Use of cell-free circulating schistosome DNA in serum, urine, semen, and saliva to monitor a case of refractory imported schistosomiasis hematobia.

This case of imported refractory schistosomiasis has highlighted the usefulness of cell-free parasite DNA as a diagnostic marker to assess active schistosome infection. In contrast to the rapid disappearance of ova in urine, parasite DNA remained persistent in several other specimen types even after the fourth treatment with praziquantel. This result was consistent with the presence of morphologically intact ova in bladder biopsy samples and with the corresponding symptoms.

Molecular factors that are associated with early developmental arrest of intraerythrocytic Plasmodium falciparum.

Malaria continues to be a devastating disease. We investigated the factors that control intraerythrocytic development of the parasite Plasmodium falciparum by using a chemically defined medium (CDM) containing non-esterified fatty acid(s) (NEFA) and phospholipids with specific fatty acid moieties, to identify substances crucial for parasite development. Different NEFAs in the CDM played distinct roles by altering the development of the parasite at various stages, with effects ranging from complete growth to growth arrest at the ring stage. We used genome-wide transcriptome profiling to identify genes that were differentially expressed among the different developmental stages of the parasite, cultured in the presence of various NEFAs. We predicted 26 transcripts that were associated with the suppression of schizogony, of which 5 transcripts, including merozoite surface protein 2, a putative DEAD/DEAH box RNA helicase, serine repeat antigen 3, a putative copper channel, and palmitoyl acyltransferase, were particularly associated with blockage of trophozoite progression from the ring stage. Furthermore, the involvement of copper ions in developmental arrest was detected by copper-ion-chelating methods, implying a critical function of copper homeostasis in the early growth stage of the parasite. These results should help to elucidate the mechanisms behind the development of P. falciparum.

Therapeutic effect of adjuvant therapy added to constraint-induced movement therapy in patients with subacute to chronic stroke: a systematic review and meta-analysis.

PURPOSE: This review investigated the effectiveness of adjuvant therapy combined with constraint-induced movement therapy (CIMT) in improving the paretic upper limb functionality in adults with stroke sequelae during the subacute to chronic rehabilitation phase. MATERIALS AND METHODS: In this systematic review and meta-analysis of randomized controlled trials (RCT), electronic databases, including PubMed, Web of Science, CINAHL, and MEDLINE, were searched. We included RCTs that investigated the outcomes of adjuvant therapy (i.e. other therapies) added to CIMT compared with CIMT alone. Key trial findings were qualitatively synthesized and analyzed. This meta-analysis examined variables, such as mean scores and standard deviations, using the following outcome measures: Fugl-Meyer Assessment (FMA) upper limb items, Action Research Arm Test (ARAT), Amount of Use (AOU) of Motor Activity Log (MAL), and Quality of Movement (QOM) of MAL. RESULTS: Eighteen eligible RCTs were included in the analysis. Adding CIMT to adjunctive therapy significantly improved FMA compared with CIMT alone (mean difference [MD] 4.02, 95% confidence interval [CI] 2.60-5.44; I2 = 85%; 15 studies; 330 participants). Similarly, the ARAT and MAL-AOU scores improved significantly. CONCLUSIONS: CIMT combined with several adjunctive therapies effectively improved upper limb function.

In recent years, clinical trials combining other therapies with Constraint-induced movement therapy (CIMT) have become increasingly common.This study shows that combining CIMT with adjuvant therapy improves upper limb function.Different protocols of the CIMT in each study could be factor that impacted the results of Motor Activity Log.In clinical practice, the findings of this study into their treatment protocols to improve patient outcomes and ensure the effective application of evidence-based rehabilitation strategies.

Development and Accuracy Evaluation of Lateral Flow Immunoassay for Rapid Diagnosis of Schistosomiasis Mekongi in Humans.

