[Show abstract][Hide abstract] ABSTRACT: In 2008 and 2009, an outbreak of desert-subtype zoonotic visceral leishmaniasis occurred in Jiashi county, Xinjiang, China. So far, no animal reservoir has been identified for this type of visceral leishmaniasis. Therefore, we surveyed the most common mammals (wild and domestic) for Leishmania infections during the outbreak in 2008 and 2009 in order to identify the source of the visceral leishmaniasis in this region. Spleen, liver, bone marrow and blood samples collected from 86 wood mice (Apodemus sylvaticus), 61midday jirds (Meriones meridianus) and 27 Yarkand hares (Lepus yarkandensis) were tested for the presence of Leishmania by microscopy, culture and PCR. All of the animals were found to be negative for Leishmania infections; On the other hand, Leishmania DNA was detected in blood samples collected from livestock reared in the outbreak area: 30.36% (17/56) of sheep, 21.57% (11/51) of goats, 17.78% (8/45) of cattle, and 21.62 (8/37) of donkeys were positive for Leishmania DNA by PCR. The amplified kDNA sequences from the livestock samples matched Leishmania DNA sequences isolated from patients with visceral leishmaniasis in the study area. We suggest that these domestic mammals are a possible reservoir host for Leishmania infantum in the outbreak area.
[Show abstract][Hide abstract] ABSTRACT: Leishmania infantum infections in dogs play a crucial role in the transmission of pathogens causing visceral leishmaniasis to humans in the Gansu province, northwest China. To be able to control zoonotic transmission of the parasite to humans, a non-invasive loop-mediated isothermal amplification (LAMP) assay to specifically detect L. infantum infections in dogs was developed.
The primers used in the LAMP assay were designed to target kinetoplast DNA minicircle sequences of the L. infantum isolate MCAN/CN/90/SC and tested using DNA isolated from promastigotes of different Leishmania species. The LAMP assay was evaluated with conjunctional swab samples obtained from 111 and 33 dogs living in an endemic and a non-endemic region of zoonotic visceral leishmaniasis in the Gansu province, respectively. The LAMP assay was also compared with conventional PCR, ELISA and microscopy using conjunctional swab, serum and bone marrow samples from the dogs, respectively.
The LAMP assay detected 1 fg of L. infantum DNA purified from cultured promastigotes which was 10-fold more sensitive than a conventional PCR test using Leishmania genus-specific primers. No cross reaction was observed with DNA isolated from promastigotes of L. donovani, L. major, L. tropica, and L. braziliensis, and the L. infantum reference strain MHOM/TN/80/IPT1. The L. infantum-positive rates obtained for field-collected samples were 61.3 %, 58.6 %, 40.5 % and 10.8 % by LAMP, PCR, ELISA and microscopy, respectively. As only one out of the 33 samples from control dogs from the non-endemic region of zoonotic visceral leishmaniasis was positive by the LAMP assay and the PCR test, the observed true negative rate (specificity) was 97 % for both methods.
This study has shown that the non-invasive, conjunctional swab-based LAMP assay developed was more sensitive in the detection of leishmaniasis in dogs than PCR, ELISA and microscopy. The findings indicate that the LAMP assay is a sensitive and specific method for the field surveillance of domestic dogs, particularly of asymptomatic canines, in ZVL-endemic areas in western China.
[Show abstract][Hide abstract] ABSTRACT: Visceral leishmaniasis (VL) is a life-threatening disease caused by protozoan parasites of the Leishmania donovani complex. Early case detection followed by adequate treatment is essential to the control of VL. However, the available diagnostic tests are either invasive and require considerable expertise (parasitological demonstration of the parasite in tissue smears) or unable to distinguish between past and active infection (serological methods). Therefore, we aimed to develop a lateral flow assay in the form of an immunochromatographic test (ICT) device based on the detection of a circulating Leishmania antigen using monoclonal antibodies (mAbs).
mAbs were produced by fusion of murine myeloma cells with splenocytes isolated from a mouse immunized with L. donovani soluble crude antigen. Out of 12 cloned hybridoma cell lines, two secreted mAbs recognizing the same leishmanial protein. These mAbs were used to produce an ICT as a sandwich assay for the detection of circulating antigen in serum and blood samples. The ICT was evaluated with 213 serum samples from VL patients living in VL endemic areas in China, and with 156 serum samples from patients with other diseases as well as 78 serum samples from healthy donors. Sensitivity, specificity and diagnostic efficiency of the new ICT was 95.8%, 98.7% and 97.3%, respectively. Compared with a commercially available antibody detecting ICT, our antigen-based ICT performed slightly better.
The newly developed ICT is an easy to use and more accurate diagnostic tool which fulfils the performance and operational characteristics required for VL case detection under field and laboratory conditions. As our ICT detects a circulating antigen, it will also be useful in monitoring treatment success and diagnosing VL in immunocompromised patients.
