Table of Content

30 October 2017, Volume 35 Issue 5

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Orginal Article

In vitro inhibitive effect of the anticancer drug sorafenib on Echinococcus multilocularis larvae

Zhi-sheng DANG, Yuzaburo OKU, Xiao-nong ZHOU, Wei HU, Ting ZHANG, Jun-hu CHEN, Bin XU, Yu-min ZHAO, Ning XIAO

2017, 35(5):  417-422. 

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Objective To evaluate the effect of an anti-cancer drug sorafenib against alveolar echinococcosis (AE) in vitro. Methods Metacestode vesicles were obtained from infected mice and cultured with rat hepatocytes in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS), 100 U/ml penicillin G and 100 mg/L streptomycin sulphate at 37 ℃ with 5% CO₂. Morphologically intact metacestode vesicles with a minimal diameter of 2-4 mm were manually picked up from the co-culture and washed with PBS. They were then allocated (n = 25-30 cysts/well) into four groups: albendazole-treated group (positive group), sorafenib-treated group (experimental group), DMSO-treated group (solvent group), and PBS group (negative control), and incubated in 24-well plates in the presence of DMEM without phenol red. Albendazole and sorafenib were dissolved in DMSO (1 ∶ 9 dissolved in PBS) and used at 10 μmol/L and 30 μmol/L respectively. The experiment was performed in triplicates per sample. The integrity of the cysts was assessed with light microscopy and scanning electron microscopy (SEM). The inhibitory effects of albendazole and sorafenib on cysts were evaluated at 48, 72, 96, 120, 144 and 168 h post-treatment. Results The cyst inhibitory rate of sorafenib was 6.6%, 42.4%, 68.5%, 77.4%, 84.0% and 89.5% at 10 μmol/L, and 7.1%, 45.6%, 70.9%, 82.6%, 84.0% and 89.9% at 30 μmol/L, respectively, at 48, 72, 96, 120, 144 and 168 h post-treatment. While the cyst inhibitory rate of albendazole was 2.6%, 6.6%, 21.4%, 47.8%, 59.9% and 70.6% at 10 μmol/L, and 3.8%, 12.7%, 27.0%, 51.4%, 54.0% and 73.0% at 30 μmol/L, respectively. DMSO and PBS showed less toxic to the cysts. Sorafenib exerted a stronger inhibitory effect than albendazole at the corresponding time points, and the difference began to be statistically significant from 72 h post-treatment. Light microscopy and SEM showed observable collapse of the germinal layer and overall damage of germinal cells in the experiment groups. Conclusion Sorafenib shows a significant inhibitory effect on Echinococcus cysts, suggesting a potential drug candidate for echinococcosis treatment.

Differential expression of circulating microRNAs in patients with cystic echinococcosis and screening for specific diagnostic biomarkers for the disease

Meng-di REN, Yu-ye NING, Ming LI, Hao WANG, Wei ZHAO

2017, 35(5):  423-428. 

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Objective To analyze the differential expression of circulating microRNAs (miRNAs) in patients with cystic echinococcosis (CE) and screen for potential diagnostic biomarkers for CE. Methods Serum miRNAs were extracted from 3 CE patients and 3 healthy controls (HCs) and analyzed with miRNA microarray using an Affymetrix miRNA4.0 chip to find miRNAs with differential expression. Results were verified on sera from 21 CE patients and 25 HCs using real-time PCR (qRT-PCR). Correlation between the miRNA and hydatid cyst weight or the number of daughter cysts was analyzed with Pearson’s correlation analysis. The IBM SPSS 19.0 software was used to generate the receiver operating characteristic curve (ROC) and analyze the area under curve (AUC). Results of microarrays revealed 172 miRNAs with an over 2-fold increase and 35 miRNAs with an over 50% decrease in CE sera as compared to the HC. qRT-PCR further quantitated the expression of hsa-miR-762, hsa-miR-3536, hsa-miR-6125, hsa-miR-6821-5P, hsa-miR-663a, hsa-miR-24-3P, hsa-miR-191-5P and hsa-miR-320a to be 10.28 ± 0.38, 5.11 ± 0.34, 11.02 ± 0.55, 11.87 ± 0.58, 5.31 ± 0.36, 5.75 ± 0.51, 5.75 ± 0.51 and 4.96 ± 0.36, respectively, all significantly higher than their HC counterparts (P < 0.05). ROC analysis showed that the AUC values of these 8 miRNA candidates were all above 8, implying their diagnostic potential for CE. Moreover, correlation analysis indicated that only miR-24-3p was positively correlated with both cyst weight (r = 0.5250, P < 0.05) and the number of daughter cysts (r = 0.6175, P < 0.05). Conclusion Eight circulating miRNAs have been identified as potential biomarkers for diagnosis of CE. MiR-24-3p may correlate with the progression and pathogenesis of CE.

Single nucleotide polymorphism analysis of cytochrome b, cytochrome c oxidaseⅠand lactate dehydrogenase genes of imported Plasmodium ovale

Ling-ling ZHANG, Li-nong YAO, Hua-liang CHEN, Qiao-yi LU, Wei RUAN

2017, 35(5):  429-433. 

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Objective To analyze the single nucleotide polymorphism (SNP) of cytochrome b (Cytb), cytochrome c oxidaseⅠ (CoxⅠ) and lactate dehydrogenase (LDH) genes of Plasmodium ovale curtisi and P. ovale wallikeri. Methods The P. ovale samples identified in Zhejiang Provincial Reference Laboratory for Malaria Diagnosis during 2011-2014 were used for nested PCR to amplify the partial fragments of Cytb, CoxⅠ and LDH genes. The amplified products were sequenced in both directions. The nucleotide sequences and their corresponding amino acid sequences were aligned and analyzed using the Mega software to find out the SNP sites and amino acid mutation sites. Results The 19 samples of P. ovale (11 curtisi and 8 wallikeri) were all from persons returning from Africa. The lengths of PCR products for Cytb, CoxⅠ and LDH were 735, 1 323 and 355 bp, respectively. There were 10, 13 and 13 single nucleotide dimorphism sites in Cytb, CoxⅠ and LDH, respectively, and most were synonymous mutation. Meanwhile, some nucleotide polymorphisms were found between the two subspecies. The CoxⅠ gene was found to harbor a 693 bp deletion mutation in one sample. Conclusion We performed SNP analysis on Cytb, CoxⅠ and LDH genes of P. ovale, which will enrich the knowledge on gene polymorphism of P. ovale.

Genotyping of merozoite surface protein 1 and 2 of imported Plasmodium falciparum in Wuhan City

Xi-shuai JIA, Shui-mao ZHOU, Yan YANG, Ming-xing XU, Kai WU

2017, 35(5):  434-439. 

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Objective To identify the genotypes of merozoite surface protein 1 (MSP1) and merozoite surface protein 2 (MSP2) of imported Plasmodium falciparum in Wuhan. Methods Blood samples were collected from returnees infected with P. falciparum from endemic areas of Africa, from 2010 to 2015 in Wuhan City, and epidemiological data were collected. The Plasmodium falciparum DNA was extracted from the blood samples. Nested PCR was used to amplify gene fragments of MSP1 and MSP2. The constituent ratios of the allelic families, the frequencies of each allelic family in patients from different countries, and the association of different allelic families with disease severity were analyzed. Analysis of proportions of severe cases with different genotypes were analyzed with χ² test. Results A total of 175 blood samples of patients with falciparum malaria were collected, which including 38 severe cases and 137 mild cases. Nested PCR for MSP1 allelic families K1, RO33 and MAD20 produced bands of 260-390, 270 and 150 bp, and MSP2 allelic families 3D7 and FC27 produced bands of 200-330 and 330-500 bp. A total of 175 patients with falciparum malaria were involved in this study, of whom 9 patients showed negative results for both MSP1 and MSP2 amplification. The MSP1 allelic families were detected in 136 patients, with a detection rate of 77.7%. The constituent ratios of MAD20, K1, RO33, MAD20 + K1, MAD20 + RO33, K1 + RO33, and MAD20 + K1 + RO33 were 5.1% (7/136), 37.5% (51/136), 20.6% (28/136), 5.9% (8/136), 4.4% (6/136), 16.9% (23/136), and 9.5% (13/136), respectively. The MSP2 allelic families were detected in 143 patients, with a detection rate of 81.7%. The constituent ratios of FC27, 3D7, and FC27 + 3D7 were 20.2% (29/143), 39.9% (57/143), and 39.9% (57/143), respectively. The MAD20 allelotype was predominantly from South Africa (10.5%, 4/38), the K1 was predominantly from South Africa (34.2%, 13/38), and the RO33 was predominantly from East Africa (2/7). The FC27 was not detected in East and North Africa, but was predominantly from South Africa (18.4%, 7/38). The 3D7 was predominantly from East Africa (4/7). The proportions of severe cases in the MAD20, K1, RO33, MAD20 + K1, MAD20 + RO33, K1 + RO33, and MAD20 + K1 + RO33 allelic families were 1/7, 19.6% (10/51), 32.1% (9/28), 1/8, 4/6, 13.0% (3/23), and 46.2% (6/13), respectively (P > 0.05 among MAD20 + RO33, MAD20 + K1 + RO33 and RO33; P < 0.05 for MAD20 + RO33, MAD20 + K1 + RO33 and RO33 versus the other four). The proportions of severe cases with 3D7, FC27, and FC27 + 3D7 were 19.3% (11/57), 20.7% (6/29), and 24.6% (14/57), respectively (P > 0.05). Conclusion There are three single allelic families of MSP1（MAD20, K1 and RO33） and four mixed allelic families in imported falciparum malaria in Wuhan, of which K1 and R033 are the dominant genotypes. There are three allelic families for MSP2（FC27, 3D7 and FC27 + 3D7）, of which 3D7 has a higher frequency than FC27.

Evaluation of soil-transmitted helminth control after cessation of intervention measures in eight demonstration plots

Ting-jun ZHU, Chang-hai ZHOU, Ju-jun WANG, Wei ZANG, Ying-dan CHEN

2017, 35(5):  439-444. 

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Objective To evaluate the control effects on soil-transmitted helminth (STH) infection after cessation of prevention measures in eight demonstration plots. Methods After cessation of intervention measures in 2009, a survey was made on the soil-transmitted helminth infection, the awareness of hygiene knowledge and the popularity of sanitary toilet and tap water in Kaiyang County in Guizhou Province, Tunchang County in Hainan Province, Guixi City in Jiangxi Province, Tongcheng City in Anhui Province, Yueyang County in Hunan Province, Xiangyun County in Yunnan Province, Rong County in Guangxi Zhuang Autonomous Region and Danling County in Sichuan Province from September to November in each year of 2011-2015. Data were analyzed with chi-square test. Results The overall infection rates of soil-transmitted helminthes in the pilot plots from 2011 to 2015 were 6.1% (490/8 005), 5.8% (474/8 146), 5.5% (447/8 066), 5.8% (477/8 164) and 4.0% (305/7 688), respectively. The infection rates of hookworms, A. lumbricoides and T. trichiura decreased from 3.8% (303/8 005), 1.6% (125/8 005) and 1.1% (84/8 005) to 2.3% (177/7 688), 1.0% (78/7 688) and 0.3% (24/7 688), respectively. From 2011 to 2015, the highest infection rate of STH was found in Tunchang County in Hainan Province (19.2%, 195/1 015; 19.7%, 209/1 063; 20.6%, 215/1 046; 22.6%, 235/1 038; and 11.2%, 114/1 022, respectively). The age group with the highest infection rate of A. lumbricoides was 5-9 years from 2011 to 2014 and 50-59 years in 2015. The age group with the highest infection rate of hookworms was > 60 years from 2011 to 2015(6.2%, 76/1 231; 5.1%, 67/1 328; 6.0%, 88/1 476; 6.2%, 118/1 894; 2.7%, 54/2 032). The infection rate of STH was 5.5% (1 090/19 805) in males and 5.3% (1 050/19 904) in females, with no significant gender difference (P > 0.05). The questionnaire pass rate on health knowledge and hygienic behavior was 93.2% (1 129/1 211), 94.9% (1 142/1 203), 94.1% (1 134/1 205), 92.3% (973/1 054) and 94.9% (854/900), respectively, from 2011 to 2015. The popularity rate of tap water increased from 33.2% (187/564) in 2011 to 59.4% (209/352) in 2015 and the popularity rate of sanitary toilet increased from 71.3% (402/564) in 2011 to 77.3% (272/352) in 2015. Conclusion The STH infection rate has remained at a low level since the termination of integrated control measures in 2009, which indicats that the control strategy has achieved sustainable effect in the pilot counties.

Surveillance results of national monitoring site for soil-transmitted nematode infections in Guizhou Province in 2011-2015

Ai-ya ZHU, Kun DU, An-mei LI, Min LUO, Hong-xia WU, Guang-chu LIN, Yue-hua PAN, Dan-ya SHE, Li-dan LU, Dan-cheng YAO

2017, 35(5):  445-450. 

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Objective To understand the infection status of soil-transmitted nematodes at the national monitoring site for soil-transmitted nematode infections in Guizhou Province during 2011-2015. Methods From 2011 to 2015, Tianguan Village of Maguan Town, Dana Village of Pingshang Township, Shuimu Village of Huachu Town, Qingshan Village of Chengguan Town and Pingshang Village of Pingshang Town were selected from the eastern, southern, western, northern, middle parts of Puding County, respectively, as the national monitoring sites for soil-transmitted nematode infections. Surveillance of soil-transmitted nematode infections was performed in one of these villages in each year. Fecal samples from residents aged over 3 were collected and eggs of intestinal nematodes were examined with the Kato-Katz method. The transparent adhesive tape test was used to examine Enterobius vermicularis eggs among children of 3-12 years. A modified floatation test with saturated sodium nitrate was used to detect Ascaris eggs in soil of vegetable field, around lavatory, courtyard and kitchen. The infection rate among residents was analyzed in aspects of gender, age and occupation with Chi square test. Results A total of 5 035 residents were examined from 2011 to 2015, with a soil-transmitted nematode infection prevalence of 14.24% (143/1 004), 15.72% (161/1 024), 12.29% (123/1 001), 15.38% (156/1 001) and 32.44% (326/1 005), respectively. The prevalence of A. lumbricoides (roundworm), Ancylostoma sp.(hookworm), T. trichiura(whipworm) and E. vermicularis (pinworm) in children were 11.40%(574/5 035), 0.62%(31/5 035), 9.33%(470/5 035) and 1.01%(14/1 381), respectively. There was no significant difference in the rate of soil-transmitted nematode infection between males (18.28%, 473/2 588) and females (17.82%, 436/2 447) (χ² = 0.15, P > 0.05). Among different age groups, the infection rate was highest in the 10- year group (23.25%, 381/1 639; χ² = 62.59, P < 0.05). Among different occupations, the infection rate was highest in students (22.46%, 524/2 333; χ² = 58.43, P < 0.05). Among different degrees of education, the infection rate was highest in those with primary school education(1.41%, 455/2 125; χ² = 39.19, P < 0.05). Among different ethnicities, the Miao ethnicity presented higher infection rate (44.99%, 220/489; χ² = 267.70, P < 0.05) than Han and other minorities. Of the 200 soil samples tested from 2011 to 2015, 86 were positive for A. lumbricoides eggs, with a positive rate of 43%. The detection rate of live fertilized Ascaris eggs was highest in soil samples of vegetable field (26%, 13/50) and around lavatory (20%, 10/50), and lowest in those of the kitchen (2%, 1/50), with a significant difference among the four types of soil (χ² = 12.71, P < 0.05). Conclusion From 2011 to 2015, the infection rate of soil transmitted nematodes was still at a high level, mostly roundworm and whipworm. The infection was more significant among people aged 10-20, students, people with primary school education, and the Miao ethnicity. The soils of the living environment showed different degrees of contamination by Ascaris eggs.

A survey on parasite infection in human intestines in the eastern part of Henan Province in 2015

Yan DENG, Ya-lan ZHANG, Wei-qi CHEN, Xi-meng LIN, Rui-min ZHOU, Ying LIU, Cheng-yun YANG, Dan QIAN, Hong-wei ZHANG, Bian-li XU

2017, 35(5):  450-455. 

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Objective To understand the endemic status of human intestinal parasitic infections in the eastern part of Henan Province, in order to facilitate control of the disease in this region. Methods According to the economic level and geographical location, stratified cluster sampling was made to randomly select 26 survey spots in 9 counties including Linying, Puyang, Taiqian, Taikang, Xiping, Xixian, Xiangcheng, Xinzheng and Yucheng, from November 2014 to June 2015. Human fecal samples were collected and examined for intestinal worm infection using Kato-Katz thick smear method. For the hookworm egg-positive samples, larvae were cultured on the filter paper to identify the species of Ancylostoma. The adhesive tape method was used to detect pinworm eggs in children aged 3 to 6, and iodine staining was used to detect protozoan infection in fecal samples. Information on the source of drinking water and the annual per capita net income of farmers was collected. The infection rate was normalized and compared among different regions. The parasitic infection rate was also analyzed in aspects of species, sex, age, and occupation using the Chi-square test. Results A total of 6 696 fecal samples were collected, from which 6 species of intestinal worms and 4 species of intestinal protozoans were found. The overall infection rate of intestinal parasites was 1.57% (105/6 696), comprising 0.91% (61/6 696) for worms and 0.66% (44/6 696) for protozoans. Concerning the infection rate, the top three intestinal parasites were the pinworms(0.36%, 24/6 696), Blastocystis hominis (0.30%, 20/6 696), and Ascaris lumbricoides (0.19%, 13/6 696). The pinworm infection rate was 2.23% (15/674) among children aged 3-6 and that revealed by the adhesive tape method was 1.93% (13/674). The hookworm larvae detected in 7 samples were all identified to be Ancylostoma duodenale. The top three counties with highest intestinal parasite infection rate were Xiangcheng (4.24%), Xinzheng (3.51%) and Taikang (3.34%). There was no significant difference in the intestinal parasite infection rate between males (1.71%, 55/3 214) and females (1.44%, 50/3 482) (χ² = 0.82, P > 0.05). Among populations with different educational levels, the infection rate was higher in those at a high school or secondary high school level (2.11%, 10/473) (χ² = 2.41, P > 0.05 vs. other groups). Among different age groups, the infection rate of intestinal parasite was highest in the 15-29 age group (2.38%, 21/884) (χ² = 9.39, P < 0.01). Among different occupations, the infection rate of intestinal parasite was significantly higher in preschool children (2.5%, 24/720) and students (2.09%, 24/1 148) (χ² = 9.93, P < 0.05). The infection rate of intestinal parasite was higher in populations in the well water-based spots (1.21%, 31/2 562) than in the tap water-based spots (0.65%, 27/4 134) (χ² = 5.71, P < 0.05). The average annual per capita net income of farmers in the survey spots was 1 200-11 600 yuan (median, 5 604.5 yuan). The intestinal parasite infection rate decreased with increasing annual per capita net income of farmers (χ² = 11.08, P < 0.01). Conclusion The intestinal parasite infection in the eastern part of Henan Province has significantly decreased than the previous two surveys. Preschool children and students are the main population targets for surveillance in the future.

Surveillance of soil-transmitted nematode infections in populations living in rural areas of Xiangcheng District in Zhangzhou City during 2011-2015

Zhi-kui ZHANG, Mei-xian WEI, Jun-ren ZHOU, wen-ge ZHOU, Bao-jian CHEN

2017, 35(5):  456-460. 

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Objective To analyze the prevalence of soil-transmitted nematode infections in populations living in rural areas of Xiangcheng District in Zhangzhou City of Fujian Province, the national surveillance site, during 2011-2015. Methods Five surveillance sites were set in Xiangcheng District during 2011-2015: Dazhai village, Qiukeng Village, Shanmei Village, Gantang Village, and Pulin Village in Eastern, Western, Southern, Northern and Central regions of Xiangcheng District, respectively. The modified Kato-Katoz’s thick smear method was used to detect the eggs of soil transmitted nematodes and the cellophane tape method was used to detect the eggs of Enterobius vermicularis in children aged 3-12 years. Seventy families were randomly selected to obtain soil samples in vegetable garden, orchard and other plantations, from which Ancylostoma sp. larvae were isolated and incubated to identify their species. Results A total of 5 352 persons were examined, of whom 572 (10.7%) were positive for nematode infections. The infection rates during 2011-2015 were 14.3% (146/1 022), 14.7% (155/1 052), 4.0% (42/1 053), 7.3% (78/1 067) and 13.0% (151/1 158), respectively. The nematode infection detected was predominated by Ancylostoma sp. infection(5.9%, 318/5 352), which was highest in 2011 (10.8%, 110/1 022) in the years (6.2%, 65/1 052; 1.4%, 15/1 053; 3.2%, 34/1 067; and 8.1%, 94/1 158 from 2012-2015, respectively), and all the infected persons showed light infection. Ascaris lumbricoides infection was only detected in 2 persons in 2012, and both had light infection. Trichuris trichiura infection was detected in 1, 1, and 2 persons in 2012, 2014 and 2015, respectively, all showing light infection. E. vermicularis infection was examined in 668 children, and 248 showed positive infection (37.1%), and the infection rate was lowest in 2014 (27.7%, 43/155) and highest in 2012 (56.5%, 87/154). The prevalence of soil-transmitted nematode infection in males (11.6%, 300/2 595) was higher than in females (9.87%, 272/2 757) (P < 0.05). The prevalence showed a trend of increase with aging except for the < 15 age group, lowest in 15-24 years (1.9%, 5/266) and highest in > 65 years (11.0%, 98/889) (P < 0.05). Most of those with infections were illiterate or semi-illiterate (23.9%, 245/1 025), or had a primary school educational level (11.9%, 238/1 995). The infection rate decreased with higher educational level (χ² = 308.16, P < 0.05). Ancylostoma sp. larvae were found in 19 of 75 soil samples, and all were identified to be Necator americanus. The number of positive soil samples was highest in Dazhai village (10 samples). Conclusion There was a trend of decrease for the soil-transmitted nematode infections during 2011-2015, but the pinworm infection in children remained high.

Endemic situation and distribution of echinococcosis in Qinghai Province in China

Jing-xiao ZHANG, Xiao MA, Yu-fang LIU, Pei-yun LIU, Jun-ying MA, Yong-shun WANG, Hui-xia CAI, Rui DU, Wei WANG, Wen LEI, Guo-ming SU

2017, 35(5):  460-465. 

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Objective To understand the endemic situation and distribution of echinococcosis in Qinghai Province. Methods Stratified random sampling was performed in 43 counties/cities/districts in Qinghai Province from March to September in 2012, according to pastoral area, pastoral-agricultural area, agricultural area, and township area. The numbers of individuals and villages for survey in each kind of area were determined based on the proportions of total number of residents in each area to the total number in the county/city/district. B ultrasound examination was performed on residents aged over 3. B ultrasound was performed among students of 6-13 years in one primary school selected from each kind of area, or in all the primary schools from the school-enriched counties. Venous blood was also collected to detect serum level of anti-echinococcus IgG by ELISA. The results were statistically analyzed using SPSS17.0 software. The detection rate of echinococcosis and positive rate of serum IgG were calculated and analyzed with chi-square test using SPSS17.0 software. Results A total of 683 villages/communities were selected from the 43 counties/cities/districts. B ultrasound was performed on 121 683 participants, of whom 1 333 were positive for echinococcosis (morbidity rate, 1.10%). The morbidity rates in the pastoral area, pastoral-agricultural area, agricultural area, and township area were 2.51% (1 101/43 822), 0.84% (111/13 228), 0.08% (30/38 882) and 0.35% (91/25 751), respectively. There was a significant difference of the morbidity rate between males（0.90%, 526/58 348） and females（1.27%, 807/63 335）（χ² = 38.93, P < 0.01）. There was also a significant difference in the morbidity rate among different age groups（χ² = 80.84, P < 0.01）, with the highest rate being in the 30-39 years（1.51%, 387/25 682). The participants were of 14 ethnicities. Of them 1 333 were positive for echinococcosis, mainly from five ethnicities, the Tibetan （morbidity rate, 2.25%, 1 277/56 752), Han （0.07%, 32/44 420）, Hui （0.01%, 1/8 229）, Mogol （0.25%, 17/6 806） and Sala ethnicity（0.04%, 1/2 558). There was a significant difference in the morbidity rate among the ethnicities（χ² = 1 354.77, P < 0.01）. Among occupations, the religious people had the highest morbidity rate (5.08%, 52/1 024), followed by herdsmen（2.42%, 1 089/44 981）. A total of 191 primary schools, all from the county towns, were selected, and 47 747 students aged 6-13 years were examined by ultrasonography, of whom 253 were detected to be positive for echinococcosis, with a morbidity rate of 0.53% （253/47 747）. ELISA results showed that 2 693 of 45 507 students were positive for serum anti-echinococcus IgG, with a positive rate of 5.92%. Conclusion There is a serious endemic situation of echinococcosis in counties of southern Qinghai.

Evaluation of the testing ability of city/prefecture-level malaria laboratories in Guizhou Province during 2014-2016

Dan-ya SHE, Jia-hong WU, Li-dan LU, Yu-ting HUANG, Yan GENG, Nian-heng ZHANG

2017, 35(5):  466-471. 

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Objective To re-examine the blood smears and the dry blood filter papers submitted by the city/prefecture-level malaria laboratories in the Malaria Reference Laboratory of Guizhou Province, to evaluate the laboratory testing ability of the city/prefecture-level malaria laboratories. Methods Microscopy and nested PCR were performed to re-examine the blood smears and dry blood filter papers in the Reference Laboratory during 2014-2016, and the blood smear quality was evaluated. Using the microscopic results of the Reference Laboratory as a standard, the malaria diagnostic coincidence concerning positive infection rate, negative rate and species identification were determined. The microscopic and nested PCR results of the Reference Laboratory were analyzed as well.Results From 2014 to 2016, a total of 163 blood smear samples, 84 positive and 79 negative tested in city/prefecture-laboratories, and 159 dry blood filter papers were submitted, with a submission rate of 100% (163/163) and 97.6% (159/163), respectively. The qualification rates of blood smear preparation, staining, cleanliness and comprehensive judgment were 58.9% （96/163）, 75.5% （123/163）, 72.4% （118/163）, and 69.9% (114/163), respect-ively. The qualification rates of blood smear preparation （71.3%） and comprehensive judgment （95.0%） in 2016 were both higher than those in 2014（46.4%,46.4%） and 2015（48.1%, 44.4%）（P < 0.05). The total coincidence rates of microscopic results of the city/prefecture-level malaria laboratories during 2014-2016 were 67.9% （38/56）, 66.7% （18/27）, and 88.8% (71/80), respectively, being highest in 2016（P < 0.05). The coincidence rates of positive infection during 2014-2016 were 81.3% (26/32), 70.0% (14/20) and 96.9% (31/32), respectively, with no significant difference among the three years (P > 0.05). The coincidence rates of species identification during 2014-2016 were 46.9% （15/32）, 65.0% （13/20） and 81.3% （26/32）, respectively （2014 vs. 2016, P < 0.05）. The coincidence rates of negative infection during 2014-2016 were 95.8% (23/24), 5/7 and 93.8% （45/48）, respectively, with no significant difference among the three years（P > 0.05). The consistency rate between microscopic and nested PCR results in the Reference Laboratory was 44.0% （70/159） with respect to positive results and species identification, and 48.43% （77/159) with respect to negative results, and the inconsistency rate was 7.6% （12/159）. Conclusion The qualification rate of comprehensive judgment and the total coincidence rate of microscopic results in the city/prefecture-level malaria laboratories was higher in 2016 than in 2014 and 2015. The results suggested that the testing ability of city/prefecture-level malaria laboratories has increased.

Investigation of Anisakis spp. larva infection in marine fish for sale in Yantai City

Wen-qian ZHANG, Xiao-tong REN, Yu-qi ZHAO, Xin-xin GAI, Yu-mei ZHANG, Xue-lian BAI

2017, 35(5):  472-477. 

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Objective To investigate the infection status of marine fish sold in Yantai City by Anisakis spp. Methods Marine fish of different species were randomly purchased from seafood markets in Yantai City from January to December 2016. The fish were dissected to find and collect Anisakis larvae from the viscera and muscle. The larvae species were identified under a light microscope. The Anisakis larvae infection status in fish was analyzed in different aspects of fish species, visceral organs and fish body weight. Results A total of 256 marine fishes of 14 species were dissected. The overall infection rate was 66.4% (170/256) and the average infection intensity was 14.8 larvae/fish. Those fishes with top infection rate included Pneumatophorus japonicas (100%, 26/26), Trichiurus lepturus (92.3%, 24/26), Platycephalus indicus (92.0%, 23/25) and Sebastodes fuscescens (85.7%, 18/21). The average infection intensity of them was 25.1, 21.1, 12.2, and 24.9 larvae/fish, respectively. Other species with comparatively lower infection rate were Monopterus albus (80.0%, 6.3 larvae/fish), Prognichthys agooz (70.0%, 3.3 larvae/fish), Larimichthys polyactis (66.0%, 11.7 larvae/fish), Scomberomorus niphonius (52.0%, 9.1 larvae/fish), Cleisthenes herzensteini (50.0%, 1.8 larvae/fish), Clupea pallasi (44.4%, 9.5 larvae/fish) and Tylosurus melanotus (38.1%, 2.0 larvae/fish). No infection was found in Pampus argenteus, Thamnaconus modestus and Perca fluviatilis. The Anisakis spp. larvae were mainly located at mesentery and intestinal walls as well as gastric wall and gonad. There were also varied relationships between infection intensity and weight among different fish species. The infection intensity of 0.25-0.35 kg P. japonicus, 0.16-0.20 kg T. lepturus, 0.16-0.25 kg P. indicus, 0.05-0.10 kg L. polyactis, 0.17-0.26 kg S. fuscescens and 0.81-1.00 kg S. niphonius was 46.5, 27.2, 18.0, 12.0, 42.7 and 68.0 larvae/fish, respectively. Among those species, the infection intensity decreased with the increase of body weight in S. fuscescens (r = 0.484, P < 0.05) and L. polyactis (r = -0.633, P < 0.01), but increased with body weight in S. niphonius (r = 0.740, P < 0.01). However, for P. japonicas, T. lepturus and P. indicus, neither relationship was found (r = -0.237, -0.080, -0.196, P < 0.05). Conclusion There is a high infection rate (above 85%) of Anisakis larvae in P. japonicas, T. lepturus, P. indicus and S. fuscescens from the seafood markets in Yantai City.

Morphological observation of Sinopotamon pinghense and investigation of its infection with metacercariae of Euparagonimus cenocopiosus in Liudou Mountain Natural Reserve in Fujian Province

Mao-rong CAI, Guo-hua LIN, Jin-zhong CHEN, Jun LUO, Ming-song HUANG, Yan DENG, You-zhu CHENG

2017, 35(5):  478-482. 

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Objective To investigate the infection of Sinopotamon pinghense with Euparagonimus metacercariae in Liudou Mountain Natural Reserve in Nanjing County and the morphology of S. pinghense. Methods Samples of S. pinghense crabs were collected from the streams outside (X1) and inside(X2) the Liudou Mountain Natural Reserve from November 2016 to May 2017, the place of collection and environmental factors of the habitats were recorded by GPS. The E. cenocopiosus metacercariae in the heart of the crabs were morphologically observed with the direct compression method. The metacercariae infection rates were compared between crabs from X1 and X2 streams, and among large, medium-sized, and small crabs. Data were analyzed with SPSS 22.0 software. The S. pinghense crabs were morphologically observed and photos were taken. Results The infection rates of E. cenocopiosus in S. pinghense from X1 and X2 streams were 17.7%（3/16) and 86.3%（63/73）, respectively, with a significant difference（χ² = 29.817, P < 0.05). The average intensity of infection was 1.7 metacercariae per crab in X1 and 11.6 in X2, with the latter being 6.9 folds of the former. A total of 742 metacercariae were collected from X2, with the maximum number of 79 metacercariae in one crab. The intensity of infection in large crabs was 2.6 and 5.8 times of those in medium-sized and small crabs, respectively. The infection rates in large, medium-sized, and small crabs in X2 were 100% (31/31), 82.6% (19/23) and 68.4% (13/19), respectively (χ² = 31.620, P < 0.05). The first male pleopod of S. pinghense reached beyond sutures of chest 5/6. Its irregularly-shaped terminal segment pointed towards the outer front direction, had a round base, and became narrower rostrally. The terminal segment was divided into 2 lobes by a longitudinal fissure, which was clearly seen from the backside. The inner margin of the subterminal segment displayed as a square angle while the outer edge was oblique. Conclusion The Liudou Mountain Natural Reserve in Nanjing County is a new natural spot with E. cenocopiosus infection. The new morphological findings of S. pinghense provides basis for identifying similar species of Sinopotamidae.

Interpertation of Technical Regulation for Application of Antimalarials

Qi GAO

2017, 35(5):  482-485. 

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This paper introduces the impotance of formulation and implement of Technical Regulation for Application of Antimalarials for treatment of malaria cases and public health in China. The paper explains how to select the anti-malaria drugs based on WHO Guidelines for The Treatment of Malaria and modified according to manufacture and supply of anti-malaria drugs in China as well as the experinces of malaria control and elimination in the country. The paper gives detailed description about the difference of used dosages between Technical Regulation for Application of Antimalarials and WHO Guidelines for The Treatment of Malaria as well as Principle and Dosage of Antimalarial Usage. It also explains how to adjust the drug dosage in special situation so that to help clinical doctors and public health workers reasonably use antimalarials for case treatment and block transmission in elimination activities.

Injection of anti-TLR9 antibody cannot efficiently block TLR9 activities in mice: a brief discussion about TLR9 location in cells

Xiao LIU, Meng-meng LI, Feng QIAN

2017, 35(5):  485-488. 

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In a paper published in this journal (No.1 Vol. 35, 2017) entitled The regulatory effect of dendritic cells on Th17 cell differentiation and function in mouse infected with Plasmodium yoelii, the authors stated in an experimental method that they used anti-TLR9 antibody to block dendritic cell function in mice. We do not think this method works out, because there have been accumulating data revealing that TLR9 is mainly an intracellular receptor, so injection of anti-TLR9 antibody cannot efficiently block TLR9 activities in vivo.

Progress in research on risk assessment approach and parameters for malaria re-emergence

Tian-mu CHEN, Shao-sen ZHANG, Shui-sen ZHOU

2017, 35(5):  489-494. 

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Risk assessment for malaria re-emergence has high public health significance in China and in other countries that have eliminated malaria or on the way of elimination. Current risk assessment approaches for malaria re-emergence are classified as classical epidemiological evaluation, mathematical modelling and infectivity-receptivity-vulnerability method (IRV). This review updates researches on risk assessment approaches and related parameters, in order to provide basis for implementation of risk assessment on malaria re-emergence in China.

Approaches to screening and identifying genes associated with drug-resistance of Plasmodium falciparum

Yi-ni TIAN, Run YE, Wei-qing PAN, Dong-mei ZHANG

2017, 35(5):  495-498. 

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Plasmodium falciparum has developed resistance to almost all currently available antimalaria drugs. The emergence of resistance to artemisinin and the alike in recent years has greatly challenged the global malaria control and elimination. Thus, screening and identifying genes associated with the drug resistance is of great significance to elucidate the action mechanism of antimalarials, monitor the occurrence and spread of resistance, and set up guidelines for antimalarial drug use. This paper summarizes the approaches to screen and identify the genes associated with drug resistance of P. falciparum.

Current status of vaccine development against leishmaniasis

Ya-shuang CAI, Zhi-gang ZHANG, Xin WANG, Qin-jian ZHAO

2017, 35(5):  499-503. 

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Leishmaniasis is a zoonotic disease caused by Leishmania parasites. Currently, leishmaniasis has affected 12 million people in over 90 countries, with 1.3 million reported cases each year and 20-30 thousand deaths. The disease causes a large social and economic burden particularly in developing countries. Current strategies for leishmaniasis control include controlling the source of infection and drug treatment, both of which, however, cannot eliminate the disease. Effective vaccination would be an ideal approach to control leishmaniasis. Here, we review the pathogenic mechanism of Leishmania, the existing vaccines for veterinary use, and human vaccine development efforts against the disease.

Research advances on the inhibition of interferon-γ-dependent cellular immunity by Toxoplasma gondii

Li-jie YAO, Hong-juan PENG

2017, 35(5):  503-508. 

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It has been reported that Toxoplasma gondii, after invading mouse cells, secrets a virulence factor ROP18 to bind and phosphorylate immunity-related p47 GTPase (IRG), inhibiting its binding to parasitophorous vacuole membrane (PVM) and thus the breakage of the parasitophorous vacuole (PV), enabling the growth and proliferation of T. gondii in the PVs. In contrast to the elucidation of immune escape mechanism of T. gondii in mouse cells, little is known in human cells. It is reported that the ubiquitin-labeled PVs can fuse with the lysosomal system in human cells, which results in death of T. gondii tachyzoits by acidification. This review summarizes the mechanisms by which T. gondii inhibits cellular immunity, especially the interferon-γ-dependent cellular immunity in host cells. In our understanding of immune evasion of T. gondii through blocking ubiquitination of PVs, recent advances are reviewed as well.

Screening of antigens specific for the anti-Cysticercus cellulosae IgG4 antibody

Jin LI, Yan WANG, Qing-kuan WEI, Feng-ju JIA, Ting XIAO, Hui SUN, Zhen-hua YU, Bing-cheng HUANG

2017, 35(5):  509-511. 

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The prepared cyst fluid antigen was separated by two-dimensional electrophoresis(2-DE). The protein spots were analyzed by ImageMaster 2D Plantinum 6.0 software after Coomassie blue staining, on which Western blotting analysis was performed using sera of patients with cerebral cysticercosis as the primary antibody. The positive protein spots were selected and analyzed by Matrix-assisted laser desorption ionization tandem mass spectrometry (GC-MS), and the peptide mass fingerprints were obtained. The specific Cysticercus cellulosae antigen for IgG4 antibody was identified with the Mascot software by searching and alignment in NCBI. 2-DE detected 201 ± 5 protein spots in the cystic acid antigen, with a relative molecular weight （M_r） of 10 000-170 000 and a pI of 3.0-10.0. Western blotting analysis showed that the antigens binding the specific IgG4 antibody were mainly small molecular-weight proteins. Fifty-one(antigen) spots were obtained from them for mass spectrometry, and 21 of them were positive(peak score>39). The positive spots were related with 10 cysticercus proteins, most closely with Ts8B2, Ts8B3, 10ku antigen and Sequence 3.

Epidemiological investigation on echinococcosis in Huangnan Tibetan Autonomous Prefecture of Qinghai Province

Xiao MA, Hu WANG, Shi-lei CHENG, Jing-xiao ZHANG, Yong-shun WANG, Yu-fang LIU, Jun-ying MA, Hui-xia CAI, Pei-yun LIU, Xiu-min HAN, Hai-qing LIU, Yan-mei ZHAO, Wei WANG, Wen LEI, Rui DU, Guo-ming SU, Na LIU, Ke-mei SHI, Xiong-ying ZHANG

2017, 35(5):  512-514. 

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To analyze the prevalence of hydatid disease in Huangnan Tibetan Autonomous Prefecture of Qinghai, two to three villages were randomly selected in each of the Henan County, Jianzha County, Tongren County, and Zeku County in Huangnan of Qinghai. Ultrasound examination was performed for local residents aged > 1 year. Animal echinococcosis was investigated by visceral anatomy, and ELISA was used to detect echinococcosis antigen in fecal samples of stray dogs. In this study, a total of 11 231 residents received ultrasound examination, of whom 77 were positive for hydatid cysts with a morbidity rate of 0.69%. The prevalence of hydatid disease was highest in Zeku County (2.12%, 68/3 213), followed by Henan County (0.26%, 9/3 430). The prevalence of hydatid disease in females was 0.72% (44/6 094), and 0.64% (33/5 137), respectively. Among various occupations, the herdsmen had the highest prevalence rate of 0.83% (61/7 338). Among age groups, the human hydatid disease prevalence rate was relatively higher in populations of 50-59 years (1.25%, 9/721), 60-69 years (1.96%, 13/663) and 30-39 years (0.91%, 24/2 637). In addition, the prevalence of hydatid disease was higher in the uneducated (0.66%) and in those at the primary school level (0.79%). The residents that settled in winter and was nomadic in summer had the highest prevalence (1.10%, 70/6 391), followed by the nomadic population (0.62%, 6/965). There were significant prevalence differences in terms of region, age, living style and educational level (P < 0.05), rather than in gender, occupation (P > 0.05). Of the 1 282 dog fecal samples collected, the fecal antigen positive rate was 11.70%（150/1 282）, with a significant regional difference (P < 0.05). Among the 3 525 intermediate host sheep, the hydatid infection rate was 2.13% (75/3 525), with a significant regional difference (P < 0.05). The results indicats a hydatid disease morbidity of 0.69% in residents and 2.13% in sheep, and the antigen positive rate in dogs feces is 11.70% in Huangnan Tibetan Autonomous Prefecture of Qinghai Province.