Seroprevalence of Bartonella henselae and Identification of Risk Factors in China.

Serum samples were tested for Bartonella henselae IgG antibodies using indirect immunofluorescence assays. We then analyzed associated risk factors. Serum samples were considered positive when reactive at a dilution of more than 1:320. Differences between groups and risk factors associated with Bartonella exposure were statistically analyzed using Chi-square tests and the generalized linear model. 122 of 1,260 samples (9.68%) were positive for B. henselae infection. The infection rate ranged from 0% to 30.43% and differed significantly among age groups ( P < 0.01); infection rate in the 50-59 years group was significantly higher than that in other age groups. The seroprevalence of Bartonella varied significantly among sites within the four provinces, and the infection rate of field workers was significantly higher than that of urban workers.

Establishment of an innovative and sustainable PCR technique for 1534 locus mutation of the knockdown resistance (kdr) gene in the dengue vector Aedes albopictus.

BACKGROUND: Mutation of the voltage-gated sodium channel (VGSC) gene, or knockdown resistance (kdr) gene, is an important resistance mechanism against DDT and pyrethroids for dengue vector Aedes albopictus. A phenylalanine to serine (F1534S), leucine (F1534L) and cysteine (F1534C) substitution were detected in many Ae. albopictus populations around the world, and the mutant allele frequencies have been increasing in recent years. Therefore, it is essential to establish a simple, time-saving and cost-effective procedure to monitor the alleles in large-scale studies. METHODS: Based on the mutation genotypes of the 1534 locus in the kdr gene, F/F, F/S, F/C, F/L, S/S, C/C, L/L and S/C, we designed specific forward and reverse primers and optimized the reaction conditions for establishing of the allele-specific PCR(AS-PCR) detection technique. DNA sequencing in this study was taken as the gold standard, and used to determine the accuracy of AS-PCR. RESULTS: The designed AS-PCR technique showed high specificity for distinguishing the mutations at the 1534 locus, as the accuracy for F/F, F/S, F/C, F/L, S/S, C/C and S/C were 100%, 95.35%, 100%, 100%, 100%, 100% and 100%, respectively. CONCLUSIONS: The designed AS-PCR technique effectively distinguished individual genotypes for the mutations at the 1534 locus in the kdr gene, which could facilitate the knockdown resistance surveillance in Ae. albopictus in large-scale studies.

Development and comparison of forecast models of hand-foot-mouth disease with meteorological factors.

Hand-foot-mouth disease (HFMD) is an acute intestinal virus infectious disease which is one of major public health problems in mainland China. Previous studies indicated that HFMD was significantly influenced by climatic factors, but the associated factors were different in different areas and few study on HFMD forecast models was conducted. Here, we analyzed epidemiological characteristics of HFMD in Yiwu City, Zhejiang Province and constructed three forecast models. Overall, a total of 32554 HFMD cases were reported and 12 cases deceased in Yiwu City, Zhejiang Province. The incidence of HFMD peaked every other year and the curve of HFMD incidence had an approximately W-shape. The majority of HFMD cases were children and 95.76% cases aged ≤5 years old from 2008 to 2016. Furthermore, we constructed and compared three forecast models using autoregressive integrated moving average (ARIMA) model, negative binomial regression model (NBM), and quasi-Poisson generalized additive model (GAM). All the three models had high agreements between predicted values and observed values, while GAM fitted best. The exposure-response curve of monthly mean temperature and HFMD was approximately V-shaped. Our study explored epidemiological characteristics of HFMD in Yiwu City and provided accurate methods for early warning which would be great importance for the control and prevention of HFMD.

Development of fluorogenic probe-based and high-resolution melting-based polymerase chain reaction assays for the detection and differentiation of Bartonella quintana and Bartonella henselae.

Bartonella henselae and Bartonella quintana are the major etiological agents of infective endocarditis, which pose a serious threat to human health. To simultaneously detect and differentiate B. henselae and B. quintana, a reliable and fast method to simultaneously detect and differentiate B. henselae and B. quintana is required. In this study, we developed and validated two rapid, highly sensitive and specific, duplex, real-time polymerase chain reaction (PCR) assays-one based on high-resolution melting (HRM) analysis, and the other on TaqMan probes-to simultaneously detect and differentiate B. henselae and B. quintana. The sensitivity of developed assays were found 100 times more sensitive than that of conventional PCR. The specificity of the assays were validated by the absence of any cross reaction with the other Bartonella species, non-Bartonella bacteria and other animals. The results indicate that the duplex HRM-based and TaqMan probe-based assays have high specificity and sensitivity, and good reproducibility for simultaneous the detection of B. henselae and B. quintana. They are cost-effective, sensitive and reliable methods; and are thus suitable for clinical diagnosis, epidemiological surveys, and disease surveillance.