Schistosoma mekongi infection is endemic in countries along the Mekong River and certain of its tributaries in the lower Mekong basin, especially in Lao People's Democratic Republic and Cambodia. Diagnosis of schistosomiasis is crucial before treatment and epidemiological surveys before and/or after an intervention, such as a mass drug administration. A newly developed immunochromatographic test (ICT) for the diagnosis of schistosomiasis mekongi, based on antiparasite antibody detection in human sera, was evaluated. The schistosomiasis mekongi-ICT (Smk-ICT) strip was developed using somatic antigen from adult S. mekongi. In total, 209 serum samples were examined, including 14 from parasitologically proven schistosomiasis mekongi patients, 30 from schistosomiasis japonica patients, other parasitosis (n = 135), and healthy volunteers (n = 30) from areas not endemic for S. mekongi. Eleven schistosomiasis mekongi samples were positive according to the Smk-ICT, whereas all healthy control samples were negative. Cross-reactions with paragonimiasis heterotremus, sparganosis, trichinellosis, and taeniasis saginata samples were observed at 2.4% (4/165). The diagnostic sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 78.6% (95% confidence interval [CI] 49.2-95.3), 97.6% (95% CI 93.9-99.3), 73.3% (95% CI 44.9-92.2), 98.2% (95% CI 94.7-99.6), and 96.1% (95% CI 92.1-98.4), respectively. The Smk-ICT kit might be useful to assess the prevalence of disease before establishing transmission control and mass deworming campaigns in countries in the Mekong River subregion.

Efficacy of sodium metaperiodate (SMP)-ELISA for the serodiagnosis of schistosomiasis mekongi.

Schistosomiasis mekongi is an important public health issue in endemic countries. In this study, we evaluated an indirect immunodiagnostic ELISA method using Schistosoma mekongi soluble egg antigen. Sodium metaperiodate (SMP)-ELISA was utilized in order to remove the glycosylated epitopes responsible for false positive reactions and the results using this method were compared with those using conventional ELISA (conv-ELISA). Forty-two serum samples from schistosomiasis mekongi egg-positive patients and 100 serum samples from schistosomiasis-negative Cambodian subjects were tested using both ELISA methods. The ranges of ELISA values for positive and negative sera were distinct on SMP-ELISA, but the ranges of the two groups of sera overlapped on conv-ELISA. Therefore, diagnostic criteria may be established based on the highest ELISA value on negative sera and the lowest ELISA value on positive sera. In the present study, both the sensitivity and specificity of SMP-ELISA reached 100% using the criteria in which an ELISA value > or = 0.2 was positive.

Risk analysis of the re-emergence of Plasmodium vivax malaria in Japan using a stochastic transmission model.

OBJECTIVES: This study analyzed the risk of infection with Plasmodium vivax in local residents through a stochastic simulation in which an infected tourist, local resident, or immigrants from an endemic area would visit Himi-shi, Toyama prefecture, which is a formerly endemic area in Japan. METHODS: In Toyama, the habitats of Anopheles sinensis, which can transmit P. vivax, have been examined previously. We constructed a stochastic model of P. vivax transmission that can handle small numbers of infected persons and infected mosquitoes. The seasonal fluctuation in the numbers of captured An. sinensis was taken into account in the model. RESULTS: Ten thousand trial simulations were carried out stochastically with a range of human blood indexes (HBI) of 1-10% for a range of months (June-September). The simulation results for a realistic assumption of a 1% HBI showed that the risk of infection for local residents was low (below 1%) except for the immigrants scenario. CONCLUSIONS: The risk of infection among local residents (second cycle) was estimated to be very low for all situations. Therefore, there is little possibility for P. vivax infection to become established in this area of Japan.

Comparison of point-of-care test and enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies in the diagnosis of human schistosomiasis japonica.

