Molecular Tools and Schistosomiasis Transmission Elimination.

Large-scale control efforts in sub-Saharan Africa may leave long-term lingering transmission. Large-scale screening of snail infection prevalence by loop-mediated isothermal amplification will enable accurate determination of man-to-snail transmission, as well as the effects of biota in snail habitat on host capacity and thus on snail-to-man transmission. Next-generation sequencing will enable identification of gut content of snails and thus their feeding preferences in hot spots and in non-hot spots, as well as for identification of attractive vegetation types for attracting snails to molluscicides.

Differentiation of Schistosoma haematobium from related schistosomes by PCR amplifying an inter-repeat sequence.

Schistosoma haematobium infects nearly 150 million people, primarily in Africa, and is transmitted by select species of local bulinid snails. These snails can host other related trematode species as well, so that effective detection and monitoring of snails infected with S. haematobium requires a successful differentiation between S. haematobium and any closely related schistosome species. To enable differential detection of S. haematobium DNA by simple polymerase chain reaction (PCR), we designed and tested primer pairs from numerous newly identified Schistosoma DNA repeat sequences. However, all pairs tested were found unsuitable for this purpose. Differentiation of S. haematobium from S. bovis, S. mattheei, S. curassoni, and S. intercalatum (but not from S. margrebowiei) was ultimately accomplished by PCR using one primer from a newly identified repeat, Sh110, and a second primer from a known schistosomal splice-leader sequence. For evaluation of residual S. haematobium transmission after control interventions, this differentiation tool will enable accurate monitoring of infected snails in areas where S. haematobium is sympatric with the most prevalent other schistosome species.

The development of a molecular approach for coprodiagnosis of Toxoplasma gondii.

Copro-diagnostic methods for Toxoplasma gondii infected cats have been traditionally based on the identification of oocysts by light microscopy or by bioassays. The first method is not sensitive and also unable to differentiate between Toxoplasma oocysts from other coccidian parasites in cats, and the second is cumbersome, time consuming and expensive. We have adapted a polymerase chain reaction (PCR) method to detect T. gondii oocyst DNA in fecal samples. Oocysts were successfully disrupted by freeze thawing coupled with mechanical means, and DNA extraction was subsequently accomplished. The test, based on amplifying a 529 bp repeated sequence, proved sensitive for detecting 1-2 oocysts in 200 microg of stool sample. The test specificity was established by showing that DNA from other cat coccidia tested negative. Specificity was reconfirmed by Southern hybridization of the PCR products with a specific probe. Of 122 stool samples from Jerusalem cats surveyed for the presence of Toxoplasma oocysts, 11 were found positive by PCR while none was detected by microscopy.

The substructure of three repetitive DNA regions of Schistosoma haematobium group species as a potential marker for species recognition and interbreeding detection.

BACKGROUND: Schistosoma haematobium is the causative agent of human urogenital schistosomiasis affecting ~112 million people in Africa and the Middle East. The parasite is transmitted by snails of the genus Bulinus, which also transmit other closely related human and animal schistosomes. The accurate discrimination of S. haematobium from species infecting animals will aid effective control and elimination programs. Previously we have shown the utility of different repetitive nuclear DNA sequences (DraI, sh73bp, and sh77bp) for the identification of S. haematobium-group species and inter-repeat sequences for discriminating S. haematobium from S. bovis. RESULTS: In this current study we clarify the structural arrangement and association between the three repetitive sequences (DraI, sh73bp, and sh77bp) in both S. haematobium and S. bovis, with a unique repeat linker being found in S. haematobium (Sh64bp repeat linker) and in S. bovis (Sb30bp repeat linker). Sequence data showed that the 3'-end of the repeat linker was connected to the DraI repetitive sequence array, and at the 5'-end of the repeat linker sh73bp and sh77bp were arranged in an alternating manner. Species-specific oligonucleotides were designed targeting the species-specific repeat linkers and used in a reverse line blot (RLB) hybridization assay enabling differentiation between S. haematobium and S. bovis. The assay was used to discriminate natural infections in wild caught Bulinus globosus. CONCLUSION: This research enabled the characterisation of species-specific DNA regions that enabled the design of species-specific oligonucleotides that can be used to rapidly differentiate between S. haematobium and S. bovis and also have the potential to aid the detection of natural hybridization between these two species.

