Genetic analysis of praziquantel response in schistosome parasites implicates a transient receptor potential channel.

Mass drug administration with praziquantel (PZQ) monotherapy is considered the mainstay for control and elimination of the parasites causing schistosomiasis in humans. This drug shows imperfect cure rates in the field, and parasites showing reduced PZQ response can be selected in the laboratory, but the extent of resistance in Schistosoma mansoni populations is unknown. We examined the genetic basis of the variation in response in a PZQ-selected S. mansoni population (SmLE-PZQ-R) in which 35% of the parasitic worms survive high-dose PZQ (73 micrograms per milliliter) treatment. We used genome-wide association to map loci underlying PZQ response and identified a transient receptor potential (Sm.TRPMPZQ) channel (Smp_246790) within the major chromosome 3 peak that is activated by nanomolar concentrations of PZQ. The PZQ response showed recessive inheritance and marker-assisted selection of parasites at a single Sm.TRPMPZQ SNP that produced populations of PZQ-enriched resistant (PZQ-ER) and PZQ-enriched sensitive (PZQ-ES) parasites, exhibiting >377-fold difference in PZQ response. The PZQ-ER parasites survived treatment in rodents at higher frequencies compared with PZQ-ES, and resistant parasites exhibited 2.25-fold lower expression of Sm.TRPMPZQ relative to sensitive parasites. Specific chemical blockers of Sm.TRPMPZQ enhanced PZQ resistance, whereas Sm.TRPMPZQ activators increased sensitivity. We surveyed Sm.TRPMPZQ sequence variations in 259 parasites from different global sites and identified one nonsense mutation that resulted in a truncated protein with no PZQ binding site. Our results demonstrate that Sm.TRPMPZQ underlies variation in PZQ responses in S. mansoni and provides an approach for monitoring emerging PZQ-resistant alleles in schistosome elimination programs.

Oxamniquine resistance alleles are widespread in Old World Schistosoma mansoni and predate drug deployment.

Do mutations required for adaptation occur de novo, or are they segregating within populations as standing genetic variation? This question is key to understanding adaptive change in nature, and has important practical consequences for the evolution of drug resistance. We provide evidence that alleles conferring resistance to oxamniquine (OXA), an antischistosomal drug, are widespread in natural parasite populations under minimal drug pressure and predate OXA deployment. OXA has been used since the 1970s to treat Schistosoma mansoni infections in the New World where S. mansoni established during the slave trade. Recessive loss-of-function mutations within a parasite sulfotransferase (SmSULT-OR) underlie resistance, and several verified resistance mutations, including a deletion (p.E142del), have been identified in the New World. Here we investigate sequence variation in SmSULT-OR in S. mansoni from the Old World, where OXA has seen minimal usage. We sequenced exomes of 204 S. mansoni parasites from West Africa, East Africa and the Middle East, and scored variants in SmSULT-OR and flanking regions. We identified 39 non-synonymous SNPs, 4 deletions, 1 duplication and 1 premature stop codon in the SmSULT-OR coding sequence, including one confirmed resistance deletion (p.E142del). We expressed recombinant proteins and used an in vitro OXA activation assay to functionally validate the OXA-resistance phenotype for four predicted OXA-resistance mutations. Three aspects of the data are of particular interest: (i) segregating OXA-resistance alleles are widespread in Old World populations (4.29-14.91% frequency), despite minimal OXA usage, (ii) two OXA-resistance mutations (p.W120R, p.N171IfsX28) are particularly common (>5%) in East African and Middle-Eastern populations, (iii) the p.E142del allele has identical flanking SNPs in both West Africa and Puerto Rico, suggesting that parasites bearing this allele colonized the New World during the slave trade and therefore predate OXA deployment. We conclude that standing variation for OXA resistance is widespread in S. mansoni.

Sleep deprivation alters neutrophil functions and levels of Th1-related chemokines and CD4+ T cells in the blood.