Analysis of a Community-based Intervention to Reduce Heat-related Illness during Heat Waves in Licheng, China: a Quasi-experimental Study.

OBJECTIVE: To reduce health-related threats of heat waves, interventions have been implemented in many parts of the world. However, there is a lack of higher-level evidence concerning the intervention efficacy. This study aimed to determine the efficacy of an intervention to reduce the number of heat-related illnesses. METHODS: A quasi-experimental design was employed by two cross-sectional surveys in the year 2014 and 2015, including 2,240 participants and 2,356 participants, respectively. Each survey was designed to include one control group and one intervention group, which conducted in Licheng, China. A representative sample was selected using a multistage sampling method. Data, collected from questionnaires about heat waves in 2014 and 2015, were analyzed using a difference-in-difference analysis and cost effectiveness analysis. Outcomes included changes in the prevalence of heat-related illnesses and cost-effectiveness variables. RESULTS: Relative to the control participants, the prevalence of heat-related illness in the intervention participants decreased to a greater extent in rural areas than in urban areas (OR=0.495 vs. OR=1.281). Moreover, the cost-effectiveness ratio in the intervention group was less than that in the control group (US$15.06 vs. US$15.69 per participant). Furthermore, to avoid one additional patient, the incremental cost-effectiveness ratio showed that an additional US$14.47 would be needed for the intervention compared to when no intervention was applied. CONCLUSION: The intervention program may be considered a worthwhile investment for rural areas that are more likely to experience heat waves. Meanwhile, corresponding improving measures should be presented towards urban areas. Future research should examine whether the intervention strategies could be spread out in other domestic or international regions where heat waves are usually experienced.

Bartonella Species Detected in the Plateau Pikas (Ochotona curzoiae) from Qinghai Plateau in China.

Bartonella species can infect a variety of mammalian hosts and cause a broad spectrum of diseases in humans, but there have been no reports of Bartonella infection in Ochotonidae. This is the first study to detect Bartonella in plateau pikas in the Qinghai plateau, providing baseline data for the risk assessment of human Bartonella infection in this area. We obtained 15 Bartonella strains from 79 pikas in Binggou and Maixiu areas of Qinghai with a positive rate of 18.99%. Based on the phylogenetic analysis of the Bartonella citrate synthase (gltA) gene sequences, most strains were closely related to B. taylorii (3/15) and B. grahamii (12/15). The latter is a pathogenic strain in humans. Our results suggest that a corresponding prevention and control strategy should be taken into consideration in the Qinghai province.

High prevalence and genetic heterogeneity of rodent-borne Bartonella species on Heixiazi Island, China.

We performed genetic analysis of Bartonella isolates from rodent populations from Heixiazi Island in northeast China. Animals were captured at four sites representing grassland and brushwood habitats in 2011 and examined for the prevalence and genetic diversity of Bartonella species, their relationship to their hosts, and geographic distribution. A high prevalence (57.7%) and a high diversity (14 unique genotypes which belonged to 8 clades) of Bartonella spp. were detected from 71 rodents comprising 5 species and 4 genera from 3 rodent families. Forty-one Bartonella isolates were recovered and identified, including B. taylorii, B. japonica, B. coopersplainsensis, B. grahamii, B. washoensis subsp. cynomysii, B. doshiae, and two novel Bartonella species, by sequencing of four genes (gltA, the 16S rRNA gene, ftsZ, and rpoB). The isolates of B. taylorii and B. grahamii were the most prevalent and exhibited genetic difference from isolates identified elsewhere. Several isolates clustered with strains from Japan and far-eastern Russia; strains isolated from the same host typically were found within the same cluster. Species descriptions are provided for Bartonella heixiaziensis sp. nov. and B. fuyuanensis sp. nov.

Optimization of Bartonella henselae multilocus sequence typing scheme using single-nucleotide polymorphism analysis of SOLiD sequence data.

