Gene characteristics of coxsackievirus A16 in hand, foot and mouth disease cases in Baoshan City, Yunnan Province, 2022 / 中国热带医学

@#Abstract: Objective　To analyze the results of the surveillance of hand, foot and mouth disease (HFMD) pathogens in Baoshan City, Yunnan Province in 2022, and to analyze the genetic characteristics of the main epidemic strain coxsackievirus A16 (CVA16), in order to provide scientific basis for the prevention and control of HFMD. Methods　Samples of hand, foot and mouth disease cases in Baoshan City submitted to Yunnan Province in 2022 were selected, the samples were processed, and the viral nucleic acid was extracted, and the Enterovirus (EV) VP4/VP2 binding region gene was amplified with primers (MD91/OL68-1) and sequenced. The sequence was BLAST searched in GenBank to determine the virus type, and then the full sequence of VP1 region was amplified with relevant primers of each type of virus and sequenced. Viruses were identified by using Enterovirus Genotyping Tool Version 1.0. according to the reference documents, the reference sequences were downloaded for making the phylogenetic tree, and the test results were statistically analyzed. Results　A total of 307 clinical samples of hand-foot-mouth disease in Baoshan City in 2022 were detected by real-time RT-PCR. There were 280 laboratory-confirmed cases (91.21%), among which the detection rate of CVA16 was 55.05% (169/307), EV-A71 was 3.58% (11/307), and other enteroviruses were 32.57% (100/307). Of the 206 HFMD test specimens, the VP4/VP2 junction and VP1 gene sequences of enterovirus were amplified and identified, and 29 enterovirus strains were obtained, with a positive virus rate of 14.08% (29/206). All viruses belonged to group A and were divided into 3 serotypes, which included 27 strains of CVA16 (93.10%, 27/29), 1 strain of EV-A71 (3.45%, 1/29), and 1 strain of CVA10 (3.45%, 1/29). Group B, C, and D viruses were not detected. The positive rate of VP4/VP2 binding region and VP1 region of coxsackievirus A16 gene in Baoshan City in 2022 was 13.11% (27/206). The genetic evolution analysis showed that the 27 strains of CVA16 belong to B1a subtype and can be divided into two evolutionary branches. Conclusions　In 2022, the main epidemic strain of HFMD in Baoshan City, Yunnan Province is CVA16 gene, belonging to B1a subtype, which can be divided into two branches, indicating 2 transmission chains prevalent in Baoshan City. Future efforts should focus on strengthening surveillance, improving the quality of monitoring, and understanding the characteristics of viral prevalence..

A natural symbiotic bacterium drives mosquito refractoriness to Plasmodium infection via secretion of an antimalarial lipase.

The stalling global progress in the fight against malaria prompts the urgent need to develop new intervention strategies. Whilst engineered symbiotic bacteria have been shown to confer mosquito resistance to parasite infection, a major challenge for field implementation is to address regulatory concerns. Here, we report the identification of a Plasmodium-blocking symbiotic bacterium, Serratia ureilytica Su_YN1, isolated from the midgut of wild Anopheles sinensis in China that inhibits malaria parasites via secretion of an antimalarial lipase. Analysis of Plasmodium vivax epidemic data indicates that local malaria cases in Tengchong (Yunnan province, China) are significantly lower than imported cases and importantly, that the local vector A. sinensis is more resistant to infection by P. vivax than A. sinensis from other regions. Analysis of the gut symbiotic bacteria of mosquitoes from Yunnan province led to the identification of S. ureilytica Su_YN1. This bacterium renders mosquitoes resistant to infection by the human parasite Plasmodium falciparum or the rodent parasite Plasmodium berghei via secretion of a lipase that selectively kills parasites at various stages. Importantly, Su_YN1 rapidly disseminates through mosquito populations by vertical and horizontal transmission, providing a potential tool for blocking malaria transmission in the field.

Molecular epidemiology of mosquito-borne viruses at the China-Myanmar border: discovery of a potential epidemic focus of Japanese encephalitis.