PURPOSE: The state of knowledge about the effect of sleep deprivation on the immune system is scarce and conflicting. It would therefore be useful to investigate the consequences of sleep deprivation on the immune system. We have studied the effect of sleep deprivation on the changes in neutrophil functions, and the ex vivo proliferative pattern of CD4+ T lymphocytes in relationship with blood cytokine and chemokine levels due to the crucial role of these cells in mounting potent immune responses. METHODS: Healthy volunteers were followed for 3 weeks. They had normal sleep in weeks 1 and 3 and they were sleep-deprived on week 2, sleeping < 6 h per 24 h, a pattern similar to sleep behaviors of many chronically sleep-deprived individuals. We assessed the levels of Th1/Th2 and inflammatory cytokines and chemokines, CD4+ T cells, and the NADPH oxidase activation and phagocytic functions in neutrophils. RESULTS: Our results suggest that sleep deprivation leads to a decreased neutrophil capacity to phagocytose bacteria and activate NADPH oxidase (p < 0.05). Sleep deprivation was associated with a potential increase in CXCL9 levels and decrease in CXCL10/CXCL9 and CCL5/CXCL9 ratios (p < 0.05). Furthermore, our results suggest that the decrease in CD4+ T cell due to sleep deprivation was not associated with changes in their proliferation as observed by Ki67 levels, but rather, it correlated with changes in CXCL10/CXCL9 ratio (p < 0.05). CONCLUSIONS: Sleep deprivation may lead to a decreased phagocytosis and NADPH oxidase activity in neutrophils and a decrease in the levels of CD4+ T cells which is related to changes in the Th1-related chemokine balance.

Host-parasite life-histories of the diurnal vs. nocturnal chronotypes of Schistosoma mansoni: adaptive significance.

OBJECTIVES: To optimise host-to-host transmission, digenean trematodes (parasites) synchronise their cercarial emission patterns with the aquatic activities of their vertebrate hosts. Schistosoma mansoni has two different circadian chronotypes: a diurnal shedding pattern with a mean peak occurring at 11:00 h, and a nocturnal shedding pattern with a mean peak occurring at 20:00 h. We analysed the life-history variations between these two chronotypes at the levels of the parasite and its hosts. METHODS: For each chronotype, we quantified three life-history traits related to the parasite (prepatent period, infection rate and cercarial production) and analysed the morphometry and the morphology of the parasite eggs; we also quantified three life-history traits related to the snail intermediate host (shell diameter, fecundity and survival rate) and one life-history trait related to the experimental definitive host (survival rate). A phylogeny based on the mitochondrial cytochrome-oxidase gene was made on samples of both chronotypes. RESULTS: Life-history analysis revealed significant variations between the two chronotypes. Life-history traits were optimal for both the parasite and the snail host for the diurnal chronotype compared to the nocturnal one. The new chronotype behaved like an allopatric population towards its snail host. Phylogenetic analysis supports the hypothesis of a lateral transfer of S. mansoni from humans to Rattus rattus. These results were interpreted in terms of an ongoing sympatric speciation. CONCLUSION: The nocturnal chronotype of S. mansoni showed non-adapted life-history traits in its relation with the snail intermediate host Biomphalaria pfeifferi. The emergence of this new phenotype is probably linked to divergent natural selection.

OBJECTIFS: Afin d'optimiser la transmission d'hôte à hôte, les trématodes digènes (parasites) synchronisent leurs schémas d'émission cercarienne avec les activités aquatiques de leurs hôtes vertébrés. Schistosoma mansoni a deux chronotypes circadiens différents: un schéma de libérations diurnes avec un pic moyen survenant à 11h00 et un schéma nocturne avec un pic moyen à 20h00. Nous avons analysé les variations de l'histoire de vie entre ces deux chronotypes aux niveaux du parasite et de ses hôtes. MÉTHODES: Pour chaque chronotype, nous avons quantifié trois traits d'histoire de vie liés au parasite (période prépatente, taux d'infection et production cercarienne) et avons analysé la morphométrie et la morphologie des œufs du parasite; nous avons également quantifié trois traits d'histoire de vie liés à l'hôte intermédiaire escargot (diamètre de la coquille, fécondité et taux de survie) et un trait d'histoire de vie lié à l'hôte définitif expérimental (taux de survie). Une phylogénie basée sur le gène mitochondrial de la cytochrome oxydase a été réalisée sur des échantillons des deux chronotypes. RÉSULTATS: L'analyse de l'histoire de vie a révélé des variations significatives entre les deux chronotypes. Les traits d'histoire de vie étaient optimaux à la fois pour le parasite et pour l'hôte escargot pour le chronotype diurne par rapport au chronotype nocturne. Le nouveau chronotype se comportait comme une population allopatrique vis-à-vis de son hôte escargot. L'analyse phylogénétique soutient l'hypothèse d'un transfert latéral de S. mansoni de l'homme à Rattus rattus. Ces résultats ont été interprétés en termes de spéciation sympatrique en cours. CONCLUSION: Le chronotype nocturne de S. mansoni montre des traits d'histoire de vie non adaptés dans sa relation avec l'hôte intermédiaire escargot, Biomphalaria pfeifferi. L'émergence de ce nouveau phénotype est probablement liée à une sélection naturelle divergente.

