Changes in peripheral blood cytokines in patients with severe fever with thrombocytopenia syndrome.

Severe fever with thrombocytopenia syndrome (SFTS) is recognized as an emerging infectious disease. This study aimed to investigate the pathogenic mechanism of SFTS. A total of 100 subjects were randomly included in the study. Cytokine levels were detected by enzyme-linked immunosorbent assay and the viral load was detected by micro drop digital PCR. The results showed that levels of interleukin-6 (IL-6), IL-8, IL-10, IFN-inducible protein-10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), transforming growth factor-ß1 (TGF-ß1), and regulated upon activation normal T cell expressed and secreted factor (RANTES) differed significantly among the SFTS patient group, healthy people group, and asymptomatic infection group (p < .05). Compared to the healthy people group, the patient group had increased cytokine levels (IL-6, IL-10, IP-10, MCP-1, and IFN-γ) but reduced levels of IL-8, TGF-ß1, and RANTES (p < .0167). IL-6, IL-8, IL-10, IP-10, MCP-1, MIP-1α, TGF-ß1, and the RANTES levels had different trends after the onset of the disease. IL-6, IL-10, IP-10, and MCP-1 levels in severe patients were higher than those in mild patients (p < .05). There was a positive correlation between viral load and IL-6 and IP-10 but a negative correlation between viral load and RANTES. SFTSV could cause a cytokine change: the cytokine levels of patients had different degrees of fluctuation after the onset of the disease. The levels of IL-6 and IL-8 in the asymptomatic infection group were found between the SFTS patients group and the healthy people group. The levels of IL-6, IL-10, IP-10, and MCP-1 in the serum could reflect the severity of the disease, and the levels of IL-6, IP-10, and RANTES were correlated with the viral load.

Severe fever with thrombocytopenia syndrome: a systematic review and meta-analysis of epidemiology, clinical signs, routine laboratory diagnosis, risk factors, and outcomes.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with the high case-fatality rate, and lack of vaccines. We aimed to systematically analysed the epidemiological characteristics, clinical signs, routine laboratory diagnosis, risk factors, and outcomes. METHODS: Documents on SFTS were collected by searching the Chinese National Knowledge Infrastructure, Wan Fang Data, PubMed, Embase, and Web of Science databases from 2011 to 2018. Meta-analysis was performed by using Review Manager and Stata software. RESULTS: Twenty-five articles involving 4143 cases were included. Diarrhea (odds ratio (OR) =1.60, 95% confidence interval (CI): 1.06 to 2.42, P = 0.02), and vomiting (OR = 1.56, 95% CI: 1.01 to 2.39, P = 0.04) on admission were associated with the fatal outcomes of SFTS. Compared to patients with mild symptoms, patients with severe symptoms had significantly elevated levels of lactic acid dehydrogenase (standard mean difference (SMD) =1.27, 95% CI: 0.59 to 1.94), alanine aminotransferase (SMD = 0.55, 95% CI: 0.24 to 0.85), aspirate aminotransferase (SMD = 1.01, 95% CI: 0.69 to 1.32), and creatine kinase (SMD = 1.04, 95% CI: 0.74 to 1.33) but had reduced platelet counts (SMD = -0.87, 95% CI: - 1.16 to - 0.58) and albumin levels (SMD = -1.00, 95% CI: - 1.32 to - 0.68). The risk factors for poor prognosis included age (mean difference (MD) =6.88, 95% CI: 5.41 to 8.35) and farming (OR = 2.01, 95% CI: 1.06 to 3.80). For the risk factors of contracting SFTS, the incidence of SFTS related to tick bites was 24% [95% CI: 0.18 to 0.31]. The pooled case-fatality rate of SFTS patients was 18% [95% CI: 0.16 to 0.21]. CONCLUSIONS: China is the country with the highest incidence of SFTS. May to July was the peak of the epidemic, and farmers were a high-risk group. The risk factor for SFTS included age (poor prognosis) and tick bites (contracting SFTS). Patients with severe diarrhea and vomiting symptoms on admission should be noted. Clinicians could use routine laboratory parameters and clinical symptoms as references for clinically suspected cases, classification of SFTS, and timely treatment, especially in basic hospitals.

Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp.

Without an approved vaccine or treatments, Ebola outbreak management has been limited to palliative care and barrier methods to prevent transmission. These approaches, however, have yet to end the 2014 outbreak of Ebola after its prolonged presence in West Africa. Here we show that a combination of monoclonal antibodies (ZMapp), optimized from two previous antibody cocktails, is able to rescue 100% of rhesus macaques when treatment is initiated up to 5 days post-challenge. High fever, viraemia and abnormalities in blood count and blood chemistry were evident in many animals before ZMapp intervention. Advanced disease, as indicated by elevated liver enzymes, mucosal haemorrhages and generalized petechia could be reversed, leading to full recovery. ELISA and neutralizing antibody assays indicate that ZMapp is cross-reactive with the Guinean variant of Ebola. ZMapp exceeds the efficacy of any other therapeutics described so far, and results warrant further development of this cocktail for clinical use.

Molecular investigation of the Pfmdr1 gene of Plasmodium falciparum isolates in Henan Province imported from Africa.

Efficacious antimalarial drugs are important for malaria control and elimination, and continuous monitoring of their efficacy is essential. The prevalence and distribution of Pfmdr1 were evaluated in African migrant workers in Henan Province. Among 632 isolates, 13 haplotypes were identified, NYSND (39.87%, 252/632), YYSND (2.85%, 18/632), NFSND (31.01%, 196/632), NYSNY (0.47%, 3/632), YFSND (13.77%, 87/632), NFSNY (0.32%, 2/632), YYSNY (2.06%, 13/632), YFSNY (0.16%, 1/632), N/Y YSND (1.90%, 12/632), N Y/F SND (6.17%, 39/632), N/Y Y/F SND (0.47%, 3/632), YYSN D/Y (0.16%, 1/632) and N/Y FSND (0.79%, 5/632). The highest frequency of NYSND was observed in individuals from North Africa (63.64%, 7/11), followed by South Africa (61.33%, 111/181), Central Africa (33.33%, 56/168), West Africa (28.94%, 68/235) and East Africa (27.03%, 10/37) (χ2 = 54.605, P < 0.05). The highest frequency of NFSND was observed in East Africa (48.65%, 18/37), followed by West Africa (39.14%, 92/235), Central Africa (26.79%, 45/168), South Africa (22.65%, 41/181) and North Africa (9.09%, 1/11) (χ2 = 22.368 P < 0.05). The mutant prevalence of codons 86 and 184 decreased. These data may provide complementary information on antimalarial resistance that may be utilized in the development of a treatment regimen for Henan Province.

Analysis of polymorphisms in the circumsporozoite protein gene of Plasmodium vivax isolates from Henan Province, China.

BACKGROUND: Plasmodium vivax malaria has historically been a major source of disease in Henan, China. In the 1970s, the morbidity of malaria was highest in the country. With support from the government and the efforts of healthcare personnel, the reported malaria cases have declined dramatically and a national elimination programme was launched in 2010. To achieve the goal, it is essential to study the diversity of autochthonous malaria and transmission of Plasmodium parasites, which will provide baseline data for disease control and management. METHODS: Thirty-two P. vivax isolates from Henan province were collected from 2008 to 2011, and circumsporozoite protein (csp) genes were analysed to estimate the genetic diversity of this parasite. RESULTS: The assessment of csp sequences indicated that all the isolates were the VK210 type, however, none of them was identical to the VK210 strain. The sequences displayed variations in the central region, and eight sub-types were observed. Among the sub-types, HN7 was the most prevalent (37.5%), followed by HN3 (34.4%). A total of 653 repeat units were discovered in 32 Henan isolates. Nucleotide sequences were grouped in 13 unique repeat nucleotide sequence allotypes that coded for 7 different repeated amino acid allotypes. B (GNGAGGQAA) and D (GDRAAGQPA) were more frequent based on the results; they represented 53.9% (352/653) of the total. In comparison to the basic repeat units of VK210, more than 75% of the central repeat units had at least one non-synonymous nucleotide change. CONCLUSIONS: Recent P. vivax populations in Henan province showed some degree of genetic diversity in csp, with 8 sub-types among 32 samples. Meantime, the results also suggested its relative conserved parasite populations. This could provide interesting baseline data that allow identifying whether potential new cases differ from the parasites already circulating in the area.