BACKGROUND: Mosquito-based arbovirus surveillance can serve as an early warning in evaluating the status of mosquito-borne virus prevalence and thus prevent local outbreaks. Although Tengchong County in Yunnan Province-which borders Myanmar-is abundant and diverse in mosquitoes, very few mosquito-based arbovirus investigations have been conducted in the recent decade. Herein, this study aims to evaluate the presence and the diffusion of mosquito-borne pathogens, currently prevalent in this region. METHODS: We collected 9486 mosquitoes, representing eight species, with Culex tritaeniorhynchus and Anopheles sinensis as the dominant species, during high mosquito activity seasons (July-October) in Tengchong, in 2018. Samples collected from 342 pools were tested using reverse-transcription PCR to determine the species, distribution, and infection rates of virus and parasite, and further analyze their genotypes, phylogenetic relationships, infection rate, and potential pathogenicity. RESULTS: Fifteen Japanese encephalitis virus (JEV) strains from Cx. tritaeniorhynchus pools were detected. Seven strains of insect-specific flaviviruses (ISFVs), including two Aedes flavivirus (AeFV) and Yunnan Culex flavivirus strains each, one Culex theileri flavivirus, Yamadai flavivirus (YDFV) and Anopheles-associated flavivirus (AAFV) strains each were detected in Aedes albopictus, Cx. tritaeniorhynchus, Cx. vagans, Cx. pseudovihnui, and An. sinensis pools, respectively. The whole-genome was successfully amplified in one strain of JEV and AeFV each. Phylogenetic analysis using the E gene placed all the newly detected JEV strains into the GI-b genotype. They showed highly nucleotide identities, and were most closely related to the strain detected in Tengchong in 2010. The comparison of the E protein of JEV strains and vaccine-derived strain, showed six amino residue differences. The bias-corrected maximum likelihood estimation values (and 95% confidence interval) for JEV in Cx. tritaeniorhynchus collected in Tengchong in 2018 were 2.4 (1.4-3.9). CONCLUSIONS: A potential Japanese encephalitis epidemic focus with the abundance of host mosquitoes and high JEV infection rate was observed in Tengchong. In addition, at least five species of ISFVs co-circulate in this area. This study highlights the importance of widespread and sustained mosquito-based arbovirus surveillance in local areas to prevent the transmission of JEV, and other emerging/re-emerging mosquito-borne pathogens.

Detection of novel piroplasmid species and Babesia microti and Theileria orientalis genotypes in hard ticks from Tengchong County, Southwest China.

To reveal the genetic diversity of Babesia microti and Theileria orientalis in Southwest China, we conducted a molecular survey of piroplasms in hard ticks in a China-Myanmar border county. Host infesting and questing ticks were collected from Tengchong County in 2013 and 2014. Piroplasm infection in ticks was detected by PCR, and then, phylogenetic analysis was conducted to study the genetic diversity of the pathogens identified in ticks. All in all, six piroplasm species comprising of B. microti; B. orientalis; a novel Babesia species designated Babesia sp. Tengchong, China; T. orientalis; T. luwenshuni; and an as yet undescribed piroplasmid species referred to as Piroplasmid sp. Tengchong, China, have been identified after screening goat- and cattle-attached ticks. In addition, B. bigemina has been identified by screening questing ticks. Phylogenetic analysis based on the 18S rRNA and partial ß-tubulin gene revealed two novel potentially zoonotic genotypes designated B. microti Tengchong-Type A and B. The T. orientalis genotypes identified in the present study represent the seven known genotypes 1-5, 7, and N3 as revealed by phylogenetic analysis of 18S rRNA and MPSP genes. Importantly, an additional genotype designated N4 has also been identified in this study, which brings the number of recognized T. orientalis genotypes to a total of twelve. Thus, besides the two novel species, Babesia sp. Tengchong, China, closely related to Babesia species isolated from yak and Piroplasmid sp. Tengchong, China, our study demonstrates that additional novel B. microti and T. orientalis genotypes exist in Southwest China.

High genetic diversity in hard ticks from a China-Myanmar border county.