Altered blood cytokines, CD4 T cells, NK and neutrophils in patients with obstructive sleep apnea.

BACKGROUND: There are contradictory reports on the effects of obstructive sleep apnea (OSA) on the immune system. In order to clarify the effect of OSA on the different components of the immune system, we studied the association of OSA with changes in cytokine and chemokine levels, proliferative patterns of CD4 and CD8 T lymphocytes as well as NK cells ex vivo and neutrophil functions. METHODS: We investigated the association of OSA with potential alterations in 14 Th1/Th2 and inflammatory cytokines and chemokines, CD4 and CD8 T cells, NK cells, and the NADPH oxidase activation and phagocytic functions in neutrophils. RESULTS: Our results suggest that the increase in CD4 T cell frequency in OSA is associated with an increased expression of the nuclear protein Ki67 (p<0.05; power>0.8), and is correlated with the levels of IL-1ß (p<0.05; power>0.8). The levels of IL-1ß as well as IL-6 showed a potential increase, while the levels of IFN-γ (p<0.05; power>0.8) and the ratio IFN-γ/IL-4 in the blood were possibly decreased in OSA. Additionally, we observed a potential increase in the expression of Ki67 in CD8hi and CD8lo NK cells (p<0.05; power>0.8). Our results also suggest that neutrophils have a decreased capacity to phagocytose bacteria and activate NADPH oxidase in OSA patients (p<0.05; power>0.8). CONCLUSION: OSA may be associated with inflammatory and pro-Th2 immune responses, an increased proliferative potential of NK and CD4 T cells and a decreased capacity of neutrophils to phagocytose bacteria and produce ROS.

Gum Arabic Reduces C-Reactive Protein in Chronic Kidney Disease Patients without Affecting Urea or Indoxyl Sulfate Levels.

INTRODUCTION: Gum Arabic (GA) is a complex polysaccharide with proven prebiotic properties and potentially beneficial systemic effects. METHODS: We randomly allocated 36 chronic kidney disease (CKD) patients to receive 10, 20, or 40 grams daily of GA for four weeks and studied the systemic effects of this intervention. RESULTS: Thirty participants completed the study with baseline glomerular filtration rate 29.1 ± 9.9 mL/min/1.7 m2. In contrast to previous observations, we found no effect on serum urea or creatinine levels. GA supplementation was associated with a small but statistically significant drop in serum sodium level (138 ± 2 to 136 ± 3 mmol/L, p = 0.002) without affecting other electrolytes, urine volume, or indoxyl sulfate (IS) levels. GA supplementation was also associated with a significant drop in C-reactive protein (CRP) level (3.5 ± 1.5 to 2.8 ± 1.6 ng/mL, p = 0.02) even in patients who received only 10 g/day (4.4 ± 1.2 to 3.2 ± 1.5 ng/mL, p = 0.03). CONCLUSIONS: Supplementing the diet of CKD patients with 10-40 g/day of GA significantly reduced CRP level which could have a positive impact on these patients' morbidity and mortality. This trial is registered with Saudi Clinical Trial Registry number 15011402.

Increased CD86 but Not CD80 and PD-L1 Expression on Liver CD68+ Cells during Chronic HBV Infection.

