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Evaluation of a real-time PCR assay for diagnosis of schistosomiasis japonica in the domestic goat.

ABSTRACT: BACKGROUND:Schistosomiasis japonica is an infectious disease caused by Schistosoma japonicum that seriously endangers human health. Domestic animals have important roles in disease transmission and goats are considered a primary reservoir host and source of infection. The prevalence and intensity of schistosomiasis infections have significantly decreased in China, and a more sensitive, specific detection method is urgently needed. The aim of this study was to develop a real-time PCR assay for accurate detection of S. japonicum infection in goats. METHODS:A real-time PCR method for detecting schistosomiasis japonica in goats was developed by amplification of a specific S. japonicum DNA fragment, and validated using a total of 94 negative and 159 positive plasma and serum samples collected in our previous study of S. japonicum infection. Both plasma and serum samples were evaluated by real-time PCR and enzyme-linked immunosorbent assay (ELISA). In addition, 120 goat plasma samples from an S. japonicum-endemic area (Wangjiang) and 33 from a non-endemic region (Weihai) were collected and evaluated using our method. RESULTS:The sensitivity and specificity of the real-time PCR for detecting infected samples were 98.74% (157/159, 95% CI: 95.53-99.85%) and 100% (94/94, 95% CI: 96.15-100%), respectively. For the ELISA, sensitivity and specificity were 98.11% (156/159, 95% CI: 94.59-99.61%) and 90.43% (85/94, 95% CI: 82.60-95.53%), respectively. Further, we found positivity rates for S. japonicum infection in Wangjiang and Weihai of 8.33% (10/120, 95% CI: 4.07-14.79%) and 0% (0/33, 95% CI: 0-10.58%), respectively. CONCLUSIONS:The results of this study indicate that our real-time PCR method exhibits higher sensitivity and specificity than ELISA and is a useful method for detection of S. japonicum infection in goats.

SUBMITTER: Guo Q

PROVIDER: S-EPMC7590668 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Evaluation of a real-time PCR assay for diagnosis of schistosomiasis japonica in the domestic goat.

Guo Qinghong Q Chen Cheng C Zhou Keke K Li Yugang Y Tong Laibao L Yue Yongcheng Y Zhou Kerou K Liu Jinming J Fu Zhiqiang Z Lin Jiaojiao J Zhao Jiaxi J Sun Pengxiang P Hong Yang Y

Parasites & vectors 20201027 1

<h4>Background</h4>Schistosomiasis japonica is an infectious disease caused by Schistosoma japonicum that seriously endangers human health. Domestic animals have important roles in disease transmission and goats are considered a primary reservoir host and source of infection. The prevalence and intensity of schistosomiasis infections have significantly decreased in China, and a more sensitive, specific detection method is urgently needed. The aim of this study was to develop a real-time PCR assa ...[more]

PMID: 33109260

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Project description:Although the prevalence of schistosomiasis japonica has declined gradually in China, more accurate and sensitive diagnostic methods are urgently needed for the prevention and control of this disease. Molecular diagnostic methods are advantageous in terms of sensitivity and specificity, but they are time-consuming and require expensive instruments and skilled personnel, which limits their application in low-resource settings. In this study, an isothermal DNA amplification assay and recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD) were set up. It was used to detect S. japonicum infections in experimental mice and domestic goats by amplifying a specific DNA fragment of S. japonicum. The lower limit of detection for the LFD-RPA assay was evaluated using dilutions of plasmid containing the target sequence. Cross-reactivity was evaluated using genomic DNA from eight other parasites. The effectiveness of the LFD-RPA assay was verified by assessing 36 positive plasma samples and 36 negative plasma samples from mice. The LFD-RPA assay and real-time PCR were also used to assess 48 schistosomiasis japonica-positive plasma samples and 53 negative plasma samples from goats. The LFD-RPA assay could detect 2.6 femtogram (fg) of S. japonicum target DNA (~39 fg genomic DNA of S. japonicum), only 10-fold less sensitive than real-time PCR assay. There was no cross-reactivity with DNA from the other eight parasites, such as Haemonchus contortus and Spirometra. The whole amplification process could be completed within 15 min at 39°C, and the results can be observed easily using the LFD. The sensitivity and specificity of the LFD-RPA assay were 97.22% (35/36, 95% CI, 85.47%-99.93%) and 100% (36/36, 95% CI, 90.26%-100%) in mice, and 93.75% (45/48, 95% CI, 82.80%-98.69%) and 100% (53/53, 95% CI, 93.28%-100%) in goats. By comparison, the sensitivity and specificity of real-time PCR were 100% (36/36, 95% CI, 90.26%-100%) and 100% (36/36, 95% CI, 90.26%-100%) for mice, and 97.92% (47/48, 95% CI, 88.93%-99.95%) and 100% (53/53, 95% CI, 93.28%-100%) for goats. The LFD-RPA assay exhibits high sensitivity and specificity for the diagnosis of schistosomiasis japonica, and it is an alternative method for diagnosis schistosomiasis japonica in low resource setting.