OBJECTIVE: Schistosomiasis japonica is an important helminthic disease in Asia. Sensitive and accurate diagnostic tools are indispensable for clinical diagnosis, screening infection and monitoring its control. In this study, we developed an immunochromatographic test (Sj-ICT) to detect anti-Schistosoma japonicum immunoglobulin G antibodies in human sera. METHODS: Somatic extract from adult S. japonicum was used as an antigen. The Sj-ICT was developed and optimized as a point-of-care test. All 214 human serum samples were evaluated for diagnostic usefulness and comparison with an enzyme-linked immunosorbent assay (ELISA). RESULTS: The diagnostic sensitivity, specificity, positive and negative predictive values, and accuracy of the Sj-ICT were 90.8%, 87.9%, 86.4%, 91.9% and 89.3%, respectively. For ELISA the values were respectively 91.8%, 87.9%, 86.5%, 92.7% and 89.7%. The concordance between both methods was 86.4 % (Cohen's kappa value = 0.729). CONCLUSIONS: The immunochromatographic test kit developed can support clinical diagnosis and large-scale surveys in endemic areas without requiring additional facilities or ancillary supplies.

Implementation of a novel PCR based method for detecting malaria parasites from naturally infected mosquitoes in Papua New Guinea.

BACKGROUND: Detection of Plasmodium species in mosquitoes is important for designing vector control studies. However, most of the PCR-based detection methods show some potential limitations. The objective of this study was to introduce an effective PCR-based method for detecting Plasmodium vivax and Plasmodium falciparum from the field-caught mosquitoes of Papua New Guinea. METHODS: A method has been developed to concurrently detect mitochondrial cytochrome b (Cyt b) of four human Plasmodium species using PCR (Cytb-PCR). To particularly discriminate P. falciparum from P. vivax, Plasmodium ovale and Plasmodium malariae, a polymerase chain reaction-repeated fragment length polymorphism (PCR-RFLP) has further been developed to use with this method. However, due to limited samples number of P. ovale and P. malariae; this study was mainly confined to P. vivax and P. falciparum. The efficiency of Cytb-PCR was evaluated by comparing it with two 'gold standards' enzyme linked immunosorbent assay specific for circumsporozoite protein (CS-ELISA) using artificially infected mosquitoes; and nested PCR specific for small subunit ribosomal RNA (SSUrRNA) using field caught mosquitoes collected from three areas (Kaboibus, Wingei, and Jawia) of the East Sepic Province of Papua New Guinea. RESULTS: A total of 90 mosquitoes were artificially infected with three strains of Plasmodium: P. vivax-210 (n = 30), P. vivax-247 (n = 30) and P. falciparum (n = 30). These infected mosquitoes along with another 32 unfed mosquitoes were first checked for the presence of Plasmodium infection by CS-ELISA, and later the same samples were compared with the Cytb-PCR. CS-ELISA for P. vivax-210, P. vivax-247 and P. falciparum detected positive infection in 30, 19 and 18 mosquitoes respectively; whereas Cytb-PCR detected 27, 16 and 16 infections, respectively. The comparison revealed a close agreement between the two assays (kappa = 0.862, 0.842 and 0.894, respectively for Pv-210, Pv-247 and P. falciparum groups). It was found that the eight CS-ELISA-positive mosquitoes detected negative by Cytb-PCR were false-positive results. The lowest detection limit of this Cytb-PCR was 10 sporozoites. A highly concordance result was also found between nested PCR and Cytb-PCR using 107 field caught mosquitoes, and both tests concordantly detected P. falciparum in an Anopheles punctulatus mosquito collected from Kaboibus. Both tests thus suggested an overall sporozoite rate of 0.9% (1/107) in the study areas. Subsequently, PCR-RFLP efficiently discriminated P. falciparum from P. vivax for all of the Cytb-PCR positive samples. CONCLUSION: A single step PCR based method has been introduced here that is highly sensitive, efficient and reliable for identifying P. vivax and P. falciparum from mosquitoes. The reliability of the technique was confirmed by its ability to detect Plasmodium as efficiently as those of CS-ELISA and nested PCR. Application of the assay offers the opportunity to detect vector species of Papua New Guinea and may contribute for designing further vector control programmes.

Plasmodium falciparum: development and validation of a measure of intraerythrocytic growth using SYBR Green I in a flow cytometer.