BACKGROUND: Many tick species have great morphological similarity and are thus grouped into species complexes. Molecular methods are therefore useful in the classification and identification of ticks. However, little is known about the genetic diversity of hard ticks in China, especially at the subspecies level. Tengchong is one of the epidemic foci of tick-borne diseases in China, but the tick species inhabiting the local area are still unknown. METHODS: Eighteen villages in Tengchong County, China, were selected for sampling carried out from September to October 2014. Infesting hard ticks were removed from the body surface of domestic animals and questing ticks were collected from grazing fields. After morphological identification, molecular characteristics of each tick species were analyzed based on both 16S rRNA and cytochrome c oxidase subunit 1 (cox1) gene fragments. RESULTS: Six tick species were identified based on morphology: Rhipicephalus microplus, R. haemaphysaloides, Ixodes ovatus, Haemaphysalis longicornis, H. shimoga and H. kitaokai. Phylogenetic analysis using the cox1 gene revealed that R. microplus ticks from the present study belong to clade C. For tick samples of both R. haemaphysaloides and I. ovatus, three phylogenetic groups were recognized, and the intergroup genetic distances exceeded the usual tick species boundaries. Haemaphysalis longicornis ticks were clustered into two separate clades based on the cox1 gene. For ticks from both H. shimoga and H. kitaokai, two phylogenetic groups were recognized based on the phylogenetic analysis of the 16S rRNA gene, and the intergroup genetic distances also exceeded the known boundaries for closely related tick species. CONCLUSIONS: According to molecular analyses, new species or subspecies closely related to R. haemaphysaloides, I. ovatus, H. shimoga and H. kitaokai probably exist in the China-Myanmar border Tengchong County, or these ticks form species complexes with highly divergent mitochondrial lineages. Morphological comparisons are warranted to further confirm the taxonomic status of these tick species.

Shifting from control to elimination: analysis of malaria epidemiological characteristics in Tengchong County around China-Myanmar border, 2005-2014.

BACKGROUND: Tengchong County experienced a decreasing malaria prevalence period in 2005-2014 but the factors contributing to the trend are unclear. Herein, the malaria epidemiological data in years of 2005-2014 were collected and analysed, in order to provide evidence for subsequent effective strategic planning of malaria elimination that may be referenced by other counties with the similar elimination programmes along the China-Myanmar border. METHODS: A retrospective study was conducted to explore malaria-endemic characteristics in years 2005-2014 in Tengchong County. All individual cases from a web-based reporting system were reviewed and analysed. Local infections and imported cases were obtained from an annual reporting system. RESULTS: In total, 8321 confirmed malaria cases were recorded in this period, and 91.5% of them were reported during 2005-2010. Plasmodium vivax was the major species (n = 5867, 70.5%). Most cases (92.9%) were found in males, mainly in the age group 30-34 years. Only five deaths resulting from Plasmodium falciparum were reported, of which three occurred in 2005. The cases were mainly reported in the townships of Wuhe (18.5%), Mangbang (12.8%) and Gudong (9.3%). In addition, 147 local malaria (1.8%) and 8174 imported malaria (98.2%) were observed during 2005-2014. However, the proportion of imported malaria was more than 95% all the time and no local transmission has been observed since 2013. Moreover, Myanmar was the main imported source, with 716 cases (94.6%, 716/757) from Myanmar in 2011-2014. CONCLUSIONS: Tengchong County has made achievements in controlling malaria, with incidence at historically its lowest level. However, imported malaria has increased and poses a great threat to malaria elimination. To achieve the elimination goal and prevent the re-introduction of malaria, surveillance systems need to be well planned and managed to ensure timely case detection and prompt response targeted to the mobile and migrate population at elimination stage.

[An Imported Case of Vivax Malaria with Co-occurrence of Different Stages of Plasmodium vivax in Erythrocyte].