Polymorphisms analysis of the Plasmodium ovale tryptophan-rich antigen gene (potra) from imported malaria cases in Henan Province.

BACKGROUND: Plasmodium ovale has two different subspecies: P. ovale curtisi and P. ovale wallikeri, which may be distinguished by the gene potra encoding P. ovale tryptophan-rich antigen. The sequence and size of potra gene was variable between the two P. ovale spp., and more fragment sizes were found compared to previous studies. Further information about the diversity of potra genes in these two P. ovale spp. will be needed. METHODS: A total of 110 dried blood samples were collected from the clinical patients infected with P. ovale, who all returned from Africa in Henan Province in 2011-2016. The fragments of potra were amplified by nested PCR. The sizes and species of potra gene were analysed after sequencing, and the difference between the isolates were analysed with the alignment of the amino acid sequences. The phylogenetic tree was constructed by neighbour-joining to determine the genetic relationship among all the isolates. The distribution of the isolates was analysed based on the origin country. RESULTS: Totally 67 samples infected with P. o. wallikeri, which included 8 genotypes of potra, while 43 samples infected with P. o. curtisi including 3 genotypes of potra. Combination with the previous studies, P. o. wallikeri had six sizes, 227, 245, 263, 281, 299 and 335 bp, and P. o. curtisi had four sizes, 299, 317, 335 and 353 bp, the fragment sizes of 299 and 335 bp were the overlaps between the two species. Six amino acid as one unit was firstly used to analyse the amino acid sequence of potra. Amino acid sequence alignment revealed that potra of P. o. wallikeri differed in two amino acid units, MANPIN and AITPIN, while potra of P. o. curtisi differed in amino acid units TINPIN and TITPIS. Combination with the previous studies, there were ten subtypes of potra exiting for P. o. wallikeri and four subtypes for P. o. curtisi. The phylogenetic tree showed that 11 isolates were divided into two clusters, P. o. wallikeri which was then divided into five sub-clusters, and P. o. curtisi which also formed two sub-clusters with their respective reference sequences. The genetic relationship of the P. ovale spp. mainly based on the number of the dominant amino acid repeats, the number of MANPIN, AITPIN, TINPIN or TITPIS. The genotype of the 245 bp size for P. o. wallikeri and that of the 299 and 317 bp size for P. o. curtisi were commonly exiting in Africa. CONCLUSION: This study further proved that more fragment sizes were found, P. o. wallikeri had six sizes, P. o. curtisi had four sizes. There were ten subtypes of potra exiting for P. o. wallikeri and four subtypes for P. o. curtisi. The genetic polymorphisms of potra provided complementary information for the gene tracing of P. ovale spp. in the malaria elimination era.

Polymorphisms of Plasmodium falciparum k13-propeller gene among migrant workers returning to Henan Province, China from Africa.

