The loop-mediated isothermal amplification (LAMP) test is a high-performance method for detecting DNA, which holds promise for use in molecular diagnosis of pathogens in the first line battle against infectious diseases, including helminths and protozoa. LAMP uses a set of primers that initiate large-scale nucleic acid synthesis by Bst DNA polymerase at isothermal conditions. Research is now focused on the identification of sensitive and specific diagnostic tests for early identification of schistosomal infection and evaluation of chemotherapy in epidemiological surveys in P.R. China . The data presented here indicate that LAMP is suitable for the detection of early infection in high-risk groups of S. japonicum, such as migrants, travellers, military personnel and school-aged children. However, it is less suitable for assessing efficacy of chemotherapy in the early stages because of its high sensitivity.
A. cantonensis is a zoonotic parasite that causes eosinophilic meningitis in humans . The most common source of infection with A. cantonensis is the consumption of raw or undercooked molluscs (e.g. snails and slugs) harbouring infectious third-stage larvae (L3). However, the parasite is difficult to identify within the snails. The purpose of this study was to develop a quick, simple molecular method to survey for A. cantonensis in intermediate host snails. LAMP is an appropriate diagnostic method for the routine identification of this parasite within its intermediate host snail Pomacea canaliculata because of its simplicity and the high sensitivity and specificity. Hence, LAMP holds promise as a useful monitoring tool for A. cantonensis in endemic regions .
P. vivax is the major cause of malaria outside Africa, mainly in Asia and the Americas . In P.R. China, more than 90% of the total malaria cases are due to P. vivax. In the central part of P.R. China, the re-emergence of malaria was considerable in provinces along the Huanghuai River, especially in Anhui and Henan provinces. As candidate methods for field malaria diagnosis, several different primer sets targeting numerous genes have been developed. It has been claimed that the LAMP method can detect as few as 100 copies of DNA template in blood samples (equal to roughly five parasites/μl of blood). This sensitivity is notably higher than any currently known immunochromatography-based malaria rapid diagnostic test (RDT) as recommended by the World Health Organization (WHO) as part of the global malaria control strategy , including PCR). A visualized LAMP method was established by the addition of a microcrystalline wax-dye capsule containing the highly sensitive DNA fluorescence dye SYBR Green I to a normal LAMP reaction prior to the initiation of the reaction . Although further validation is needed and indeed ongoing, we can conclude that this novel, cheap and quick visualized LAMP method is feasible for malaria diagnosis under resource-constrained field settings in rural parts of P.R. China. Another novel LAMP targeting the 529 bp repeat element (529 bp-LAMP) was established to detect T. gondii DNA in blood samples of experimental mice infected with tachyzoites of the RH strain. Due to its simplicity, high sensitivity and cost-effectiveness for common use, we suggest that this assay should be used as an early diagnostic tool for health control of toxoplasmosis .
Early identification of the infection is essential to provide timely and appropriate chemotherapy to patients. Multiplex ligation-dependent probe amplification (MLPA) is a simple, robust and fast method designed for simultaneous detection of specific genomic sequences targeting multiple mutations to amplify specific MLPA probes rather than target DNA. The flexibility and specificity make MLPA a potential tool for specific identification of infections by opisthorchid liver flukes in endemic areas. MLPA has been used for rapid and specific detection of single nucleotide acid differences between C. sinensis, O. viverrini and O. felineus. The flexibility and specificity make MLPA a potential tool for specific identification of infections by opisthorchid liver flukes in endemic areas.
Liver flukes belonging to the genus Fasciola are among the causes of human food-borne parasitic diseases . It is important to characterize this species and the disease it causes, which has resulted in substantial economic losses to the livestock industry and has been increasingly observed to be of risk for humans as well. Therefore, current phenotypic techniques fail to reflect the full extent of the diversity of Fasciola spp. In this respect, the use of molecular techniques to identify and differentiate various species of Fasciola spp. offers considerable advantages. The advent of a variety of molecular genetic techniques also provides a powerful method to elucidate many aspects of Fasciola biology, epidemiology and genetics. However, the discriminatory power of these molecular methods varies, as do the speed, ease of performance and cost. There is a definite need for the development of new methods to identify the mechanisms underpinning the origin and maintenance of genetic variation within and among Fasciola populations. The increasing application of current and new methods, such as PCR- and LAMP-based approaches, will no doubt yield a much improved understanding of Fasciola epidemiology and evolution as well as more effective means of parasite control .