BACKGROUND: The failure to establish potent anti-HBV T cell responses suggests the absence of an effective innate immune activation. Kupffer cells and liver-infiltrating monocytes/macrophages have an essential role in establishing anti-HBV responses. These cells express the costimulatory molecules CD80 and CD86. CD80 expression on antigen-presenting cells (APCs) induces Th1 cell differentiation, whereas CD86 expression drives the differentiation towards a Th2 profile. The relative expression of CD80, CD86 and PD-L1 on APCs, regulates T cell activation. Few studies investigated CD80 and CD86 expression on KCs and infiltrating monocytes/macrophages in HBV-infected liver and knowledge about the expression of PD-L1 on these cells is controversial. The expression of these molecules together in CD68+ cells has not been explored in HBV-infected livers. METHODS: Double staining immunohistochemistry was applied to liver biopsies of HBV-infected and control donors to explore CD80, CD86 and PD-L1 expression in the lobular and portal areas. RESULTS: Chronic HBV infection was associated with increased CD68+CD86+ cell count and percentage in the lobular areas, and no changes in the count and percentage of CD68+CD80+ and CD68+PD-L1+ cells, compared to the control group. While CD68+CD80+ cell count in portal areas correlated with the fibrosis score, CD68+CD80+ cell percentage in lobular areas correlated with the inflammation grade. CONCLUSION: The upregulation of CD86 but not CD80 and PD-L1 on CD68+ cells in HBV-infected livers, suggests that these cells do not support the induction of potent Th1. Moreover, the expression of CD80 on CD68+ cells correlates with liver inflammation and fibrosis.

A Potential Inhibitory Profile of Liver CD68+ Cells during HCV Infection as Observed by an Increased CD80 and PD-L1 but Not CD86 Expression.

AIM: The lack of potent innate immune responses during HCV infection might lead to a delay in initiating adaptive immune responses. Kupffer cells (KCs) and liver-infiltrating monocytes/macrophages (CD68+ cells) are essential to establish effective anti-HCV responses. They express co-stimulatory molecules, CD80 and CD86. CD86 upregulation induces activator responses that are then potentially regulated by CD80. The relative levels of expression of CD80, CD86 and the inhibitory molecule, PD-L1, on CD68+ cells modulate T cell activation. A few studies have explored CD80 and PD-L1 expression on KCs and infiltrating monocytes/macrophages in HCV-infected livers, and none investigated CD86 expression in these cells. These studies have identified these cells based on morphology only. We investigated the stimulatory/inhibitory profile of CD68+ cells in HCV-infected livers based on the balance of CD80, CD86 and PD-L1 expression. METHODS: CD80, CD86 and PD-L1 expression by CD68+ cells in the lobular and portal areas of the liver of chronic HCV-infected (n = 16) and control (n = 14) individuals was investigated using double staining immunohistochemistry. RESULTS: The count of CD68+ KCs in the lobular areas of the HCV-infected livers was lower than that in the control (p = 0.041). The frequencies of CD68+CD80+ cells and CD68+PD-L1+ cells in both lobular and total areas of the liver were higher in HCV-infected patients compared with those in the control group (p = 0.001, 0.031 and 0.007 respectively). Moreover, in the lobular areas of the HCV-infected livers, the frequency of CD68+CD80+ cells was higher than that of CD68+CD86+ and CD68+PD-L1+ cells. In addition, the frequencies of CD68+CD80+ and CD68+CD86+ cells were higher in the lobular areas than the portal areas. CONCLUSIONS: Our results show that CD68+ cells have an inhibitory profile in the HCV-infected livers. This might help explain the delayed T cell response and viral persistence during HCV infection.

Plasmodium falciparum population structure in Sudan post artemisinin-based combination therapy.

Over the past decade, Sudan has stepped up malaria control backed by WHO, and this has resulted in significant reduction in parasite rate, malaria morbidity and mortality. The present study analyzed Plasmodium falciparum parasites in four geographical separated areas, to examine whether the success in malaria control following the use of artemisinin-based combination therapy (ACT) has disrupted the population structure and evolution of the parasite. We examined 319 P. falciparum isolates collected between October 2009 and October 2012 in four different areas in Sudan (Jazira [central Sudan], Southern Darfur [western Sudan], Upper Nile [southern Sudan] and Kasala [eastern Sudan]). Twelve microsatellites were analyzed for allelic diversity, multi-locus haplotype and inter-population differentiation. Level of diversity was compared to that detected for three of the above microsatellites among P. falciparum parasites in central and eastern Sudan in 1999, prior to introduction of ACT. Diversity at each locus (unbiased heterozygosity [H]) was high in all areas (Jazira, H=0.67), (Southern Darfur, H=0.71), (Upper Nile, H=0.71), and (Kasala, H=0.63). Microsatellites were distributed widely and private alleles, detected in a single population, were rare. The extent of diversity in the above sites was similar to that seen, in 1999, in central (Khartoum, H=0.73) and eastern Sudan (Gedaref, H=0.75). Significant Linkage disequilibrium (LD) was observed between the microsatellites in all populations. Pairwise FST analysis revealed that parasites in the four areas could be considered as one population. However, the parasites in Sudan clustered away from parasites in West Africa and the Arabian Peninsula. Despite marked reduction in malaria risk in Sudan, the extent of diversity and parasite genetic structure are indicative of a large population size. Further considerable reduction in transmission would be needed before fragmented sub-population can be seen. In addition, the large divergence of P. falciparum in Sudan from West Africa and Arabian Peninsula populations may result from differential evolutionary pressures acting at the population level, which shall be considered in eradication plans.