BACKGROUND: Multi-locus sequence typing (MLST) is widely used to explore the population structure of numerous bacterial pathogens. However, for genotypically-restricted pathogens, the sensitivity of MLST is limited by a paucity of variation within selected loci. For Bartonella henselae (B. henselae), although the MLST scheme currently used has been proven useful in defining the overall population structure of the species, its reliability for the accurate delineation of closely-related sequence types, between which allelic variation is usually limited to, at most, one or two nucleotide polymorphisms. Exploitation of high-throughput sequencing data allows a more informed selection of MLST loci and thus, potentially, a means of enhancing the sensitivity of the schemes they comprise. METHODS: We carried out SOLiD resequencing on 12 representative B. henselae isolates and explored these data using single nucleotide polymorphism (SNP) analysis. We determined the number and distribution of SNPs in the genes targeted by the established MLST scheme and modified the position of loci within these genes to capture as much genetic variation as possible. RESULTS: Using genome-wide SNP data, we found the distribution of SNPs within each open reading frame (ORF) of MLST loci, which were not represented by the established B. henselae MLST scheme. We then modified the position of loci in the MLST scheme to better reflect the polymorphism in the ORF as a whole. The use of amended loci in this scheme allowed previously indistinguishable ST1 strains to be differentiated. However, the diversity of B. henselae was still rare in China. CONCLUSIONS: Our study demonstrates the use of SNP analysis to facilitate the selection of MLST loci to augment the currently-described scheme for B. henselae. And the diversity among B. henselae strains in China is markedly less than that observed in B. henselae populations elsewhere in the world.

Phylogenetic analysis of Bartonella detected in rodent fleas in Yunnan, China.

Previous studies have demonstrated a diversity of Bartonella spp. in rodent populations in Yunnan Province, China. Although Bartonella spp. have been isolated from cat fleas and cattle ticks collected from their animal hosts, little is known about Bartonella carried by rodent fleas. In this study, Bartonella DNA was detected by polymerase chain reaction (PCR) in two of five species of rodent fleas. These included Xenopsylla cheopis and Ctenophthalmus lushuiensis, which were collected from Rattus tanezumi flavipectus and from the nests of voles, respectively, during 1997 from two sites in western Yunnan Province, China. Sequence analysis of the Bartonella citrate synthase gene (gltA) amplicons obtained from six of 65 grouped flea samples showed that Bartonella genetic variants were clustered in four groups. One from Xenopsylla cheopis was identical to Bartonella tribocorum, whereas the other three genotypes from Ctenophthalmus lushuiensis were related to the vole-associated Bartonella isolates and cat-associated Bartonella clarridgeiae. This is the first detection of this Bartonella variant from fleas in China. Therefore, further investigations are needed to clarify the distribution of Bartonella in rodents and their ectoparasites in China to define the role of these arthropods in the transmission routes of Bartonella.

[Study on Bartonella vinsonii berkhoffii isolated from blood of native dogs in China].

OBJECTIVE: To isolate and identify Bartonella strains from native dogs in Shandong province in China. METHODS: EDTA-anticoagulated blood samples were collected from 71 native dogs in Yanggu county of Shandong province in March 2005. All isolates were grown on brain heart infusion agar plates containing 5% defibrinated rabbit blood. The agar plates were incubated at 37 degrees C in a humidified with 5% CO2 environment for 4 weeks or longer. All Bartonella-like isolates were examined by routine Gram and Giménez staining and then followed by polymerase chain reaction (PCR) and PCR-RFLP analysis for identification and differentiation of the isolates. Sequencing 16S rRNA, citrate synthase (gltA) gene and 16S-23S rRNA ITS were carried out and sequential similarities were calculated using the DNASTAR5 software package. The phylogenetic tree was inferred from each bootstrap sample, using the neighbor-joining methods as executed in the MEGA 3.1 software. The translation from DNA to protein were determined by DNASIS 2.5. RESULTS: The two Bartonella-like organisms (strains Q52SHD and Q64SHD) were isolated from the blood of 71 dogs. Light microscopic examination of the Gram and Giménez-stained micro-organisms showed small, short and slightly curved pleomorphic gram-negative bacilli. Amplified products of the three pairs of Bartonella genus-specific primers carried the same size as the predicted of those Bartonella species. Data from PCR-RFLP analysis showed that the two strains that having the same profiles were all different from the B. henselae type strain-16S rRNA, gltA and 16S-23S rRNA ITS sequences from the two isolates were 100.0%, 99.7% and 97.2% homologous to B. vinsonii berkhoffii. CONCLUSIONS: Based on these findings, the two isolates Q52SHD and Q64SHD were demonstrated as B. vinsonii berkhoffii. To our knowledge, this was the first report on the presence of Bartonella infection in native dogs from China, which constituted a large reservoir of Bartonella species in this country.