Development and evaluation of a loop-mediated isothermal amplification (LAMP) diagnostic test for detection of whipworm, Trichuris trichiura, in faecal samples.

Whipworm infection or trichuriasis caused by Trichuris trichiura is of major public health concern in sub-Saharan Africa, particularly among pre-school and school-going children. It is among the neglected tropical diseases targeted for elimination through mass drug administration (MDA). One of the outcomes of MDA is a rapid decline in levels of infection intensity, making it difficult to monitor effectiveness of control measures using the conventional Kato-Katz procedure, which relies on the microscopic detection of parasite ova in faecal samples. In the present study, a loop-mediated isothermal amplification (LAMP) test was developed for the detection of T. trichiura infection in faecal samples. LAMP technology offers greater sensitivity and specificity than the microscopy-based tests. A set of four specific primers targeting the internal transcribed spacer 2 region of the ribosomal DNA were designed using Primer Explorer software. DNA was extracted from faecal samples using the alkaline lysis method (HotSHOT) and the LAMP reaction performed at 63°C for 1 h. The amplicons were visualized by both gel electrophoresis and with the naked eye following staining with SYBR green dye. Sensitivity and specificity tests were determined using the standard Kato-Katz diagnostic procedure as a reference test. The developed LAMP assay reliably detected T. trichiura DNA in faecal samples, with a specificity and sensitivity of 88% and 77%, respectively. No cross-reactivity was observed with several common helminth parasites. The developed LAMP assay is an appropriate diagnostic method for the detection of T. trichiura DNA in human faecal samples due to its simplicity, low cost, high sensitivity and specificity.

Schistosomes of small mammals from the Lake Victoria Basin, Kenya: new species, familiar species, and implications for schistosomiasis control.

Recent schistosomiasis control efforts in sub-Saharan Africa have focused nearly exclusively on treatment of humans with praziquantel. However, the extent to which wild mammals act as reservoirs for Schistosoma mansoni and therefore as sources of renewed transmission following control efforts is poorly understood. With the objective to study the role of small mammals as reservoir hosts, 480 animals belonging to 9 rodent and 1 insectivore species were examined for infection with schistosomes in Kisumu, in the Lake Victoria Basin, Kenya. Animals were collected from 2 sites: near the lakeshore and from Nyabera Marsh draining into the lake. A total of 6.0% of the animals captured, including 5 murid rodent species and 1 species of shrew (Crocidura olivieri) were infected with schistosomes. Four schistosome species were recovered and identified using cox1 DNA barcoding: S. mansoni, S. bovis, S. rodhaini and S. kisumuensis, the latter of which was recently described from Nyabera Marsh. Schistosoma mansoni and S. rodhaini were found infecting the same host individual (Lophuromys flavopunctatus), suggesting that this host species could be responsible for the production of hybrid schistosomes found in the area. Although the prevalence of S. mansoni infection in these reservoir populations was low (1.5%), given their potentially vast population size, their impact on transmission needs further study. Reservoir hosts could perpetuate snail infections and favour renewed transmission to humans once control programmes have ceased.

Schistosoma kisumuensis n. sp. (Digenea: Schistosomatidae) from murid rodents in the Lake Victoria Basin, Kenya and its phylogenetic position within the S. haematobium species group.

Schistosoma kisumuensis n. sp. is described based on 6 adult males and 2 adult females collected from the circulatory system of 3 murid rodent species, Pelomys isseli, Mastomys natalensis, and Dasymys incomtus. Specimens were collected from a single location, Nyabera Swamp, in Kisumu, Kenya in the Lake Victoria Basin. This new species is morphologically similar to members of the S. haematobium group, currently represented by 8 species parasitizing artiodactyls and primates, including humans. Schistosoma kisumuensis differs from these species by producing relatively small Schistosoma intercalatum-like eggs (135.2 x 52.9 microm) with a relatively small length to width ratio (2.55). Comparison of approximately 3000-base-pair sequences of nuclear rDNA (partial 28S) and mtDNA (partial cox1, nad6, 12S) strongly supports the status of S. kisumuensis as a new species and as a sister species of S. intercalatum. The cox1 genetic distance between these two species (6.3%) is comparable to other pairwise comparisons within the S. haematobium group. Separation of the Congo River and Lake Victoria drainage basins is discussed as a possible factor favoring the origin of this species.

Genetic diversity and population structure of Schistosoma mansoni within human infrapopulations in Mwea, central Kenya assessed by microsatellite markers.