Genetic diversity of Plasmodium falciparum and distribution of drug resistance haplotypes in Yemen.

BACKGROUND: Despite evident success of malaria control in many sites in the Arabian Peninsula, malaria remains endemic in a few spots, in Yemen and south-west of Saudi Arabia. In addition to local transmission, imported malaria sustains an extra source of parasites that can challenge the strengths of local control strategies. This study examined the genetic diversity of Plasmodium falciparum in Yemen and mutations of drug resistant genes, to elucidate parasite structure and distribution of drug resistance genotypes in the region. METHODS: Five polymorphic loci (MSP-2, Pfg377 and three microsatellites on chromosome 8) not involved in anti-malarial drug resistance, and four drug resistant genes (pfcrt, pfmdr1, dhfr and dhps) were genotyped in 108 P. falciparum isolates collected in three sites in Yemen: Dhamar, Hodeidah and Taiz. RESULTS: High diversity was seen in non-drug genes, pfg377 (He = 0.66), msp-2 (He = 0.80) and three microsatellites on chr 8, 7.7 kb (He = 0.88), 4.3 kb (He = 0.77) and 0.8 kb (He = 0.71). There was a high level of mixed-genotype infections (57%), with an average 1.8 genotypes per patient. No linkage disequilibrium was seen between drug resistant genes and the non-drug markers (p < 0.05). Genetic differentiation between populations was low (most pair-wise FST values <0.03), indicating extensive gene flow between the parasites in the three sites. CONCLUSION: The high diversity of P. falciparum in Yemen is indicative of a large parasite reservoir, which represents a challenge to control efforts. The presence of two distinct pfcrt genotype, CVIET and SVMNT, suggests that chloroquine resistance can possibly be related to a migratory path from Africa and Asia. The absence of the triple mutant dhfr genotype (IRN) and dhps mutations supports the use of artesunate + sulphadoxine-pyrimethamine as first-line therapy. However, the prevalent pfmdr1 genotype NFSND [21%] has previously been associated with tolerance/resistance response to artemisinin combination therapy (ACT). Regular surveys are, therefore, important to monitor spread of pfmdr1 and dhfr mutations and response to ACT.

Genetic diversity, fixation and differentiation of the freshwater snail Biomphalaria pfeifferi (Gastropoda, Planorbidae) in arid lands.

The freshwater snail Biomphalaria pfeifferi is the main intermediate host of human intestinal Bilharziasis. It is widely distributed in Africa, Madagascar and middle-eastern countries, and its habitat includes wetlands, and arid to semi-arid areas. Based on analysis of 18 microsatellites, we investigated reference allelic variation among 30 populations of B. pfeifferi from three drainage basins in Dhofar, Oman (the eastern limit of its distribution). This is an arid to semi-arid region, with a 9,000-year history of very low rainfall, but is subject to unpredictable and destructive flash floods. In this context we showed that genetic fixation was very high compared to genetic differentiation which was moderate and, that, relative to B. pfeifferi populations from wetlands, the populations in Dhofar show evidence of lower levels of genetic diversity, a higher degree of genetic fixation, a quasi-absence of migration, and a higher level of genetic drift. Despite the extreme conditions in the Dhofar habitat of this species, it is able to survive because of its very high self-fertilization (approaching 100 %) and fecundity rates.

Source of drug resistant Plasmodium falciparum in a potential malaria elimination site in Saudi Arabia.

