatellite (MS) markers in samples collected from P. falciparum- infected children (< five years) before introduction of ITNs (1996, baseline, n = 69) and five years after intervention (2001, follow-up, n = 74).

Results

There were no significant changes in overall high mixed infections and unbiased expected heterozygosity between baseline (%M_A = 94% and H_e = 0.75) and follow up (%M_A = 95% and H_e = 0.79) years. However, locus specific analysis detected significant differences for some individual loci between the two time points. Pfg377 loci, a gametocyte-specific MS marker showed significant increase in mixed infections and H_e in the follow up survey (%M_A = 53% and H_e = 0.57) compared to the baseline (%M_A = 30% and H_e = 0.29). An opposite trend was observed in the erythrocyte binding protein (EBP) MS marker. There was moderate genetic differentiation at the Pfg377 and TAA60 loci (F_ST = 0.117 and 0.137 respectively) between the baseline and post-ITN parasite populations. Further analysis revealed linkage disequilibrium (LD) of the microsatellites in the baseline (14 significant pair-wise tests and I ^S _A = 0.016) that was broken in the follow up parasite population (6 significant pairs and I ^S _A = 0.0003). The locus specific change in H_e, the moderate population differentiation and break in LD between the baseline and follow up years suggest an underlying change in population sub-structure despite the stability in the overall genetic diversity and multiple infection levels.

Conclusions

The results from this study suggest that although P. falciparum population maintained an overall stability in genetic diversity after five years of high ITN coverage, there was significant locus specific change associated with gametocytes, marking these for further investigation.

     

Differential association of gene content polymorphisms of killer cell immunoglobulin-like receptors with placental malaria in HIV- and HIV+ mothers

Pregnant women have abundant natural killer (NK) cells in their placenta, and NK cell function is regulated by polymorphisms of killer cell immunoglobulin-like receptors (KIRs). Previous studies report different roles of NK cells in the immune responses to placental malaria (PM) and human immunodeficiency virus (HIV-1) infections. Given these references, the aim of this study was to determine the association between KIR gene content polymorphism and PM infection in pregnant women of known HIV-1 status. Sixteen genes in the KIR family were analyzed in 688 pregnant Kenyan women. Gene content polymorphisms were assessed in relation to PM in HIV-1 negative and HIV-1 positive women, respectively. Results showed that in HIV-1 negative women, the presence of the individual genes KIR2DL1 and KIR2DL3 increased the odds of having PM, and the KIR2DL2/KIR2DL2 homozygotes were associated with protection from PM. However, the reverse relationship was observed in HIV-1 positive women, where the presence of individual KIR2DL3 was associated with protection from PM, and KIR2DL2/KIR2DL2 homozygotes increased the odds for susceptibility to PM. Further analysis of the HIV-1 positive women stratified by CD4 counts showed that this reverse association between KIR genes and PM remained only in the individuals with high CD4 cell counts but not in those with low CD4 cell counts. Collectively, these results suggest that inhibitory KIR2DL2 and KIR2DL3, which are alleles of the same locus, play a role in the inverse effects on PM and PM/HIV co-infection and the effect of KIR genes on PM in HIV positive women is dependent on high CD4 cell counts. In addition, analysis of linkage disequilibrium (LD) of the PM relevant KIR genes showed strong LD in women without PM regardless of their HIV status while LD was broken in those with PM, indicating possible selection pressure by malaria infection on the KIR genes.     

Prevalence and identity of Cryptosporidium spp. in pig slurry

Cryptosporidium spp. were detected in 25 of 56 pig slurry samples from 33 Irish farms by PCR and DNA sequencing. The organisms detected included C. suis, Cryptosporidium pig genotype II, and C. muris. We concluded that Cryptosporidium oocysts can persist in treated slurry and potentially contaminate surface water through improper discharge or uncontrolled runoff.     

Determination of ciprofloxacin in human plasma using high-performance liquid chromatography coupled with fluorescence detection: application to a population pharmacokinetics study in children with severe malnutrition

