Application of an innovative grid-based surveillance strategy to ensure elimination and prevent reintroduction of malaria in high-risk border communities in China.

Grid management is a grassroots governance strategy widely implemented in China since 2004 to improve the government's efficiency to actively find and solve problems among populated regions. A grid-based strategy surveillancing high-risk groups, including mobile and migrant populations (MMPs), in the China-Myanmar border region has played an indispensable role in promoting and consolidating the malaria elimination efforts by tracking and timely identification of potential importation or re-establishment of malaria among MMPs. A sequential mixed methods was implementated to explore the operational mechanism and best practices of the grid-based strategy including through the focus group discussions (FGDs), comparison of before and after the implementation of a grid-based strategy in the field sites, and data collection from the local health system.This paper distills the implementation mechanism and highlights the role of the grid-based strategy in the elimination and prevention of re-establishment of malaria transmission.

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.

[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.

[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.

A molecular survey of febrile cases in malaria-endemic areas along China-Myanmar border in Yunnan province, People's Republic of China.

BACKGROUND: Imported malaria is a major threat to neighboring malaria-eliminating countries such as P.R. China and is difficult to monitor. A molecular survey of febrile patients with a history of traveling abroad along the Myanmar-China endemic border areas from January 2008 to August 2012 was carried out. The rates of infection with species of Plasmodium and compliance of microscopy diagnosis with nested PCR (Polymerase Chain Reaction) results were calculated. RESULTS: Plasmodium genus-specific nested PCR confirmed that 384 cases were positive. Further species-specific nested PCR showed that the rate of Plasmodium vivax infection was 55% (213/384); that of Plasmodium falciparum was 21% (81/384) and 17% (67/384) of cases were co-infection cases of P. vivax and P. falciparum; the remaining 6% (23/384) of cases were caused by other species, such as Plasmodium ovale, P. malaria, P. knowlesi or mixed infections of Plasmodium. In total there was 13% (50/384) false microscopy diagnosis including 6% (22/384) error in species diagnosis and 7% (28/384) undiagnosed cases in co-infection or low parasitemia malaria cases. CONCLUSIONS: This study indicates that there are considerable numbers of malaria cases in the China-Myanmar endemic border areas that remain undiagnosed or misdiagnosed by microscopy, especially in low-level and/or complex co-infection cases. It is urgent to develop accurate rapid diagnostic tests and apply PCR confirmation for efficient surveillance.

Emergence of human babesiosis along the border of China with Myanmar: detection by PCR and confirmation by sequencing.

Babesiosis is a tick-borne, zoonotic disease caused by Babesia spp. Two cases of babesiosis were detected by nested polymerase chain reaction (PCR) in Yunnan province, China, and further confirmed by molecular assay. The blood smears showed intraerythrocytic ring form and tetrads typical of small B. microti. In both cases, the rapid diagnostic test (RDT) ruled out the possibility of co-infections with malaria. Neither case was initially diagnosed because of the low Babesia parasitemia. These two cases of babesiosis in areas along the Myanmar-China border pose the question of the emergence of this under recognized infection in countries or areas where malaria is endemic.

Co-infections with Babesia microti and Plasmodium parasites along the China-Myanmar border.

BACKGROUND: Babesiosis is an emerging health risk in several parts of the world. However, little is known about the prevalence of Babesia in malaria-endemic countries. The area along the China-Myanmar border in Yunnan is a main endemic area of malaria in P.R. China, however, human infection with Babesia microti (B. microti) is not recognized in this region, and its profile of co-infection is not yet clear. METHODS: To understand its profile of co-infections with B. microti, our investigation was undertaken in the malaria-endemic area along the China-Myanmar border in Yunnan between April 2012 and June 2013. Four parasite species, including B. microti, Plasmodium falciparum (P. falciparum), P. vivax, and P. malariae, were identified among 449 suspected febrile persons detected by nested polymerase chain reaction (PCR) assay based on small subunit ribosomal ribonucleic acid (RNA) genes of B. microti and Plasmodium spp. RESULTS: Of all the collected samples from febrile patients, mono-infection with B. microti, P. vivax, P. falciparum, and P. malariae accounted for 1.8% (8/449), 9.8% (44/449), 2.9% (13/449), and 0.2% (1/449), respectively. The rate of mixed infections of B. microti with P. falciparum or P. vivax are both 0.2% (1/449), and mixed infections of P. falciparum and P. vivax accounted for 1.1% (5/449). CONCLUSIONS: This report supports the hypothesis that babesiosis caused by B. microti is emerging along the China-Myanmar border in the Yunnan province, P.R. China, but it was ignored because of low parasitemia or mixed infection with Plasmodium spp. More sensitive and specific diagnosis methods are needed to find the rapid response mechanism of emergency for babesiosis and malaria co-prevalence areas.

[Survey on human rabies cases and its viral molecular biological features in Baoshan city, Yunnan province].

OBJECTIVE: To understand the epidemiological features of two rabies cases in Baoshan city year 2006 and 2007 and to analyze its source of infection. METHODS: Questionnaires were used to do the epidemiological survey on each of the rabies cases. Brain tissue samples of rabies patients were collect to detect the rabies virus by direct immunofluorescence assay (DFA) and RT-PCR assay. Homology and phylogenetic tree were analyzed, based on the whole nucleotide and deduced amino acid sequence of P, M and N gene of rabies virus followed by molecular epidemiological analysis. RESULTS: In July 2006, one human rabies case was identified in Longyang district, and another one in Tengchong county in Baoshan city in 2007. The degrees of exposure of these two patients was all at degree III. Two brain tissue samples among the dead patients (No. CYN0601H and CYN0701H) were confirmed positive by both DFA and RT-PCR assay. The homology analysis of P, M and N gene sequences among CYN0601H, CYN0701H and other rabies strains isolated from other provinces and other counties, showed that the samples in Baoshan city shared the highest homology with the strains in Thailand. Phylogenetic analysis indicated that the two samples were very close and all belonged to genetype 1 Lyssavirus, with the closest relationship between samples in Baoshan city and strains in Thailand. CONCLUSION: It was confirmed on the virus molecular level that the two patients in Baoshan city were both suffered from rabies. The prevalent strains in Baoshan city was probably imported from foreign country, suggesting that prevention and control measures on rabies virus in the boarder areas of Yunnan should be strengthened.