A major challenge to the success of malaria control program in Saudi Arabia is the high influx of expatriates and holy visitors from malaria endemic countries. In the present study we examined whether drug resistant parasite genotypes reported in Jazan region, southwest of Saudi Arabia are imported or developed locally. We examined 178 Plasmodium falciparum isolates for alleles of dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr), associated with Sulfadoxine-Pyrimethamine (SP) resistance, and three microsatellites flanking each gene. In addition, we examined a neutral polymorphic gene (Pfg377). We compared the dhfr and dhps haplotypes in Jazan, using network analysis, to an existing similar data set of 94 P. falciparum isolates from eastern Sudan. In Jazan, double mutant dhfr allele (51I, 108N) occurred with a prevalence of 33%. The vast majority (99%) of dhps were wild-type alleles. The mean expected heterozygosity (H(e)) of microsatellites around mutant dhfr alleles (H(e)=0.312; n=60) was lower (P ≤ 0.05) than that around the wild-type allele (H(e)=0.834; n=116). Also, the mutant dhfr isolates showed high H(e) for dhps (H(e)=0.80) and the non-drug resistance locus Pfg377 (H(e)=0.63) indicative of selection for mutant dhfr only. The predominant double mutant dhfr haplotype in Jazan (73%), was prevalent among P. falciparum in east Africa. Network analysis suggests the mutant haplotype of dhfr gene was possibly introduced into Jazan from East Africa. The absence of mutations in dhps as well as triple mutant dhfr haplotype associated with SP failure support the current use of SP as a partner with artesunate as a first line therapy in Saudi Arabia. However, the close relationship between the major mutant dhfr haplotype in Sudan and Saudi Arabia, favour the hypothesis of recent migration as a source of the major resistant dhfr lineage. Thus, regular monitoring of the dhfr and dhps haplotypes is of high priority to guard possible importation of high level SP resistant lineages.

A new chronotype of Schistosoma mansoni: adaptive significance.

OBJECTIVES: To optimise host-to-host transmission, digenean trematodes (parasites) synchronize their cercarial emission patterns with the aquatic activities of their vertebrate hosts. Schistosoma mansoni has a strictly diurnal shedding pattern involving two circadian chronotypes: an early shedding pattern with a mean peak occurring at 11:00 h and a late pattern with a mean peak occurring at 16:00 h. We analysed the cercarial emergence pattern of three schistosome populations from Oman where S. mansoni is resurgent. METHODS: For each schistosome population, the cercarial emergence pattern was assessed hourly over several days. Because we identified a new chronotype hitherto unknown in S. mansoni, we undertook taxonomic characterisation based on egg morphology and mitochondrial DNA sequence (COX1). RESULTS: Taxonomic characterisation revealed that the three schistosome populations belong to the species S. mansoni. Hence, this is the first report of this species exhibiting a nocturnal chronotype, with the mean peak occurring at 20:00 h. We interpreted the new chronotype as being the result of a lateral transfer of S. mansoni from humans to Rattus rattus. CONCLUSION: The cercarial emergence pattern of S. mansoni from Oman is circadian, exhibiting either a diurnal or a nocturnal phenotype.

The phylogeography of Indoplanorbis exustus (Gastropoda: Planorbidae) in Asia.

BACKGROUND: The freshwater snail Indoplanorbis exustus is found across India, Southeast Asia, central Asia (Afghanistan), Arabia and Africa. Indoplanorbis is of economic importance in that it is responsible for the transmission of several species of the genus Schistosoma which infect cattle and cause reduced livestock productivity. The snail is also of medical importance as a source of cercarial dermatitis among rural workers, particularly in India. In spite of its long history and wide geographical range, it is thought that Indoplanorbis includes only a single species. The aims of the present study were to date the radiation of Indoplanorbis across Asia so that the factors involved in its dispersal in the region could be tested, to reveal potential historical biogeographical events shaping the phylogeny of the snail, and to look for signs that I. exustus might be polyphyletic. RESULTS: The results indicated a radiation beginning in the late Miocene with a divergence of an ancestral bulinine lineage into Assam and peninsular India clades. A Southeast Asian clade diverged from the peninsular India clade late-Pliocene; this clade then radiated at a much more rapid pace to colonize all of the sampled range of Indoplanorbis in the mid-Pleistocene. CONCLUSIONS: The phylogenetic depth of divergences between the Indian clades and Southeast Asian clades, together with habitat and parasitological differences suggest that I. exustus may comprise more than one species. The timescale estimated for the radiation suggests that the dispersal to Arabia and to Southeast Asia was facilitated by palaeogeographical events and climate change, and did not require human involvement. Further samples from Afghanistan, Africa and western India are required to refine the phylogeographical hypothesis and to include the African Recent dispersal.

Laboratory Studies on the Prevalence and Cercarial Rhythms of Trematodes from Bulinus truncatus and Biomphalaria Pfeifferi Snails from Khartoum State, Sudan.