Transmission control for schistosomiasis - why it matters now.

Current population-based schistosomiasis treatment programs are a first step to reducing the global burden of Schistosoma-related disease; however, they might not dramatically reduce parasite transmission in highly endemic areas. Consequently, the benefits of these programs remain in doubt because recurring low-level reinfection is likely to be associated with subtle but persistent morbidities such as anemia, undernutrition and diminished performance status. The real health benefits of transmission control need to be reconsidered and attention given to more aggressive and, ultimately, more affordable parasite elimination strategies. The next generation of schistosomiasis control can be optimized using new monitoring tools and effective transmission containment.

Large-scale, polymerase chain reaction-based surveillance of Schistosoma haematobium DNA in snails from transmission sites in coastal Kenya: a new tool for studying the dynamics of snail infection.

Levels of prepatent Schistosoma haematobium infection were monitored in intermediate host snails (Bulinus nasutus) collected from transmission sites in coastal Kenya, using a polymerase chain reaction (PCR) assay amplifying the Dra I repeated sequence of S. haematobium. The timing and number of prepatent and patent infections were determined for each site and, where the time of first appearance was clear, the minimal prepatent period was estimated to be five weeks. High, persistent, prepatency rates (range = 28-54%), indicated a significant degree of repeated area contamination with parasite ova. In contrast, rates of cercarial shedding proved locally variable, and were either low (range = 0.14-3.4%) or altogether absent, indicating that only a small proportion of infected snails reach the stage of cercarial shedding. Given the apparently strong focal effects of environmental conditions, implications of these new data are discussed regarding the estimation of local force of transmission and the design of control activities.

Copro-diagnosis of Echinococcus granulosus infection in dogs by amplification of a newly identified repeated DNA sequence.

Diagnosis of Echinococcus granulosus infection in dogs by detecting adult worms recovered post mortem or purged from the intestines after treatment with arecoline is not suitable for mass screening. Large-scale diagnosis by detection of copro-antigens is useful but only with relatively high intensity infections, and only by genus. To provide a more sensitive and specific diagnosis, a polymerase chain reaction (PCR) assay was developed, that amplified a target repeated sequence (EgG1 Hae III) newly identified in the genome of the common sheep strain of E. granulosus. This repeated sequence consists of approximately 6,900 copies, arranged in tandem, in groups of 2-6 repeats. The corresponding primers used in the PCR easily detected a single egg with no cross-amplification of DNA from closely related cestodes, including E. multilocularis and Taenia spp. Fecal samples from naturally infected dogs, with 2-10,000 E. granulosus worms at necropsy, were all PCR positive, while E. multilocularis or Taenia spp. positive controls as well as non-endemic controls were all PCR negative. This copro-PCR assay was demonstrated to be 100% specific and also detected all necropsy-positive E. granulosus-infected dogs. It is suggested that this copro-PCR assay has the potential for pre-mortem diagnosis of E. granulosus infection even in areas where E. granulosus and E. multilocularis are co-endemic.

Distribution patterns and cercarial shedding of Bulinus nasutus and other snails in the Msambweni area, Coast Province, Kenya.

In the Msambweni area of the Kwale District in Kenya, an area endemic for Schistosoma haematobium, potential intermediate-host snails were systematically surveyed in water bodies associated with human contact that were previously surveyed in the 1980s. Bulinus (africanus) nasutus, which accounted for 67% of the snails collected, was the only snail shedding S. haematobium cercariae. Lanistes purpureus was the second most common snail (25%); lower numbers of Bulinus forskalii and Melanoides tuberculata were also recovered. Infection with non-S. haematobium trematodes was found among all snail species. Rainfall was significantly associated with the temporal distribution of all snail species: high numbers of Bulinus nasutus developed after extensive rainfall, followed, in turn, by increased S. haematobium shedding. Spatial distribution of snails was significantly clustered over a range of up to 1 km, with peak clustering observed at a distance of 400 meters. Water lily (Nymphaea spp.) and several aquatic grass species appeared necessary for local colonization by B. nasutus or L. purpureus.