Reliable analytical techniques to test growth-promoting and antimalarial efficacy on plasmodia are very important. Flow cytometry (FCM) offers the possibility to study developmental stages of intraerythrocytic growth of malaria parasites using nucleic acid staining. To analyze the growth of Plasmodium falciparum SYBR Green I was introduced as an intercalating dye with FCM for the 488nm line of an argon laser. Procedures employing FCM, including fixatives, dye concentrations, dilution buffer, and staining period, were optimized to simplify the method. FCM as described here allows parasitemia and parasites of different stages to be quantified according to the DNA content. The proportion of parasitized erythrocytes estimated by FCM and the Giemsa method agreed with determination by parasite lactate dehydrogenase. The protocol was extended to merozoite counting as a sensitive assay of growth inhibition of the parasite.

Modeling the dynamics and control of transmission of Schistosoma japonicum and S. mekongi in Southeast Asia.

A mathematical model for transmission of schistosomes is useful to predict effects of various control measures on suppression of these parasites. This review focuses on epidemiological and environmental factors in Schistosoma japonicum and Schistosoma mekongi infections and recent advances in mathematical models of Schistosoma transmission.

Elimination of Schistosomiasis Mekongi from Endemic Areas in Cambodia and the Lao People's Democratic Republic: Current Status and Plans.

The areas endemic for schistosomiasis in the Lao People's Democratic Republic and in Cambodia were first reported 50 and 60 years ago, respectively. However, the causative parasite Schistosoma mekongi was not recognized as a separate species until 1978. The infection is distributed along a limited part of the Mekong River, regulated by the focal distribution of the intermediate snail host Neotricula aperta. Although more sensitive diagnostics imply a higher figure, the current use of stool examinations suggests that only about 1500 people are presently infected. This well-characterized setting should offer an exemplary potential for the elimination of the disease from its endemic areas; yet, the local topography, reservoir animals, and a dearth of safe water sources make transmission control a challenge. Control activities based on mass drug administration resulted in strong advances, and prevalence was reduced to less than 5% according to stool microscopy. Even so, transmission continues unabated, and the true number of infected people could be as much as 10 times higher than reported. On-going control activities are discussed together with plans for the future.

Allelic dimorphism-associated restriction of recombination in Plasmodium falciparum msp1.

Allelic dimorphism is a characteristic feature of the Plasmodium falciparum msp1 gene encoding the merozoite surface protein 1, a strong malaria vaccine candidate. Meiotic recombination is a major mechanism for the generation of msp1 allelic diversity. Potential recombination sites have previously been mapped to specific regions within msp1 (a 5' 1-kb region and a 3' 0.4-kb region) with no evidence for recombination events in a central 3.5-kb region. However, evidence for the lack of recombination events is circumstantial and inconclusive because the number of msp1 sequences analysed is limited, and the frequency of recombination events has not been addressed previously in a high transmission area, where the frequency of meiotic recombination is expected to be high. In the present study, we have mapped potential allelic recombination sites in 34 full-length msp1 sequences, including 24 new sequences, from various geographic origins. We also investigated recombination events in blocks 6 to 16 by population genetic analysis of P. falciparum populations in Tanzania, where malaria transmission is intense. The results clearly provide no evidence of recombination events occurring between the two major msp1 allelic types, K1-type and Mad20-type, in the central region, but do show recombination events occurring throughout the entire gene within sequences of the Mad20-type. Thus, the present study indicates that allelic dimorphism of msp1 greatly affects inter-allelic recombination events, highlighting a unique feature of allelic diversity of P. falciparum msp1.

Antibody isotype responses to paramyosin, a vaccine candidate for schistosomiasis, and their correlations with resistance and fibrosis in patients infected with Schistosoma japonicum in Leyte, The Philippines.

We examined whether antibody isotype responses to paramyosin (PM), a vaccine candidate for schistosomiasis, are associated with age-dependent resistance and pathology in liver fibrosis using human sera collected from 139 individuals infected with Schistosoma japonicum in Leyte, The Philippines. We report that IgA and IgG3 responses to PM showed a positive correlation with age and that the epitopes responsible were localized predominantly within the N-terminal half of PM. In addition, the IgG3 response to PM was associated with serum level of procollagen-III-peptide (P-III-P), an indicator of progression of liver fibrosis. These results imply that IgG3 against PM may not only provoke age-dependent resistance to S. japonicum infection but also enhance liver fibrosis. In contrast, levels of IgE to PM and to multiple PM fragments showed a negative correlation with P-III-P level. Thus, in contrast to IgG3, increases in PM-specific IgE may contribute to suppression of liver pathogenesis in schistosomiasis.