OBJECTIVES: (a) To determine the natural infection rate of Bulinus truncatus and Biomphalaria pfeifferi snails with trematodes' cercariae. (b) To determine the emergence and rhythmicity of cercariae. (c) To elucidate the high-risk time for man and other animals to acquire infection. METHODS: Snails were collected from Dawar El Mahadi Agricultural Scheme, Khartoum State, identified in the laboratory, kept at room temperature and fed on lettuce. The snails were screened weekly for six weeks for natural infection and infected snails were kept in the dark. The swimming patterns and resting position of the freshly emerged cercariae were studied using a stereomicroscope. The rhythmicity of the different types of cercariae was studied by screening three sets of 5 naturally infected snails under fluorescent light from 07.00 to 19.00 and similar sets from 19.00 to 07.00. RESULTS: Out of 1,257 screened Bulinus truncatus, 187 (14.9%) shed four types of cercariae. The highest prevalence of natural infection (9.5%) was by schistosome cercariae followed by amphistome (2.5%), xiphidiocercariae (2.4%) and lastly by avian cercariae (0.5%). However, out of 200 screened B. pfeifferi, 22 (11%) shed only xiphidiocercariae. The rhythmicity studies showed that the emergence of schistosome cercariae increased steadily from 07.00 to reach its peak at 11.00-13.00. The emergence rhythms of avian cercariae are similar to those of the schistosome, but with an early peak at 09.00-11.00. The xiphidiocercariae and amphistome cercariae started with high rate of emergence at 07.00. and decreased gradually to very low levels or complete disappearance, respectively, around sunset. CONCLUSION: Information on cercarial rhythmicity and chronobiological characteristics are thought to be useful in avoiding water contact during high-risk time of infection and may be helpful in the identification of closely related species and strains of cercariae.

Vaginal hirudiniasis from Dhaherah Province, Oman: With a Note on Internal Hirudiniasis.

A 75 years old lady from the Dhaherah Province, Oman, presented at Ibri Hospital, complaining from abnormal vaginal bleeding. Histopathological investigations of a cervical biopsy revealed no atypical features. A worm-like organism was removed from the vagina. Histocytological investigations of the organism revealed typical structures of a leech. The case was identified as vaginal hirudiniasis, probably due to Limnatis nilotica that necessitated a note on internal hirudiniasis.

Origin and diversification of the human parasite Schistosoma mansoni.

Schistosoma mansoni is the most widespread of the human-infecting schistosomes, present in 54 countries, predominantly in Africa, but also in Madagascar, the Arabian Peninsula, and the Neotropics. Adult-stage parasites that infect humans are also occasionally recovered from baboons, rodents, and other mammals. Larval stages of the parasite are dependent upon certain species of freshwater snails in the genus Biomphalaria, which largely determine the parasite's geographical range. How S. mansoni genetic diversity is distributed geographically and among isolates using different hosts has never been examined with DNA sequence data. Here we describe the global phylogeography of S. mansoni using more than 2500 bp of mitochondrial DNA (mtDNA) from 143 parasites collected in 53 geographically widespread localities. Considerable within-species mtDNA diversity was found, with 85 unique haplotypes grouping into five distinct lineages. Geographical separation, and not host use, appears to be the most important factor in the diversification of the parasite. East African specimens showed a remarkable amount of variation, comprising three clades and basal members of a fourth, strongly suggesting an East African origin for the parasite 0.30-0.43 million years ago, a time frame that follows the arrival of its snail host. Less but still substantial variation was found in the rest of Africa. A recent colonization of the New World is supported by finding only seven closely related New World haplotypes which have West African affinities. All Brazilian isolates have nearly identical mtDNA haplotypes, suggesting a founder effect from the establishment and spread of the parasite in this large country.

The parasitic trematode Fasciola hepatica exhibits mammalian-type glycolipids as well as Gal(beta1-6)Gal-terminating glycolipids that account for cestode serological cross-reactivity.