[Show abstract][Hide abstract] ABSTRACT: Human cystic and alveolar echinococcoses are zoonotic diseases caused by the larval stages of Echinococcus granulosus and Echinococcus multilocularis, respectively. As the diseases are co-endemic in many areas of the world, a simple and rapid test for the differential diagnosis of cystic echinococcosis (CE) and alveolar echinocoocosis (AE) is needed. Here, we describe the development of an immunochromatographic test (ICT) using crude hydatid cyst fluid and a recombinant 18-kDa protein (rEm18) as antigens for the detection of E. granulosus and E. multilocularis antibodies in serum samples. The ICT was evaluated with serum samples from 195 echinococcosis patients from different endemic areas in northwestern China. These included 144 from CE patients, 51 from AE patients, 67 from patients with other parasitic diseases, 13 from patients with serous hepatic cysts, and 60 from healthy individuals. The sensitivity and specificity of the ICT for CE were 91.0 and 96.9 % and for AE were 98.0 and 99.3 % with diagnostic efficiencies of 94.1 and 99.1 %, respectively. No significant differences and high degrees of agreement were found between the ICT and an enzyme-linked immunosorbent assay for both CE and AE. Five serum samples from cysticercosis patients and one serum sample from a healthy control were found positive for CE with the ICT. These findings indicate that this test allows for discrimination between both forms of human echinococcosis. In conclusion, the ICT developed in this study is a promising tool for the simultaneous detection and discrimination of CE and AE. This test will be useful for serodiagnosis of CE and AE in clinical settings and screening programs.
No preview · Article · Aug 2013 · Parasitology Research
[Show abstract][Hide abstract] ABSTRACT: To establish and evaluate a colloid gold immunochromatographic strip test for the diagnosis of alveolar echinococcosis.
Total RNA was prepared from Echinococcus multilocularis protoscoleces collected from Xinjiang Uygur Autonomous Region. Em18 gene was obtained by reverse transcription-polymerase chain reaction (RT-PCR). The PCR product was sequenced and cloned into pGEX-3X vector. The recombinant plasmid was expressed and induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) to obtain recombinant protein. The anti-human IgG monoclonal antibodies was conjugated with colloid gold as detecting reagent; the recombinant Em18 antigen and goat anti-mouse IgG were immobilized on nitrocellulose in proper position. The prepared immunochromatographic strip was evaluated using serum samples from patients with alveolar echinococcosis (56), cystic echinococcosis (87), cysticercosis (30), schistosomiasis japonica (10), toxoplasmosis (10) and healthy subjects (50) . Comparison between the immunochromatographic strip test and ELISA was made by kappa statistics.
Sensitivity detected by the immunochromatographic strip test was 92.9% (52/56). The cross-reactivity to cystic echinococcosis and cysticercosis was 9.2% (8/87) and 3.3% (1/30), respectively. There was no cross reactivity with schistosomiasis japonica and toxoplasmosis. 4 samples out of 50 healthy people showed false positive reaction. The overall specificity was 93.0 (174/187). Sensitivity and specificity both showed no statistical difference between immunochromatographic strip test and ELISA. High degree of agreement was observed between the strip test and ELISA (kappa = 0.98).
The developed immunochromatographic strip test using recombinant Em18 antigen as coated antigen is a sensitive, specific, simple and rapid assay for diagnosing alveolar echinococcosis.
No preview · Article · Apr 2012 · Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases
[Show abstract][Hide abstract] ABSTRACT: Visceral leishmaniasis (VL) is still an important public health problem in China. In recent years endemic regions spread, prevalence increased, and even an outbreak of the disease occurred in China due to global warming and population movement. It is essential to elucidate the current epidemic situation and epidemiological characteristics of VL for designing control policy. In the present study we describe the current epidemiological profile and characteristics of VL in China based on retrospectively reviewing of VL cases reported between 2005 and 2010 by a passive surveillance system.
The present study was a retrospective review of VL cases notified between 2005 and 2010 based on the passive surveillance data. The data were tabulated, diagrammatized and analyzed through descriptive statistics in a Microsoft Excel spreadsheet.
A total of 2450 VL cases were notified, with a mean of 408 cases per year. 61 counties were identified as endemic area with 2224 autochthonous cases, and the other 118 counties as non-endemic areas with 226 imported cases. 97.71% of cases were concentrated in Xinjiang, Gansu and Sichuan Provinces. 9 major counties reported a mean of > 10 cases per year, with a total of 1759 cases reported. Different types of VL revealed distinct epidemiological characteristics.
The number of VL cases and endemic counties both increased in the period 2005-2010 in China. Different type or sub-type of VL revealed distinct epidemiological characteristics. Therefore, differential control measures must be taken in different endemic areas against incidence increase and endemic area spread.