The blood sample from a case of vivax malaria in Tengchong City, Yunnan Province, was tested with CareStartTM malaria rapid diagnostic test, Giemsa staining and nested PCR in 2012. The case was determined to be infected with Plasmodium vivax, P. malariae or P. ovale other than P. falciparum by CareStartTM malaria rapid diagnostic test. Microscopic results revealed multinuclear P. vivax ring form and multi-infections with P. vivax in blood slides, with occurrence of two or more nuclei in one ring form accounting for 14.68%（188/1 280）, and parasitism of two or more P. vivax rings in one erythrocyte accounting for 22.50%（288/1 280）. In addition, co-occurrence of ring form and trophozoite, and ring form and gametophyte was found in erythrocytes. Nested PCR revealed P. vivax-specific amplification products. Combining the results with epidemiological information and clinical symptoms, this case was finally diagnosed as imported vivax malaria, and the erythrocytes of the case harbored different stages of P. vivax parasites.

[Evaluation of Measures and Achievements of Malaria Control in Tengchong City, Yunnan Province during 2010-2015].

Objective: To evaluate the measures and achievements of malaria control in Tengchong City during 2010-2015. Methods: The malaria control information on epidemiology, foci disposal, blood detection of febrile patients, and medical treatment during 2010-2015 in Tengchong City was collected and analyzed using Microsoft Excel 2010. Results: In 2010-2015, 1 654 malaria cases were reported in Tengchong City, including 18 indigenous cases, 22 domestically mobile cases, and 1 614 imported cases from abroad, of whom 1 584 cases （98.1%） were imported from Myanmar. Most of the cases were vivax malaria（76.2%, 1 261/1 654）. No indigenous malaria cases were reported from 2013 to 2015. Blood test was conducted for 80 655 febrile patients, with a positive detection rate of 2.1%（1 654/80 655）. The positive detection rate was highest in 2010 （2.8%, 700/24 861）, lowest in 2011（1.4%, 341/23 623）, and decreased from 2012 to 2015. In addition, 1 654 cases were directly reported through online system. The 24-h case report rate during 2013-2015 was 100%. A total of 1 191 cases were investigated. The 3-day case investigation rate during 2013-2015 was 100%. A total of 1 351 endemic foci were investigated. The 7-day foci disposal rate during 2014-2015 was 100%. Conclusion: No indigenous transmission has been reported for three years in Tengchong City. However, the imported malaria remains an important problem.

Transmission Risk from Imported Plasmodium vivax Malaria in the China-Myanmar Border Region.

Malaria importation and local vector susceptibility to imported Plasmodium vivax infection are a continuing risk along the China-Myanmar border. Malaria transmission has been prevented in 3 border villages in Tengchong County, Yunnan Province, China, by use of active fever surveillance, integrated vector control measures, and intensified surveillance and response.

[Malaria surveillance in Tengchong County of Yunnan Province in 2013].

OBJECTIVE: To analyze the surveillance data of malaria in Tengchong County of Yunnan Province in 2013, so as to provide the evidence for carrying out the malaria elimination in the future. METHODS: The data of epidemic situation and surveillance of malaria in Tengchong County in 2013 were collected and analyzed for the prevalence state as well as and the monitoring indicators including the blood examination of fever patients of unknown origin, initiative detection of cases, under-reporting survey, sentinel surveillance and species and density investigation of Anopheles mosquitoes. RESULTS: Totally 138 malaria cases were reported in Tengchong County in 2013, among which 118 cases were infected with Plasmodium vivax and 20 cases with P. falciparum, and all the reported cases were imported. The completion rates of blood examinations, case reports and case investigations all reached 100%. A total of 57 cases were involved in initiative detection, but no positive cases were found. The twice under-reporting of malaria case surveys were conducted and 1 case had been under-reported. The sentinel surveillance was carried out both in the domestic and overseas sites at the same time to detect the suspected malaria cases, 172 cases were screened totally, and 15 ones were detected as positives. Totally 528 returnees were screened, and there were no positive case found. A. sinensis and A. liangshanensis were the dominant species, followed by A. minimus, A. maculatus and others. CONCLUSIONS: The malaria epidemic situation is stable in Tengchong County in 2013, and all the work in surveillance has been conducted successfully, but there still exist some difficulties in the process of malaria elimination. The local health departments should further strengthen the surveillance on imported cases and the management on migrant population as well as the capability building for health workers in malaria control in the future.