Clinical pharmacokinetic studies of ciprofloxacin require accurate and precise measurement of plasma drug concentrations. We describe a rapid, selective and sensitive HPLC method coupled with fluorescence detection for determination of ciprofloxacin in human plasma. Internal standard (IS; sarafloxacin) was added to plasma aliquots (200 μL) prior to protein precipitation with acetonitrile. Ciprofloxacin and IS were eluted on a Synergi Max-RP analytical column (150 mm×4.6 mm i.d., 5 μm particle size) maintained at 40°C. The mobile phase comprised a mixture of aqueous orthophosphoric acid (0.025 M)/methanol/acetonitrile (75/13/12%, v/v/v); the pH was adjusted to 3.0 with triethylamine. A fluorescence detector (excitation/emission wavelength of 278/450 nm) was used. Retention times for ciprofloxacin and IS were approximately 3.6 and 7.0 min, respectively. Calibration curves of ciprofloxacin were linear over the concentration range of 0.02-4 μg/mL, with correlation coefficients (r(2))≥0.998. Intra- and inter-assay relative standard deviations (SD) were <8.0% and accuracy values ranged from 93% to 105% for quality control samples (0.2, 1.8 and 3.6 μg/mL). The mean (SD) extraction recoveries for ciprofloxacin from spiked plasma at 0.08, 1.8 and 3.6 μg/mL were 72.8±12.5% (n=5), 83.5±5.2% and 77.7±2.0%, respectively (n=8 in both cases). The recovery for IS was 94.5±7.9% (n=15). The limits of detection and quantification were 10 ng/mL and 20 ng/mL, respectively. Ciprofloxacin was stable in plasma for at least one month when stored at -15°C to -25°C and -70°C to -90°C. This method was successfully applied to measure plasma ciprofloxacin concentrations in a population pharmacokinetics study of ciprofloxacin in malnourished children.     

Towards a shared understanding of sustainability for neglected tropical disease programs

BackgroundSustainability within neglected tropical disease (NTD) programs is a complex and challenging issue. The need for a shared understanding about what sustainability means for NTD programs is more important than ever as stakeholders are currently realigning for the next decade of NTD programming with the launch of WHO’s new NTD roadmap for 2012–2030. The aim of this paper is to assess different perspectives to generate a working definition of sustainability for NTD programs.Methodology/Principal findingsThis study surveyed affiliates of the NTD NGO Network (NNN) about their definitions of sustainability and then analyzed the data using an inductive and deductive process. The research team drafted a sustainability statement based on the survey findings and then solicited and incorporated feedback on the statement from a diverse group of expert reviewers. The final statement includes a working definition of sustainability for NTD programs that highlights three key essential components to sustainability: domestic commitment, responsive resource mobilization, and accountability.Conclusions/SignificanceThis research resulted in a sustainability statement, based on a survey and extensive consultation with stakeholders, that represents a starting point for shared understanding around the concept of sustainability for NTD programs. Future collaborative work should build off this definition and seek to incorporate indicators for sustainability into programmatic decision-making.     

Prevalence and Identity of Cryptosporidium spp. in Pig Slurry

Cryptosporidium spp. were detected in 25 of 56 pig slurry samples from 33 Irish farms by PCR and DNA sequencing. The organisms detected included C. suis, Cryptosporidium pig genotype II, and C. muris. We concluded that Cryptosporidium oocysts can persist in treated slurry and potentially contaminate surface water through improper discharge or uncontrolled runoff.     

Effect of transmission reduction by insecticide-treated bednets (ITNs) on antimalarial drug resistance in western Kenya

Despite the clear public health benefit of insecticide-treated bednets (ITNs), the impact of malaria transmission-reduction by vector control on the spread of drug resistance is not well understood. In the present study, the effect of sustained transmission reduction by ITNs on the prevalence of Plasmodium falciparum gene mutations associated with resistance to the antimalarial drugs sulfadoxine-pyrimethamine (SP) and chloroquine (CQ) in children under the age of five years was investigated during an ITN trial in Asembo area, western Kenya. During the ITN trial, the national first line antimalarial treatment changed from CQ to SP. Smear-positive samples collected from cross sectional surveys prior to ITN introduction (baseline, n = 250) and five years post-ITN intervention (year 5 survey, n = 242) were genotyped for single nucleotide polymorphisms (SNPs) at dhfr-51, 59, 108, 164 and dhps-437, 540 (SP resistance), and pfcrt-76 and pfmdr1-86 (CQ resistance). The association between the drug resistance mutations and epidemiological variables was evaluated. There were significant increases in the prevalence of SP dhps mutations and the dhfr/dhps quintuple mutant, and a significant reduction in the proportion of mixed infections detected at dhfr-51, 59 and dhps-437, 540 SNPs from baseline to the year 5 survey. There was no change in the high prevalence of pfcrt-76 and pfmdr1-86 mutations. Multivariable regression analysis further showed that current antifolate use and year of survey were significantly associated with more SP drug resistance mutations. These results suggest that increased antifolate drug use due to drug policy change likely led to the high prevalence of SP mutations 5 years post-ITN intervention and reduced transmission had no apparent effect on the existing high prevalence of CQ mutations. There is no evidence from the current study that sustained transmission reduction by ITNs reduces the prevalence of genes associated with malaria drug resistance.