Since it is difficult to monitor the susceptibility of P. vivax to antimalarial drugs by in vitro tests, molecular markers of drug resistance are useful tools for mapping the current and changing pattern of pyrimethamine resistance of P. vivax isolates. P. vivax isolates were collected from four different sites in central P.R. China, and the sequence of the entire dhfr domain was determined to investigate genetic variation in P. vivax dihydrofolate reductase . This study suggested that P. vivax in central P.R. China may be relatively susceptible to pyrimethamine. It also highlights that genotyping in the pvdhfr genes remains a useful tool to monitor the emergence and spread of P. vivax pyrimethamine resistance. Thus, genetic markers of parasite isolates are a potentially important part of the arsenal.
Transmission-blocking vaccines (TBVs) have been considered an important strategy for disrupting the malaria transmission cycle, especially for P. vivax malaria, which undergoes gametocytogenesis earlier during infection. Pvs25 and Pvs28 are transmission-blocking vaccine candidates for P. vivax malaria. Assessment of genetic diversity of the vaccine candidates will provide necessary information for predicting the performance of vaccines, which will guide us during the development of malaria vaccines. Genetic analysis revealed limited genetic diversity of pvs25 and pvs28, suggesting antigenic diversity may not be a particular problem for Sal I based TBVs in most P. vivax-endemic areas of P.R. China .
At present, selective chemotherapy with praziquantel is one of the main strategies in P.R. China's national schistosomiasis control programme, and thus diagnosis of infected individuals is of central importance. Thus far, a simple, affordable, sensitive and specific assay for field diagnosis of schistosomiasis japonica is not available, and this poses a great barrier towards full control let alone elimination of the disease . Hence, a search for a diagnostic approach, which delivers these characteristics, is essential and should be given high priority .
Recently, a rapid and simple test for the detection of human antibodies against S. japonicum (i.e. the dipstick dye immunoassay (DDIA)) has been made commercially available in P.R. China. This assay produces results within 5–10 min without any more specialized instrument than a micropipette. The performance of DDIA was compared with stool examination evaluating its accuracy as a primary approach for screening the population in seven villages of low endemicity for schistosomiasis japonica . Using stool examination as reference standard, DDIA performed with a high overall sensitivity of 91.3% and also a high negative predictive value, with a mean value of 99.3%. Hence, it was concluded that DDIA is a sensitive, rapid, simple and portable diagnostic assay and can be used as a primary approach for screening schistosome infection in areas of low endemicity. However, more sensitive and specific confirmatory assays need to be developed and combined with DDIA for targeting chemotherapy accurately. In addition to DDIA, another rapid dipstick assay (with the same specificity) based on latex particles and immunochromatography (DLIA), has been introduced . The results show that DLIA is a simple, rapid, convenient, sensitive and specific assay for the diagnosis of schistosomiasis japonica and is therefore very suitable for large-scale field applications and clinical detection.
In addition to the above-mentioned assays, an innovative test for the diagnosis of schistosomiasis, a time-resolved fluoroimmunoassay (TRFIA), has been developed for detecting the signal transduction protein 14-3-3, a circulating antigen of S. japonicum. The results demonstrate that it has the potential of becoming a useful diagnostic method for schistosomiasis . EWS with respect to the risk of infection is an important preventive measure against schistosomiasis. The study published here demonstrates that antibody responses to the Sj23HD antigen could be monitored for early detection of schistosome infection in mice, especially by immunoblot, which demonstrated greater sensitivity and specificity than an enzyme-linked immunosorbent assay (ELISA) for detection Sj23HD antibodies .
The detection of schistosome circulating antigens (CAs) is an effective approach to discriminate between previous exposure and current infection. Anti-adult worm antigen (AWA) IgY was produced by immunization of the hyline hens with AWA. Then purified IgY was immobilized onto the resin as a capture antibody to immune-precipitate CAs from patients' serum samples. The precipitated proteins were separated by one-dimensional electrophoresis and analyzed by LC-MS/MS. Four proteins including protein BUD31 homolog, ribonuclease, SJCHGC06971 protein and SJCHGC04754 protein were identified among the CAs, which could be used for biomarkers of diagnostics .
Cathepsin cysteine proteases play multiple roles in the life cycle of parasites such as food uptake, immune invasion and pathogenesis, making them valuable targets for diagnostic assays, vaccines and drugs. To find out whether cathepsin B could be applied for serodiagnosis in C. sinensis infection, a gene encoding this enzyme in C. sinensis (Cs CB) was identified and investigated with regard to its diagnostic value . It was revealed that secreted CsCB may play an important role in the biology of C. sinensis and could be a diagnostic candidate for helminthiases.