A recently developed high-throughput technique that allows multi-locus microsatellite analysis of individual miracidia of Schistosoma mansoni was used to assess the levels of genetic diversity and population structure in 12 infrapopulations of the parasite, each infrapopulation derived from an infected school child from the Mwea area, central Kenya. The mean number of alleles per locus was in the range 8.22-10.22, expected heterozygosity in Hardy-Weinberg equilibrium was 0.68-0.70, and pairwise F(ST) values ranged from 0.16% to 3.98% for the 12 infrapopulations. Although the genetic diversity within each infrapopulation of S. mansoni in this area was generally high, low levels of genetic structure were observed, suggestive of high levels of gene flow among infrapopulations. Private alleles were found in 8 of the 12 infrapopulation, the highest number of private alleles recorded per infrapopulation was 3. Our data suggest that the level of gene flow among infrapopulations of S. mansoni in Mwea is extremely high, thus providing opportunity for spread of rare alleles, including those that may confer character traits such as drug resistance and virulence.

A new approach to characterize populations of Schistosoma mansoni from humans: development and assessment of microsatellite analysis of pooled miracidia.

OBJECTIVES: To develop and assess a microsatellite technique to characterize populations of Schistosoma mansoni from humans. METHODS: For each of five patients, we calculated the allele count and frequency at 11 loci for several pools of miracidia (50 and 100), and compared these to population values, determined by amplifying microsatellites from 186 to 200 individual miracidia per patient. RESULTS: We were able to detect up to 94.5% of alleles in pools. Allele count and frequency strongly and significantly correlated between singles and pools; marginally significant differences (P < 0.05) were detected for one patient (pools of 50) for allele frequencies and for two patients (pools of 100) for allele counts. Kato-Katz egg counts and number of alleles per pool did not co-vary, indicating that further direct comparisons of the results from these two techniques are needed. CONCLUSIONS: Allele counts and frequency profiles from pooling provide important information about infection intensity and complexity, beyond that obtained from traditional methods. Although we are not advocating use of pooling to replace individual genotyping studies, it can potentially be useful in certain applications as a rapid and cost effective screening method for studies of S. mansoni population genetics, or as a more informative way to quantify and characterize human worm populations.

A single dose of intramuscular sulfadoxine-pyrimethamine as an adjunct to quinine in the treatment of severe malaria: pharmacokinetics and efficacy.

It has been suggested that sulfadoxine-pyrimethamine (SD/PM) may be useful in the treatment of severe malaria since it could enhance the killing of parasites by quinine (QN) and it can be given as a single intramuscular injection. Eighty Kenyan children with severe malaria were allocated at random to receive either intramuscular QN alone (quinine dihydrochloride 20 mg salt/kg as a loading dose, followed by 10 mg salt/kg 12 hourly for a total of 6 doses) or the same QN regimen plus one intramuscular injection of SD/PM (sulfadoxine 25 mg/kg, pyrimethamine 1.25 mg/kg). There was no difference in time to defervescence, aparasitaemia, or 50% reduction in parasitaemia, parasite elimination half-life, or mortality between the 2 groups. In addition, the concentrations of SD and PM were measured in 14 children and of QN in 8 of these children. Concentrations needed to achieve synergy against PM-resistant strains of Plasmodium falciparum were achieved in all of the children with severe malaria within the first hour and maintained for more than 72 h. SD/PM did not perturb the pharmacokinetics of QN.

The disposition of oral and intramuscular pyrimethamine/sulphadoxine in Kenyan children with high parasitaemia but clinically non-severe falciparum malaria.

1. H.p.l.c. methods are described for the measurement of pyrimethamine and sulphadoxine in small volumes of plasma dried on filter paper strips. 2. Pyrimethamine/sulphadoxine (Fansidar, Hoffman LaRoche) was given by mouth and by intramuscular injection to children with uncomplicated falciparum malaria but with high parasitaemia (n = 8 for both routes; pyrimethamine 1.25 mg kg-1, sulphadoxine 25 mg kg-1). 3. Plasma concentrations of pyrimethamine and sulphadoxine associated with synergistic effects against pyrimethamine-resistant strains of Plasmodium falciparum in vitro were achieved within 1 h of administration and were maintained beyond the end of sampling. 4. After both oral and parenteral administration the plasma concentrations of both compounds were lower than those predicted by data from healthy subjects. 5. Areas under the plasma concentration-time curves of sulphadoxine after oral and i.m. administration did not differ significantly, although maximum plasma drug concentrations were higher after the i.m. route (P = 0.03). 6. The AUC values of pyrimethamine did not differ significantly between the two routes of administration. However, after i.m. administration AUC(0,24 h) values were smaller (P = 0.03), and the time to maximum plasma drug concentration (tmax) was longer (P = 0.004) than when the drug was given orally.