Full-text · Article · Feb 2012 · Parasites & Vectors
[Show abstract][Hide abstract] ABSTRACT: To diagnose and identify pathogen of two suspected cases of cutaneous leishmaniasis.
Two cases of dermatosis with several major ulcers on the skin were examined, who worked and returned from Algeria (case 1) and Saudi Arabia (case 2), respectively. The stained smears of skin tissue from lesions were observed by microscope. Extravasate from lesions was cultured in NNN medium to search protozoan parasites, which were obtained by centrifugation. Two pairs of species-specific primers, ITS1-ITS2 and K13A-K13B, were used to amplify inter-nal transcribed spacer of rDNA and kinetoplast DNA, respectively. The products were sequenced and analyzed by Blast.
There were Leishmania amastigotes in the tissue smear of case 2, while none in that of case 1. Promastigotes were found in culture medium of both cases. The PCR products of ITS1-ITS2 and K13A-K13B from 2 cases were about 330 bp and 120 bp with respective homology of 100% and 96% to corresponding sequences of Leishmania major. The accession numbers of 4 sequences were JF831924-JF831927.
Two cases of dermatosis are diagnosed as imported cutaneous leishmaniasis and the pathogen is L. major.
No preview · Article · Dec 2011 · Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases
[Show abstract][Hide abstract] ABSTRACT: Canine leishmaniasis (CanL) is endemic in western China, resulting in important public health problem. It is essential to evaluate the prevalence of canine Leishmania infantum infection for designing control policy. In the present study we report for the first time prevalence of Leishmania infection in dogs living in Jiuzhaigou County (Sichuan Provence, China), which is not only an important endemic area of CanL but also a tourism scenic spot, detected by PCR, ELISA and dipstick test. The results could provide key information for designing control programs against canine and human leishmaniasis. In addition, the complete sequence of the Leishmania isolate from Sichuan Province has not been reported to date and we present the sequences of 116 base-pair (bp) fragment of the conserved region in the minicircle kinetoplast DNA (kDNA) and the results of phylogenetic analyses based on the sequence of the amplified fragment.
The proportion of dogs infected with Leishmania in Jiuzhaigou County was 36.79%, 9.43%, and 51.88% detected by ELISA, dipstick test, and PCR, respectively. The ELISA and PCR tests were more sensitive than dipstick test. The PCR method is the most sensitive way to detect dogs infected with Leishmania parasites. The total positive rate for infected dogs in the area was 59.43% by the three methods. The PCR products of 116-bp fragment amplified from the kDNA conserved region of dog blood samples and laboratory maintained L. infantum were DNA sequenced and the variation of the sequences was observed. The phylogenetic tree based on the sequences of 116-bp fragment reveals that L. infantum is more genetically related to visceralizing species L. donovani than to the Leishmania species associated with cutaneous disease.
More than half of dogs living in the endemic Jiuzhaigou County were infected by L. infantum. Control measures, such as treatment or eradication of infected dogs, or prohibition of maintaining dogs, must be taken against these infected dogs due to their role in the transmission of the infection to vectors. The phylogenetic tree based on the sequences of conserved region in kDNA of Leishmania can effectively distinguish species of Leishmania.
Preview · Article · May 2011 · Parasites & Vectors
[Show abstract][Hide abstract] ABSTRACT: Few outbreaks of the desert sub-type of zoonotic visceral leishmaniasis (VL) have been described worldwide. In 2008, the incidence rate of VL in Jiashi County, Xinjiang Uygur Autonomous Region in the western part of the People's Republic of China, increased more than twenty-folds compared to the average annual incidence rate. The majority of the cases (96.6%) occurred among <2 year-old infants. For the first time in the desert area of Xinjiang, the parasites were isolated from bone marrow aspirates, using the NNN medium culture approach. The genetic analysis of the ITS-1 nucleotide sequence indicated that three isolates from eastern Jiashi County were genetically closely related and belonged to the Leishmaniainfantum group. However, they differed from an isolate from Kashi city which was classified as a member of the Leishmaniadonovani group.
No preview · Article · Apr 2010 · Parasitology International
[Show abstract][Hide abstract] ABSTRACT: To evaluate a gold-immunochromatographic assay (GICA) for malaria diagnosis in an endemic area of vivax malaria.
Blood samples were collected from febrile patients in 5 township-hospitals of Mengcheng County, Anhui Province, between September and October 2008. The samples were examined by GICA and microscopy under double blind condition and the results were compared.
Among 292 blood samples, 181 were found P. vivax-positive by microscopy, and 163 were positive by GICA. Altogether, the coincidence of the two methods stood for 92.8% (271/292), including 108 negatives and 163 positives. 21 samples with discrepancy covered 18 microscopy positive but GICA negative, 3 microscopy negative but GICA positive. The GICA positive rate in patients with a parasitaemia of > 1,000 parasites/microl, 100-1,000 parasites/microl, and < 100 parasites/microl was 93.5% (115/123), 86.0% (43/50), and 62.5% (5/8), respectively.
