Real-time PCR diagnosis of Schistosoma japonicum in low transmission areas of China

Background:Schistosomiasis in the People’s Republic of China(PRC)can be traced back to antiquity.In the past 60 years,the Chinese government has made great efforts to control this persistent disease with elimination slated by 2020 through the implementation of a comprehensive control strategy.This strategy aims to reduce the role of bovines and humans as sources of infection as a pre-requisite for elimination through transmission interruption.The goal of elimination will be achievable only by the implementation of a sustainable surveillance and control system,with sensitive diagnosis a key feature so that the true disease burden is not underestimated.Currently used diagnostics lack the necessary sensitivity to accurately determine the prevalence of Schistosoma japonicum infection in areas with low infection intensities.It is of critical importance to find and treat people and to identify animals with low-level infections if the National Control Programme for China is to achieve schistosomiasis elimination.Methods:We evaluated a real-time polymerase chain reaction(qPCR)assay using 633 human stool samples collected from five villages in Hunan,Anhui,Hubei,and Jiangxi provinces,and 182 bovine(70 cattle and 112 buffalo)stool samples obtained from four villages in Hunan,Anhui,and Jiangxi provinces in the PRC.All stool samples were subjected to the miracidium hatching test(MHT,a diagnostic procedure used in the National Schistosomiasis Control Programme)and the qPCR assay.Samples positive by MHT were subjected to either the Kato-Katz technique for humans,or the formalin-ethyl acetate sedimentation-digestion(FEA-SD)procedure for bovines,to determine infection intensities.Results:The qPCR assay exhibited a high level of sensitivity in the detection of S.japonicum infections.With both the human and bovine samples,a significantly higher prevalence was determined using the qPCR assay(11.06%humans,24.73%bovines)than with the MHT(0.93%humans,7.69%bovines).The animal contamination index(calculated using data obtained with the qPCR technique)for all positive bovines was 27618000 eggs per day,indicating a considerable amount of environmental egg contamination that would be underestimated using less sensitive diagnostic procedures.Conclusions:The qPCR assay we have evaluated will be applicable as a future field diagnostic and surveillance tool in low-transmission zones where schistosomiasis elimination is targeted and for monitoring post-intervention areas to verify that elimination has been maintained.

Distribution pattern of the snail intermediate host of schistosomiasis japonica in the Poyang Lake region of China

Background:With the closure of the Three Gorges Dam in 2003 the hydrology of Poyang Lake was altered dramatically leading to significant changes in the environment.In order to assess the impact on schistosomiasis this study assessed the spatial and temporal patterns of the snail intermediate host,Oncomelania hupensis in the Poyang Lake tributaries.The results of the study have important implications for future snail control strategies leading to disease elimination.Methods:The marshland area surrounding Poyang Lake was divided randomly into 200×200 m vector grids using ArcGIS software,and the surveyed grids were randomly selected by the software.The snail survey was conducted in each selected grid using a survey frame of 50×50 m with one sideline of each grid serving as the starting line.No less than ten frames were used in each surveyed grid with Global Positioning System(GPS)recordings for each.All snails in each frame were collected to determine infection status by microscopy.Altitude data for all frames were extracted from a lake bottom topographic map in order to analyze the average altitude.All snail survey data were collected and statistically analyzed with SPSS 20.0 software in order to determine the difference of the percentage of frames with living snails and mean density of living snails in different regions of Poyang Lake.The altitude of the snail-infested marshlands and snail dens were subsequently identified.Results:A total of 1159 potential snail sampling grids were surveyed,of which 15231 frames(0.1 m2/frame)were investigated.1241 frames had live Oncomelania snails corresponding to 8.15%of the total number of frames.The mean density of living snails was 0.463/0.1 m2 with a maximum of 57 snails per frame.The percent of frames with snails in the southern sector(8.13%)of Poyang Lake did not differ statistically from the north(8.21%).However,the mean density of live snails in the northern sector(0.164/0.1 m2)of the lake was statistically higher(F=6.727;P=0.010)than the south(0.141/0.1 m2).In the south of the lake,the elevation of snail-inhabited marshland ranged between 11-16 m,and could be further subdivided into two snail-concentrated belts at 12-13 m of elevation and 15-16 m of elevation respectively.In the north of the lake,the elevation of snail-inhabited marshland ranged between 9-16 m with the elevation of 12-14 m being the snail-concentrated zone.Conclusions:The elevation of snail-infested marshlands in the Poyang Lake region ranged from 9 to 16 m.The snail distribution and habitat has moved north of the lake and to a lower altitude due to changes in the water level post dam closure.Based on the current geological features of the snail habitant focused mollusciciding should occur in snail dense northern regions with frequent bovine and human traffic.Targeting these identified'hotspots'of transmission will assist in elimination efforts.