High susceptibility of Neotricula aperta gamma-strain from Krakor and Sdau in Cambodia to Schistosoma mekongi from Khong Island in Laos.

Neotricula aperta gamma-strain snails collected from Krakor and Sdau in Cambodia were found to have the same or higher susceptibility to Schistosoma mekongi as N. aperta originally isolated from Khong in Laos. Infection rates of N. aperta gamma-strain snails exposed to 3 miracidia at week 8 were: Khong gamma-strain, 22.6%; Krakor gamma-strain, 33.3%; and Sdau gamma-strain, 67.4%. At week 10, the Sdau gamma-strain showed the highest infection rate of 83.3%. We thus found significantly high susceptibility of the Sdau gamma-strain to S. mekongi originally isolated from Khong. However, in another experiment, susceptibility of the Sdau gamma-strain was rather comparable to that of Khong and Krakor gamma-strain. We also found no significant differences in infection and survival rates between the Khong and Krakor gamma-strain when the snails were exposed to 3 or 6 miracidia. This is the first report to confirm the high susceptibility in the laboratory of N. aperta gamma-strain snails from endemic areas in Cambodia to S. mekongi originally isolated from Laos. The high susceptibility of N. aperta gamma-strain snails to S. mekongi in distant areas may be an important factor in the endemic transmission of human schistosomiasis.

Limited allelic diversity of Plasmodium falciparum merozoite surface protein 1 gene from populations in the Solomon Islands.

Meiotic recombination generates allelic diversity in the Plasmodium falciparum merozoite surface protein 1 (msp1) gene. In this study, we monitored recombination-based diversity of msp1 in Guadalcanal, the Solomon Islands, where malaria transmission is high. We identified 5' recombinant types, 3' sequence types, and msp1 haplotypes (unique associations of 5' recombinant types and 3' sequence types), and compared them with those from areas of low transmission in Thailand and Vanuatu. The mean number of 5' recombinant types per person (multiplicity) was lower in Guadalcanal than in Thailand. Guadalcanal populations had 6-8 msp1 haplotypes; the numbers are comparable to Vanuatu but much lower than in Thailand. There were marked geographic differences in distribution of msp1 haplo-types. Linkage disequilibrium in msp1 was stronger in Guadalcanal than in Thailand, suggesting limited recombination events in the Solomon Islands. We suggest that the frequency of recombination events in msp1 is determined not only by transmission intensity but by the number of msp1 alleles prevalent and multiplicity of infections.

Modeling the dynamics and control of Schistosoma japonicum transmission on Bohol island, the Philippines.

We have investigated a mathematical model for the transmission of Schistosoma japonicum in the infested region of northeastern Bohol island in the Philippines. The development of transmission models is important for planning control strategies. Since S. japonicum has a complicated mode of transmission, the rates of transmission among its hosts cannot be measured directly by field observation. Instead, they have been estimated through model analysis. The model takes into account the seasonal variations and includes a function of control measures. In 1981, a project to eliminate schistosomiasis started on Bohol island. The prevalence decreased dramatically and has kept low level less than 1%. The simulations based on the model predicted that there is little probability of resurgence of an epidemic in the northeastern endemic villages of Bohol island due to the fact that the project has attained a high coverage of selective mass treatment based on stool examination accompanied by a successful snail control operation.

Effects of repeated praziquantel treatment on schistosomiasis mekongi morbidity as detected by ultrasonography.