Schistosoma mansoni and Schistosoma haematobium: identification and characterization of glycoconjugate antigens in the hemolymph of infected vector snails.

Two carbohydrate epitopes were identified by monoclonal antibodies (KCS and E2) and characterized with respect to their immunoreactivity, monosaccharide structure, and location. Immunofluorescence demonstrated the presence of both epitopes on the surfaces of sporocysts, cercariae, and miracidia of Schistosoma mansoni, Schistosoma haematobium, and Schistosoma japonicum. However, spatial distribution and density of expression varied among species and developmental stages, and neither epitope was detectable on adult worm surfaces. Both glycans were found in the hemolymph of infected, but not uninfected, intermediate snail hosts. The presence of epitopes in hemolymph, as well as in schistosome eggs, is species-specific for KCS, recognizing only S. mansoni, and partly specific for E2, which reacted predominantly with S. haematobium. Immunoaffinity purification of target antigens for KCS and E2 from hemolymph of infected Biomphalaria and Bulinus, respectively, followed by carbohydrate composition analysis revealed a high content of fucose in both glycans. Methylation analysis demonstrated exclusively terminal fucose for the target antigen of KCS and terminal as well as internal fucose for the one of E2. Removal of terminal fucose abolished reactivity with both monoclonal antibodies. Both glycans are different from previously characterized schistosome carbohydrates. Their biological function(s) remain to be defined.

Evaluation of loop-mediated isothermal amplification suitable for molecular monitoring of schistosome-infected snails in field laboratories.

We previously described loop-mediated isothermal amplification (LAMP) for detection of Schistosoma haematobium and S. mansoni DNA in infected snails. In the present study, we adapted the LAMP assay for application in field laboratories in schistosomiasis-endemic areas. Isolation of DNA was simplified by blotting snail tissue (extracted in NaOH/sodium dodecyl sulfate) onto treated membranes, which enabled preservation at ambient temperatures. A ready-mix of LAMP reagents, suitable for shipment at ambient temperature and storage in minimal refrigeration, was used. Local survey teams without experience in molecular biology acquired operational expertise with this test within a few hours. Fifty-four field-caught snails were tested locally by LAMP and 59 were tested at similar conditions in Jerusalem. The LAMP results were consistent with those of a polymerase chain reaction; only four samples showed false-negative results. Results indicate that LAMP assays are suitable for detection of S. haematobium and S. mansoni in low-technology parasitology laboratories in which schistosomiasis elimination activities are undertaken.

The development of a loop-mediated isothermal amplification method (LAMP) for Echinococcus granulosus [corrected] coprodetection.

We have previously developed a polymerase chain reaction (PCR) assay for detection of Echinococcus granulosus infection, which proved very sensitive and specific for identification of infected dogs. We have now developed a loop-mediated isothermal amplification (LAMP) assay, which amplifies the same genomic repeated sequences of E. granulosus for coprodetection. This assay enabled detection of a single egg in fecal samples and showed high species specificity for E. granulosus with no cross-amplification of DNA from closely related helminths, including Echinococcus multilocularis. Because the method does not require thermocycling for DNA amplification, or electrophoresis for amplicon detection, it can potentially be used for premortem identification of E. granulosus-infected dogs to enable large-scale surveys in endemic countries where highly specialized equipment to undertake PCR analysis is rare.

Differentiating Schistosoma haematobium from related animal schistosomes by PCR amplifying inter-repeat sequences flanking newly selected repeated sequences.