Co-parasitism of intestinal protozoa and Schistosoma japonicum in a rural community in the Philippines

Background:Co-parasitism is a frequent occurrence in impoverished communities in the tropics resulting in a considerable disease burden.While there are extensive reports of intestinal helminthiases,including schistosomiasis japonica,the occurrence and extent of diseases caused by intestinal protozoa(IP)have yet to be investigated in depth in the Philippines.We present a detailed analysis of polyparasitism in a rural community of Northern Samar,focusing on co-infections of IP with Schistosoma japonicum.Methods:A descriptive cross sectional study was carried out in 2015 across 18 barangays(villages)endemic for S.japonicum in Northern Samar,the Philippines to assess the burden of human schistosomiasis and IP infections.Faecal samples collected from 412 participants from the 18 barangays were included in the final molecular analysis.A multiplex quantitative PCR assay was developed and used for the detection of Blastocystis spp.,Entamoeba histolytica,Cryptosporidium spp.and Giardia duodenalis in stool samples.The findings were combined with previous results of droplet digital PCR diagnosis of individuals from the same 18 barangays infected with S.japonicum determined using the same stool samples for analysis.Results:Mean age of the study participants was 40.3 years(95%CI:38.8-41.8)with 53%(n=218)being males.Prevalence of S.japonicum(74.5%)and Blastocystis spp.(58.7%)was significantly higher compared to other infections,with E.histolytica having the lowest prevalence(12.1%).A majority of individuals were infected with more than one parasite with two infections being most common(n=175,42.5%).The prevalence of individuals with two parasites was significantly higher than all others with 27.9%(n=115)subjects harbouring a single parasite species.Of individuals with two infections,S.japonicum and Blastocystis spp.were the most common combination(n=110,62.9%).Examining age within the population,58.5%(n=38)of school-aged children and 60.1%(n=14)of women of child bearing age harboured at least two parasite species.Conclusions:The study revealed that polyparasitism with IP infections and schistosomiasis japonica is highly prevalent in individuals in Northern Samar which likely contributes to the significant public health and socio-economic burden suffered by this population.More generally,the findings are of relevance when considering implementation of integrated control strategies for intestinal parasites.

Performance of the point-of-care circulating cathodic antigen test in the diagnosis of schistosomiasis japonica in a human cohort from Northern Samar, the Philippines

Background:Zoonotic schistosomiasis,caused by Schistosoma japonicum,remains a major public health problem in the Philippines.This study aimed to evaluate the commercially available rapid diagnostic point-of-care circulating cathodic antigen(POC-CCA)test in detecting individuals infected with S.japonicum in a human cohort from an endemic area for schistosomiasis japonica in the Philippines.Methods:Clinical samples were collectedin 18 barangays endemic for S.japonicum infection in Laoang and Palapag municipalities,Northern Samar,the Philippines,in 2015.The presence of CCA in flter-concentrated urine samples(n=412)was evaluated using the commercial kits and the results were converted to images,which were further analyzed by ImageJ software to calculate R values.The diagnostic performance of the immunochromatographic POCCCA test was compared using the Kato-Katz(KK)procedure,in-house enzyme-linked immunosorbent assays(ELISAs)and droplet digital(dd)PCR assays as reference.Results:The POC-CCA test was able to detect S.japonicum-infected individuals in the cohort with an eggs per gram of faeces(EPG)more than or equal to 10 with sensitivity/specifcity values of 63.3%/93.3%.However,the assay showed an inability to diagnose schistosomiasis japonica infections in all cohort KK-positive individuals,of which the majority had an extremely low egg burden(EPG:1–9).The prevalence of S.japonicum infection in the total cohort determined by the POC-CCA test was 12.4%,only half of that determined by the KK method(26.2%).When compared with the ELISAs and ddPCR assays as a reference,the POC-CCA assay was further shown to be a test with low sensitivity.Nevertheless,the assay exhibited signifcant positive correlations with egg burden determined by the KK technique and the target gene copy number index values determined by the ddPCR assays within the entire cohort.Conclusions:By using in silico image analysis,the POC-CCA cassette test could be converted to a quantitative assay to avoid reader-variability.Because of its low sensitivity,the commercially available POC-CCA assay had limited potential for determining the status of a S.japonicum infection in the target cohort.The assay should be applied with caution in populations where schistosome parasites(especially S.japonicum)are present at low infection intensity.