Schistosomiasis mekongi is endemic in the Mekong River basin; about 80,000 people are at risk of infection in Cambodia. We conducted ultrasonographic studies of patients with schistosomiasis mekongi in Kratie province, Cambodia, focusing especially on the relationship between the frequency of praziquantel treatment and findings of ultrasonographic imaging. The frequency of praziquantel treatment in the period from 1995 to 2002 was classified into four groups: 1-2, 3-4, 5-6, and 7-8 times. Ultrasonographic images were examined to determine the presence of thickening of the portal vein wall and formation of meandering collateral circulation of the splenic vein. We selected these parameters because they are unaffected by interobserver bias. The results showed that thickening of the portal vein wall may have potential to improve with frequent praziquantel treatment. On the other hand, established hard splenomegaly and meandering collateral circulation of the splenic vein, improved very little with praziquantel treatment.

Determination of the period for establishment of a liver network echogenic pattern in Schistosoma japonicum infection.

Schistosomiasis is caused by infection with Schistosoma haematobium, S. mansoni, S. japonicum, or S. mekongi. S. japonicum infection results in liver cirrhosis at the final stage. A "network" (NW) echogenic pattern on hepatic ultrasonography appears to be specific to S. japonicum infection. The principal aim of the present study was to determine the exact year(s) or even month(s) required for the establishment of the liver NW echogenic pattern from the initial infection in young patients with schistosomiasis japonica since there are few data on this important point. We conducted yearly ultrasonographic, serologic, coprologic, and physical examinations of schistosomiasis patients in the Philippines from 1996 up to the present. During that period, the total number of patients examined was approximately 2,000, among whom we selected 2 patients for determination of the duration required for NW establishment, when they were 10 years old. Although the exact time of initial exposure to schistosomes cannot be determined, the duration for the establishment of NW was definitively confirmed in patient no. 1 to be between 19-24 months based on the results of serologic and coprologic examinations. For patient no. 2, the circumstantial evidence suggested that the establishment of a NW might require 5 to 6 years at maximum. To the best of our knowledge, this is the first evidence-based report on the determination of the period required for the establishment of a liver NW echogenic pattern in S. japonicum infection in the Philippines.

Evaluation of the anthelmintic effects of artesunate against experimental Schistosoma mansoni infection in mice using different treatment protocols.

The therapeutic effects of artesunate against experimental Schistosoma mansoni infection in mice were analyzed. Previous studies showed that artesunate is highly effective against S. japonicum infection, but the action of this drug against S. mansoni remained uncovered. The present study examines the optical conditions for artesunate against S. mansoni and evaluates the effects of inhibiting the sexual maturation of adult worms. Mice infected with S. mansoni were orally administered with artesunate according to different schedules. Four consecutive administrations of 300 mg/kg of artesunate at 2-week intervals conferred almost total protection without the development of pathological lesions in the liver. The significant reduction in the number of eggs produced by surviving worms and the status of egg maturation suggested that artesunate inhibits sexual maturation. Electron microscopy revealed that artesunate caused morphological damage, especially on the worm tegument. Artesunate was also very effective in iron-deficient mice. Furthermore, the efficacy of artesunate was equal to or better than that of artemether against S. japonicum infection. Considering that artemether is more toxic, artesunate is currently one of the most efficient drugs against immature S. mansoni.

Detection of active schistosome infection by cell-free circulating DNA of Schistosoma japonicum in highly endemic areas in Sorsogon Province, the Philippines.

The current status of schistosomiasis in highly endemic areas is difficult to determine by ovum detection because of the superficially low parasite load after mass drug administration, whereas the parasite transmission rates are still high. Cell-free parasite DNA is fragments of parasite-derived DNA existing in the host's body fluids. We conducted population-based studies to test the presence of cell-free schistosome DNA in endemic areas of Sorsogon Province, the Philippines. Schistosome DNA in the serum and urine of Kato-Katz (KK)-positive subjects was detected by PCR (100% sensitivity). Schistosome DNA was also detected from KK-negative subjects (9/22 serum and 10/41 urine samples). Schistosome DNA was found to be network echogenic pattern (NW)-positive (serum 53.3%, urine 42.9%) or NW-negative (serum 25.5%, urine 20.8%) and enzyme-linked immunosorbent assay (ELISA)-positive (serum 47.1%, urine 40%) or ELISA-negative (serum 33.3%, urine 13.3%). These results indicate that cell-free schistosome DNA is a promising diagnostic marker for active schistosome infection in the case of light infection.