In schistosomiasis elimination programs, successful discrimination of Schistosoma haematobium from the related animal Schistosoma parasites will be essential for accurate detection of human parasite transmission. Polymerase chain reaction assays employing primers from two newly selected repeated sequences, named Sh73 and Sh77, did not discriminate S. haematobium when amplifying Sh73-77 intra- or inter-repeats. However, amplification between Sh73 and the previously described DraI repeat exhibited discriminative banding patterns for S. haematobium and Schistosoma bovis (sensitivity 1 pg and 10 pg, respectively). It also enabled banding pattern discrimination of Schistosoma curassoni and Schistosoma intercalatum, but Schistosoma mattheei and Schistosoma margrebowiei did not yield amplicons. Similar inter-repeat amplification between Sh77 and DraI yielded amplicons with discriminative banding for S. haematobium, and S. bovis; however, S. mattheei was detected only at low sensitivity (1 ng). The Sh73/DraI assay detected snails infected with S. haematobium, S. bovis, or both, and should prove useful for screening snails where discrimination of S. haematobium from related schistosomes is required.

A comparative analysis of coprologic diagnostic methods for detection of Toxoplama gondii in cats.

The relative role of transmission of Toxoplasma gondii infection from cats to humans appears to have recently increased in certain areas. Large-scale screening of oocyst shedding in cats cannot rely on microscopy because oocyst identification lacks sensitivity and specificity, or on bioassays, which require test animals and weeks before examination. We compared a sensitive and species-specific coprologic-polymerase chain reaction (copro-PCR) for detection of T. gondii infected cats with microscopy and a bioassay. In experimentally infected cats followed over time, microscopy was positive occasionally, and positive copro-PCR and bioassay results were obtained continuously from days 2 to 24 post-infection. The copro-PCR is at least as sensitive and specific as the bioassay and is capable of detecting infective oocysts during cat infection. Therefore, this procedure can be used as the new gold standard for determining potential cat infectivity. Its technologic advantages over the bioassay make it superior for large-scale screening of cats.

Detection of Schistosoma mansoni and Schistosoma haematobium DNA by loop-mediated isothermal amplification: identification of infected snails from early prepatency.

Monitoring post-control transmission of schistosomes by examining humans becomes less effective as infection rates among humans decrease. Molecular monitoring of prepatent schistosome infection in snails by the polymerase chain reaction (PCR) has been used for studying human-to-snail transmission, and snail prepatent infection rates were found to correspond to infection prevalence and average intensity in human populations contacting the sites studied. We have now developed loop-mediated isothermal amplification (LAMP) assays for identifying Schistosoma mansoni and S. haematobium to facilitate large-scale evaluation of post-intervention transmission potential. LAMP primers were designed based on the Sm1-7 and DraI repeated sequences of the corresponding schistosomes, and amplification by LAMP of these 121-basepair highly abundant sequences provided a detection sensitivity of 0.1 fg of genomic DNA. When these LAMP assays were applied for examining infected laboratory snails, it was possible to identify infection from the first day after exposure to miracidia. The potential advantages of these assays are discussed.

Polymerase chain reaction-based detection of lymphatic filariasis.

PCR-based diagnostic assays are promising tools for the monitoring and evaluation of the Global Programme for Elimination of Lymphatic Filariasis. Sensitive and specific assays have been described for the detection of Wuchereria bancrofti, Brugia malayi, or Brugia timori infection in blood, sputum, and vectors. These techniques can be most cost-effective when employed for pool screening, which is important in the later stages of control programs when infection rates of humans and vectors are low, and large numbers of samples must be examined.

Detection of bird schistosomes in lakes by PCR and filter-hybridization.

Many lakes around the world are contaminated with bird schistosome cercariae, which penetrate into human skin, causing an itching dermatitis called "swimmers' itch." Bathers could be forewarned from exposure to the larvae and ecological examinations could be performed, when a sensitive method to detect the parasites in aquatic systems, where lots of organisms hinder microscopic examinations, would be available. For this purpose we cloned, sequenced, and analyzed a 396 bp tandem repeated DNA sequence from Trichobilharzia ocellata (ToSau3A), and employed it for developing molecular detection assays. It hybridized with less than 100 pg DNA from different Trichobilharzia species (T. ocellata, Trichobilharzia franki, and Trichobilharzia regenti), but not with 10 ng DNA from other related or sympatric trematodes. A PCR assay, amplifying this sequence with the same specificity, detected 100 fg T. ocellata DNA, 1 cercaria in 0.5 g plankton, and 2 cercariae in 0.5 g host snail tissues.