Neutral glycosphingolipids from sheep-derived Fasciola hepatica liver flukes were isolated and characterized both structurally and serologically. After HPLC fractionation, glycolipids were analyzed by linkage analysis, enzymatic cleavage, and MALDI-TOF as well as electrospray ionization mass spectrometry. Obtained results revealed the presence of two types of neutral glycolipids. The first group represented mammalian-type species comprising globo- and isoglobotriaosylceramides (Gal(alpha1-4)Gal(beta1-4)Glc(1-1)ceramide and Gal(alpha1-3)Gal(beta1-4)Glc(1-1)ceramide, respectively) as well as Forssman antigen (GalNAc(alpha1-3)GalNAc(beta1-3/4)Gal(alpha1-4/3)Gal(beta1-4)Glc(1-1)ceramide). Applying Helix pomatia agglutinin, recognizing terminal alpha-linked GalNAc, to cryosections of adult flukes, the latter glycolipid could be localized to the F. hepatica gut. As Forssman antigen from the parasite and sheep host led to identical MALDI-TOF MS profiles, this glycolipid might be acquired from the definitive host. As a second group, highly antigenic glycolipids were structurally characterized as Gal(beta1-6)Gal(beta1-4)Glc(1-1)ceramide, Gal(beta1-6)Gal(alpha1-3/4)Gal(beta1-4)Glc(1-1)ceramide and Gal(beta1-6)Gal(beta1-6)Gal(alpha1-3/4)Gal(beta1-4)Glc(1-1)ceramide, the latter two structures of which exhibited both isoglobo- or globo-series core structures. Terminal Gal(beta1-6)Gal1-motifs have previously been shown to represent antigenic epitopes of neogala-series glycosphingolipids from tape worms. Using human Echinococcus granulosus infection sera, Gal(beta1-6)Gal-terminating glycolipids could be allocated to the gut in adult liver fluke cryosections. Corresponding neogala-reactive antibodies in F. hepatica infection serum were detected by their binding to E. granulosus and Taenia crassiceps neogala-glycosphingolipids. These antibodies might contribute to the known serological cross-reactivity between F. hepatica and parasitic cestode infections.

Emergence of infections with Schistosoma mansoni in the Dhofar Governorate, Oman.

Infections with Schistosoma mansoni were identified in an area of Dhofar (Oman), where this parasite had been virtually absent during recent years and was reported only very sporadically before 1992. In the present survey, performed late in 2001, between 1 and 13% of children (n=519) were found to excrete eggs (one Kato-Katz-smear) in four schools, from four different villages, but no infections were detected in additional five schools (n=281). Infections were light (<72 eggs/g of faeces) in 19 of the 36 children found infected. Serologic examination of sera (n=511) was done by ELISA (based on soluble worm antigen) and immunofluorescence tests (IFT, based on cryostat sections of adult S. mansoni). The prevalence according to serological tests was between 3 and 43% in the four schools with infected children. Positive test results were taken to reflect active infections, since false positive reactions could largely be excluded. According to ultrasound (US) examinations performed on 96 individuals (children and adults) from the four villages, livers were normal in all except three cases of mild pathology, which could be assigned to schistosomiasis mansoni (pattern C, ages 32-40 years). All data suggest that transmission of S. mansoni has been re-introduced only recently in Dhofar and that this emergence of schistosomiasis is limited to at most a few foci.

Ecological and molecular studies on emerging schistosomiasis mansoni in Dhofar Governorate, Sultanate of Oman.

The recent detection of some cases of autochtonous schistosomiasis mansoni in Dhofar, Oman, prompted a search for the transmission sites. The five field surveys we conducted from November 2000 to February 2002 provided ecological data on schistosomiasis in Dhofar. Twenty-eight water bodies situated within 8-160 km from Salalah, the largest city of Dhofar and at altitudes of up to 900 m, were surveyed for freshwater snails. Biomphalaria arabica was found in 15 of them. Three sites (Tibraq, Siginitti and Arazat) had Schistosoma infected snails, the first snails shedding cercariae of this parasite ever collected in Oman. The parasite from Dhofar was analysed by Random Amplified Polymorphic DNA comparisons using 11 primers and 167 polymorphic fragments and had 87-88% similarity with Schistosoma mansoni from Guadeloupe, but only 37-38% similarity with S. rodhaini from Burundi. Thus, it is a strain of S. mansoni. During the November 2000 survey, the prosobranch snail Melanoides tuberculata was associated with B. arabica in 10 of the 13 B. arabica sites. Cercariae from other species of Digenea emerged from five of the B. arabica sites, including the three named above. This paper presents the first finding of S. mansoni in the Dhofar Governorate and represents an initial study of the biology of S. mansoni transmission. This parasite and its cycle need further biological and molecular characterization, and the clarification of its epidemiological status in Dhofar Governorate is an urgent task.