GICA is a useful diagnosis method for endemic area of vivax malaria.
No preview · Article · Dec 2009 · Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases
[Show abstract][Hide abstract] ABSTRACT: To establish and evaluate a gold immunochromatographic strip test for detection and differentiation of Plasmodium vivax and P. falciparum.
The monoclonal antibodies, F4H12, G4C9 and D8F7, were conjugated with colloid gold as detecting reagent; monoclonal antibody B2G10 (against P. vivax/ P. falciparum) and D6A7 (only against P. falciparum) were immobilized on nitrocellulose in proper position. Blood samples from 107 febrile patients from endemic area of malaria and 17 patients with visceral leishmaniasis were used for evaluating the specificity. Blood samples of malaria patients (110 with P. vivax and 54 with P. falciparum) were used for evaluating the sensitivity.
5 samples out of 107 febrile patients and 17 patients with visceral leishmaniasis showed false positive reaction with a specificity of 96.0% (119/124), all the 17 samples from patients with visceral leishmaniasis were negative. 164 blood samples of malaria patients showed a sensitivity of 92.3% (153/164), 92.7% (102/110)and 94.4% (51/ 54) for patients infected with P. vivax or P. falciparum, respectively.
The immunochromatographic strip test based on antigen-capturing is a sensitive, specific, simple and rapid assay for malaria diagnosis.
No preview · Article · Nov 2007 · Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases
[Show abstract][Hide abstract] ABSTRACT: To analyze the status of Leishmania infantum asymptomatic infection in human population of a Kala-azar endemic area in Wenxian County, Gansu Province, and to evaluate the tests used.
Blood samples were tested by PCR using two pairs of primers, RV1-RV2 and K13A-K13B, for detecting Leishmania-specific DNA. ELISA and rK39-dipstick were used to detect Leishmania-specific antibodies.
The positive rate of PCR, ELISA and rK39-dipstick was 30.9%(83/269). 24.2%(65/269) and 0 (0/269) respectively.
The prevalence of asymptomatic infection of L. infantum in humans is high in the area. PCR test based on RV1-RV2 and K13A-K13B primer pairs is a sensitive and specific method for detecting the asymptomatic infection.
No preview · Article · Mar 2007 · Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases
[Show abstract][Hide abstract] ABSTRACT: To establish PCR method for the detection of the asymptomatic infection of Leishmania infantum.
Six primer pairs were selected for detecting Chinese strain of L. infantum by optimizing conditions which affect amplification. Their sensitivity and specificity were compared by using DNAs extracted from human blood seeded with cultured L. infantum promastigotes (MHOM/CN/86/GS) as template. Blood samples of the inhabitants without symptoms of visceral leishmaniasis in the endemic area were analyzed with two selected primer pairs with good sensitivity and specificity.
The specificity of all six primer pairs reached 100%, and the sensitivity varied among the primer pairs. The primer pairs RV1-RV2 (0.1 parasite/ml blood) and K13A-K13B (1 parasite/ml blood) were most sensitive. Leishmania DNA was detected in 33% (33/100) and 30% (30/100) human blood samples by RV1-RV2 and K13A-K13B primer pairs respectively.
This study suggests that RV1-RV2 and K13A-K13B primer pairs are suitable in detecting the asymptomatic infection of L. infantum, and the prevalence of the asymptomatic infection is high in human population in the endemic area.
No preview · Article · May 2006 · Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases
[Show abstract][Hide abstract] ABSTRACT: To prepare monoclonal antibodies specific to lactate dehydrogenase of Plasmodium falciparum.
The Plasmodium falciparum lactate dehydrogenase (pLDH) gene was amplified from whole blood of malaria patients by PCR and cloned into expression vector pGEX-3X. Recombinant pLDH protein was expressed and purified, and used for immunizing mice to prepare monoclonal antibodies (McAbs). The McAbs were characterized by Western blotting analysis.
The Plasmodium falciparum lactate dehydrogenase gene was amplified and cloned into ex pression vector pGEX-3X. The recombinant pLDH plasmid was expressed in E. coli) BL-21 cells. 15 cell lines of McAbs with high titer against pLDH were obtained using the recombinant pLDH as immunogen. Western blotting analysis showed that these McAbs recognized a Mr 33,000 of native Plasmodium falci parvum protein without cross reaction with constituents of red blood cell of febrile patients from endemic area of malaria.
Fifteen hybridoma cell lines secreting high titer of McAb specific to Plasmodium falciparum LDH were established based on the recombinant pLDH.
No preview · Article · Sep 2005 · Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases