A comparative study of lung-microscopy and tissue homogenate in detecting Angiostrongylus cantonensis in the Pomacea canaliculata / 中国热带医学

@#Abstract: Objective　To compare the efficiency of lung-microscopy and tissue homogenate in the detection of Angiostrongylus cantonensis larvae from Pomacea canaliculate, with the amin of finding a simple and rapid method suitable for different scenarios. Methods　Pomacea canaliculata was caught and collected from ponds, ditches, rivers and other environments in the confirmed epidemic areas reported by predecessors. After each snail was weighed one by one, and dissected into two parts: lung sac and muscle. Firstly, each snail lung sac was check for nodules with lung-microscopy, and the nodules were picked out with anatomical needle and press them separately. Then, the worm was found and identified for worm species under microscope, and the lung sac and snail meat then was rechecked with tissue homogenate. Results　A total of 330 snails were detected, with 19.1% (63/330) snails with Angiostrongylus cantonensis stage Ⅲ larvae were detected by tissue homogenate and 15.8% (52/330) snails with nodules were detected by lung-microscopy. Among them, 36 snails with nodules and larvae were detected by the lung-microscopy, and all of them were positive by the tissue homogenate, with a coincidence rate of 100% (36/36); 16 snails with nodules but no larvae, among which 6 snails were positive and 10 snails were negative by the tissue homogenate, The false detection rate was 19.2% (10/52). 278 snails with no nodules, but 21 of them were detected by the tissue homogenate, the missing rate was 7.6% (21/278). There was no significant difference between tissue homogenate and lung-microscopy (taking the positive determination of nodules as the standard) (χ2=1.27, P=0.26, P>0.05). There was significant difference between tissue homogenate and lung-microscopy (taking the detection of larvae as the standard)(χ2=8.66, P = 0.003, P<0.01). There was no significant difference between the two methods and tissue homogenate in the detection rate of large snails ( ≥25 g, χ2=0.08，P=0.777; χ2=2.58, P=0.108), but there was significant difference between the two methods and tissue homogenate in the detection rate of small snail (≤10 g, χ2=5.63, P=0.02). Conclusions　Compared with the tissue homogenate, lung-microscopy is simple in the detection of large snails, requires less instruments and equipment, and its detection speed is faster. It is suitable for the field investigation of the natural focus of Angiostrongylus cantonensis. The tissue homogenate has high sensitivity, can directly display the insect state and activity, has strong insect vitality and high detection rate, it's more suitable for food safety risk monitoring.

Taxonomic integrative and phylogenetic identification of the first recorded Triatoma rubrofasciata in Zhangzhou, Fujian Province and Maoming, Guangdong Province, China.

BACKGROUND: Most species of Triatominae live exclusively in Latin America. However, one species, Triatoma rubrofasciata, has been recorded in the Americas as well as in various port areas in Africa and Asia. An increasing number of T. rubrofasciata have been reported in southern China in recent years. However, the origin of this invasive insect vector in China remains unknown, therefore, accurate identification and phylogenetic analysis of the bugs are urgently needed. METHODS: A total of seven triatomine insect specimens were found and collected from Maoming City, Guangdong Province, China (GDMM) and Zhangzhou City, Fujian Province, China (FJZZ), respectively. The obtained insect vector specimens were observed under a dissecting microscope for morphological classification and then the genomic DNA was extracted, and the 16S ribosomal RNA (rRNA), 28S rRNA as well as cytochrome oxidase subunit I (COI) genes of the species were amplified and sequenced. Subsequently, molecular phylogenetic analyses based on multiple alignments of the above genes were conducted in order to identify the species and determine the phylogenetic origin approximation accurately. RESULTS: The triatomine insects collected from GDMM and FJZZ were identified as Triatoma rubrofasciata using morphological and genetic analyses. All of the Chinese T. rubrofasciata captured in FJZZ, GDMM and other localities in southern China, together with a Vietnamese and Brazilian strain, formed a new, cohesive clade. T. rubrofasciata in GDMM and FJZZ are likely derived from strains found in Vietnam or Brazil. CONCLUSIONS: To the best of our knowledge, this is the first record of the invasive insect T. rubrofasciata, which is likely derived from strains native to Vietnam or Brazil, in both Maoming City, Guangdong Province and Zhangzhou City, Fujian Province of China. A comparison of the DNA sequences of the 16 s rRNA, 28 s rRNA and COI genes confirmed the specific identification of T. rubrofasciata, and its potential origin in China is based on the phylogenetic analyses undertaken in this study. More targeted interventions and improved entomological surveillance are urgently needed to control the spread of this haematophagous insect in China.

Trypanosoma brucei rhodesiense infection in a Chinese traveler returning from the Serengeti National Park in Tanzania.

BACKGROUND: Human African trypanosomiasis (HAT) is one of the most complex parasitic diseases known to humankind. It usually occurs in endemic areas in Africa, but is occasionally detected in returning travelers and migrants in non-endemic countries. CASE PRESENTATION: In August 2017, a case of HAT was diagnosed in China in a traveler returning from the Masai Mara area in Kenya and the Serengeti area in Tanzania. The traveler visited Africa from 23 July to 5 August, 2017. Upon return to China, she developed a fever (on 8 August), and Trypanosoma brucei rhodesiense infection was confirmed by laboratory tests (on 14 August) including observation of parasites in blood films and by polymerase chain reaction. She was treated with pentamidine followed by suramin, and recovered 1 month later. CONCLUSIONS: This is the first imported rhodesiense HAT case reported in China. This case alerts clinical and public health workers to be aware of HAT in travelers, and expatriates and migrants who have visited at-risk areas in Africa.

Surveillance and response strategy in the malaria post-elimination stage: case study of Fujian province.

Malaria used to be a serious health problem in Fujian province in the past, but no local malaria transmission has been found since 2000. In order to eliminate the potential residual cases and prevent re-introduction of malaria so as to achieve the final goal of malaria elimination in Fujian province, various strategy and intervention approaches were tailored to the local settings. For instance, the monitoring of febrile patients by blood smear examinations and vector surveillance and control were strengthened in addition to the routine intervention in the mountainous area of Fujian province, where malaria was highly endemic and the mosquito Anopheles anthropophagus distributed with a high vectorial capacity. There were two local cases who got infected due to imported cases found in the building site of an expressway in 2004 and 2005, respective. All other imported malaria cases were detected during post-elimination stage through surveillance system. Based on results from post-transmission surveillance, malaria transmission has been interrupted in Fujian province for 13 years. Therefore, post-transmission surveillance and response is an important intervention to maintain the malaria elimination achievements in Fujian province.

The effect of temperature on the extrinsic incubation period and infection rate of dengue virus serotype 2 infection in Aedes albopictus.

Dengue fever is an acute mosquito-borne viral disease caused by dengue virus (DENV). Temperature may affect the efficiency of the mosquito vectors in spreading DENV. Aedes albopictus mosquitoes were infected orally with a DENV2 suspension and incubated at different temperatures. Subsequently, DENV2 antigen was collected from salivary gland and thorax-abdomen samples on different days postinfection and tested using an immunofluorescence assay to determine the extrinsic incubation period and infection rate. As the temperature increased, the extrinsic DENV2 incubation period in Ae. albopictus gradually shortened, and infection rates showed a tendency to initially increase, followed by a subsequent decrease.

[Genetic polymorphism of the gene encoding the apical membrane antigen-1 of Plasmodium falciparum].

OBJECTIVE: To study the genetic diversity of apical membrane antigen-1 gene from Plasmodium falciparum (PfAMA-1). METHODS: Filter paper blood samples were collected from 23 imported P. falciparum malaria patients who returned to Fujian Province from 2006 to 2012. Nested PCR were used to amplify the PfAMA-1 gene. The umplified fragments were sequenced, and analyzed by bioinformatic software. RESULTS: All 23 samples were amplified a 505 bp band. Thirty-two nucleotides were found to be variable, resulting in 18 haplotypes. Eight of these 18 halotypes were being reported here for the first time. The parasites collected from Africa showed the higher level of variability [haplotypes diversity (Hd)= 0.0985, nucleotide diversity (π)=0.0258] as compared to the isolates from Asia (Hd=0.909, π=0.0221). The average difference of dN-dS for all 23 PfAMA-1 sequences was 0.031±0.006. Sequence-based neutrality tests were not significant in Africa and Asia (P>0.05). The minimum number of recombination events (Rm) was 10, and the linkage disequilibrium index (R2) evidently declined with the increase of nucleotide distance. A molecular phylogenetic tree constructed using the neighbor-joining method showed that the 23 isolates were assigned to three clades (G1, G2 and G3). Most samples from Africa formed G1, and G3 contained most of Asian isolates. CONCLUSION: Plasmodium falciparum isolates from Africa show a higher genetic diversity than the isolates from Asia for PfAMA-1 gene.

[A case of imported schistosomiasis haematobia first reported in Fujian Province].

The first case of imported schistosomiasis haematobia in Fujian Province was detected and the patient was cured following treatment with praziquantel.

[Prevalence of Angiostrongylus cantonensis infection in snails for sale in Fuzhou and Xiamen].

OBJECTIVE: To investigate the prevalence of Angiostrongylus cantonensis infection in snails for sale from Fuzhou and Xiamen of Fujian Province. METHODS: During 2009-2012, two markets and five restaurants from each city were selected as surveillance sites. A. cantonensis infection rate in Pomacea canaliculata, Bellamya aeruginosa and Cipangopaludina cathayensis was examined two times per month. More than 50 P. canaliculata or C. cathayensis, and about 500 g B. aeruginosa were collected in each site. A. cantonensis larvae infection was determined by lung-microscopy in P. canaliculata, and by tissue homogenate method in C. cathayensis and B. aeruginosa, respectively. RESULTS: In markets, a total of 5,744 P. canaliculata were collected, and the infection rate of A. cantonensis larvae was 13.8% (753/5 744) with the lowest prevalence in 2009 (8.4%, 28/334) and the highest one in 2011 (16.7%, 361/2,160). The overall infection rate of A. cantonensis showed an increasing trend over the past years (P < 0.05). In restaurants, 879 P. canaliculata snails were examined in Xiamen City, and the infection rate was 12.8% (877/6 879). No significant difference was found among years (P > 0.05). A. cantonensis larvae were found from P. canaliculata for sale in different seasons with no statistical difference (P > 0.05). 19,843 B. aeruginosa snails were collected in markets from the two cities, and the infection rate was 0.2% (31/19 843). The infection rate was highest in 2011 (0.3%, 16/5,953) and lowest in 2010 (0.04%, 2/4,706). All the 361 C. cathayensis snails were negative. CONCLUSION: A. cantonensis larvae are found in P. canaliculata and B. aeruginosa from markets and restaurants of Fuzhou and Xiamen in different seasons during the years.

[Genotype I Japanese encephalitis virus is the main genotype in mosquito in Fujian province].

OBJECTIVE: To grasp the infection rate and genotypes of Japanese encephalitis virus (JEV) in mosquito in Fujian province. METHODS: Mosquito specimens in Sanming city, Jianyang city and Fuzhou city in Fujian province were collected in 2010. RT-PCR was used to detect the JEV sequence from the mosquitoes by specific primers. The sequence splicing and the differentiation analysis for nucleotides, deduced amino acid sequence and phylogenetic tree were performed by the software of ATGC, Clustal X (1.83), MegAlign, GeneDoc 3.2 and Mega (4.0). RESULTS: Totally 6987 mosquitoes were collected and main species was Culex tritaeniorhynchus and Anopheles sinensis. The infection rate of JEV in mosquitoes in Sanming, Jianyang and Fuzhou were 1.25%, 1.76% and 0.65%, respectively. One full genome in the positive specimens was sequenced. And further study showed that the positive JEV sequences belonged to genotype I. CONCLUSION: Genotype I Japanese encephalitis virus is the main genotype in mosquitos in Fujian province.

[DNA amplification of Plasmodium vivax parasites from Giemsa-stained blood smears].

OBJECTIVE: To develop methods of extracting DNA from malaria parasites on Giemsa-stained blood smears. METHODS: Improved Na2HPO4 method and Chelex-100 ion-exchange technique were used to extract DNA from Giemsa-stained or unstained blood smears. Nested PCR was employed for amplification and identification of allelotype in the Plasmodium vivax merozoite surface protein-1 (PvMSP-1). RESULTS: Target DNA bands appeared in all samples of unstained thick blood smears, while no DNA bands were visible in the fixed and stained thin smears. Both methods identified PvMSP-1 alleles from smears with parasitemia of > or = 0.01%. CONCLUSION: It is feasible to identify PvMSP-1 alleles from Giemsa-stained blood smear.