Project description:BackgroundSchistosomiasis is a huge threat to human and animal health. Apart from bovines, goats play an important role in the transmission of schistosomiasis in some endemic areas of China. An accessible, quality-assured goat schistosomiasis diagnostic technique is needed. Recently, our laboratory identified two recombinant diagnostic antigens, SjPGM and SjRAD23 via an immuno-proteomic method. The application of these two recombinant antigens to develop a higher sensitivity and specificity technique for the sheep schistosomiasis diagnosis is urgently needed.MethodsEpitopes of SjPGM and SjRAD23 were predicted and three polypeptides, two from SjRAD23 and one from SjPGM, were selected. Recombinant plasmids containing two to three DNA sequences encoding predicted polypeptides or large hydrophilic region of Sj23 (LHD-Sj23) were constructed and expressed. Eight recombinant schistosome antigens including four multi-epitope proteins and four recombinant single-molecule antigens as well as SEA, were assessed by ELISA in 91 sera from schistosome-infected goats, 44 sera from non-infected goats, 37 sera from Orientobilharzia-infected goats, and 12 from Haemonchus contortus-infected goats.ResultsELISA tests showed that three multi-epitope proteins had higher sensitivity than the four single-molecule antigens (rSjRAD23, rSjPGM, rBSjRAD23-1, rBSj23) and the multi-epitope protein rBSjPGM-BSjRAD23-1-BSj23 had the highest sensitivity (97.8 %, 89/91) and maintained good specificity (100 %, 44/44) as well as low cross-reactivity with haemonchosis (8.33 %, 3/12) and orientobilharziasis (13.51 %, 5/37) in the diagnosis of goat schistosomiasis. In contrast, when SEA was applied as a diagnosis antigen, it had 100 % (91/91) sensitivity, 75 % (33/44) specificity, 25 and 83.78 % cross-reactivity with haemonchosis (3/12) and orientobilharziasis (31/37), respectively.ConclusionsThe application of recombinant multi-epitope proteins may increase the sensitivity of diagnosis technique and retain high specificity of single-molecule antigens for schistosomiasis, and the recombinant antigen rBSjPGM-BSjRAD23-1-BSj23 has the potential to be used as a diagnosis antigen for goat schistosomiasis.

Project description:The laboratorial diagnosis of the intestinal schistosomiasis is always performed using Kato-Katz technique. However, this technique presents low sensitivity for diagnosis of individuals with low parasite burden, which constitutes the majority in low endemicity Brazilian locations for the disease. The objective of this study was developed and to validate a real-time PCR assay (qPCR) targeting 121 bp sequence to detect Schistosoma spp. DNA for the diagnosis of intestinal schistosomiasis and a sequence of the human β-actin gene as internal control. Firstly, the qPCR was standardized and next it was evaluated for diagnosis and cure assessment of intestinal schistosomiasis in the resident individuals in Tabuas and Estreito de Miralta, two locations in Brazil endemic for intestinal schistosomiasis. The qPCR assay results were compared with those of the Kato-Katz (KK) test, examining 2 or 24 slides, Saline Gradient (SG) and "reference test" (24 KK slides + SG). The cure assessment was measured by these diagnostic techniques at 30, 90, and 180 days post-treatment. In Tabuas, the positivity rates obtained by the qPCR was 30.4% (45/148) and by "reference test" was of 31.0% (46/148), with no statistical difference (p = 0.91). The presumed cure rates at 30, 90, and 180 days post-treatment were 100, 94.4, and 78.4% by the analysis of 24 KK slides, 100, 94.4, and 78.4% by the SG, and 100, 83.3, and 62.1% by the qPCR assay. In Estreito de Miralta, the positivity obtained by qPCR was 18.3% (26/142) and with "reference test" was 24.6% (35/142), with no statistical difference (p = 0.20). The presumed cure rates were 93.3, 96.9, and 96.5% by the KK, 93.3, 96.9, and 100% by the SG, and 93.3, 93.9, and 96.5% by the qPCR at 30, 90, and 180 days post-treatment, respectively. This study showed that the diagnostic techniques presented different performance in the populations from the two districts (Tabuas and Estreito de Miralta) and reinforces the need of combining techniques to improve diagnosis accuracy, increasing the detection of individuals with low parasite burden. This combination of techniques consists an important strategy for controlling the disease transmission.

Project description:In an increasingly globalized and interconnected world, the outbreak of an infectious disease in one country can become a worrying health emergency for the whole world. A current example is the 2022 monkeypox virus (mpox) outbreak affecting multiple areas across the world. In this context, strategies to interrupt transmission as soon as possible by identifying cases, clusters, and sources of infection should be developed around the world to prevent these crises. The aim of this retrospective and collaborative study was to perform external clinical validation of the VIASURE monkeypox virus real-time PCR detection kit (CerTest Biotec, Spain) with ready-to-use reagents designed for the rapid detection of mpox. A total of 165 samples with suspected infection were used for this analysis. The standard procedures of the clinical microbiology laboratory of the Miguel Servet University Hospital, using the RealStar Orthopoxvirus PCR kit v1.0 (Altona Diagnostics) and bidirectional Sanger sequencing (STAB VIDA, Caparica, Portugal), were considered reference techniques. Furthermore, a subset of 67 mpox-negative samples and 13 mpox-positive samples were routinely tested for clinical diagnosis of other rash/ulcerative pathologies. Accuracy testing resulted in appropriate clinical validation values, as follows: sensitivity, 1 (95% confidence interval [CI], 0.97 to 1); specificity, 1 (95% CI, 0.98 to 1); positive predictive value, 1 (95% CI, 0.93 to 1); negative predictive value, 1 (95% CI, 0.95 to 1). The strength of agreement between assays was almost perfect. The added value is the useful support for the specific diagnosis of mpox infections due to the diagnostic specificity data obtained. IMPORTANCE Given that a large number of mpox outbreaks have been reported worldwide since 2022 in countries in which the disease is not endemic, the main concern for clinicians and global health systems should be to develop effective, available, and easy-to-implement diagnostic strategies to interrupt mpox transmission as soon as possible. This retrospective study demonstrates the satisfactory clinical parameters of a commercially available molecular diagnostic kit for routine testing for mpox in clinical diagnostic laboratories.

Dataset Information

Evaluation of a real-time PCR assay for diagnosis of schistosomiasis japonica in the domestic goat.

Publications

Evaluation of a real-time PCR assay for diagnosis of schistosomiasis japonica in the domestic goat.

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