BACKGROUND: Henan Province has been in the malaria elimination stage, with all reports of the disease being imported since 2012 and over 90% coming from Africa. Surveillance and population studies are essential for the early detection and subsequent prevention of the spread of drug resistance. The K13-propeller gene was recently identified as a proposed molecular marker of artemisinin (ART) resistance. In this study, we detected mutations of the K13-propeller gene in samples taken from imported malaria cases in Henan Province from 2012 to 2015. METHODS: There were 483 samples that were obtained from Plasmodium falciparum-infected malaria migrant workers who returned to Henan Province from Africa between 2012 and 2015. The single nucleotide polymorphisms in the K13-propeller gene were assessed by nested PCR with DNA sequencing. Frequency and geographic difference of K13-propeller gene mutant types were analyzed. RESULTS: Of 483 patients, 476 were cured and 7 died. There were no K13-propeller mutations in the blood samples from the 7 patients who died, but there were 23 different genotypes of the K13-propeller that were observed in 24 (4.97%) of the samples. C580Y, which was the predominant one in the resistance of ART, was not detected in the samples, but R539T and P574L which have also been associated with ART resistance, were observed in two samples from Angola and Equatorial Guinea. No mutations were detected in 11 samples from North Africa. The frequency of the K13-propeller was 6.50% (8/123) in Central Africa, followed by East Africa (1/19, 5.26%), West Africa (9/198, 4.55%) and South Africa (6/132, 4.55%). There was no significant difference among these four areas (P = 0.795). CONCLUSION: R539T and P574L were found in migrant workers who traveled from Africa to Henan Province, although the frequency of the K13-propeller mutants was low. These data may enrich the molecular surveillance of antimalarial resistance and will be helpful for developing and updating the antimalarial policy in Henan Province.

[Polymorphisms of tryptophan-rich antigen gene of Plasmodium ovale subspecies from imported malaria cases in Henan Province].

Objective: To analyze the genetic sequence of tryptophan-rich antigen (PoTRA) gene of Plasmodium ovale subspecies from imported malaria cases in Henan Province in 2015. Methods: Blood samples were collected from 22 imported ovale malaria cases in Henan Province in 2015. After DNA extraction, PoTRA was amplified by nested PCR, and was inserted into the pMD18-T vector. The plasmid was extracted and sequenced, and the results were blasted in GenBank to determine the subspecies of P. ovale. The sizes and species of the PoTRA gene were analyzed. The amino-acid sequence of PoTRA was also aligned to analyze the difference in amino acid sequence. The phylogenetic tree was constructed to analyze the genetic relationship among the samples by neighbor-joining. Results: Of the 22 imported cases, eight (36.4%) were infected with P. ovale wallikeri, which had two sizes, the predominant 245 and 299 bp. The other 14 cases (63.6%) were infected with P. ovale curtisi, which had three sizes, the predominant 299, 317 and 335 bp. Amino-acid sequence alignment revealed that the two types of P. ovale wallikeri differed in two amino-acid units, MANPINMANPIN and AITPIN, while the three types of P. ovale curtisi differed in amino-acid units TITPIS and TINPIN. The phylogenetic tree showed that the 22 samples belonged to two subpopulations of P. ovale curtisi and P. ovale wallikeri, wherein the P. ovale curtisi was further divided into two sub-branches, and samples with sizes of 317 and 335 bp were in the same sub-branch with a closer genetic relationship. Conclusion: Two subspecies, P. ovale curtisi and P. ovale wallikeri, are identified from the imported ovale malaria cases in Henan Province in 2015. The P. ovale curtisi has three genetic types and P. ovale wallikeri has two genetic types of PoTRA gene, revealing genetic polymorphisms of PoTRA.

Phenotypic H-Antigen Typing by Mass Spectrometry Combined with Genetic Typing of H Antigens, O Antigens, and Toxins by Whole-Genome Sequencing Enhances Identification of Escherichia coli Isolates.

Mass spectrometry-based phenotypic H-antigen typing (MS-H) combined with whole-genome-sequencing-based genetic identification of H antigens, O antigens, and toxins (WGS-HOT) was used to type 60 clinical Escherichia coli isolates, 43 of which were previously identified as nonmotile, H type undetermined, or O rough by serotyping or having shown discordant MS-H and serotyping results. Whole-genome sequencing confirmed that MS-H was able to provide more accurate data regarding H antigen expression than serotyping. Further, enhanced and more confident O antigen identification resulted from gene cluster based typing in combination with conventional typing based on the gene pair comprising wzx and wzy and that comprising wzm and wzt The O antigen was identified in 94.6% of the isolates when the two genetic O typing approaches (gene pair and gene cluster) were used in conjunction, in comparison to 78.6% when the gene pair database was used alone. In addition, 98.2% of the isolates showed the existence of genes for various toxins and/or virulence factors, among which verotoxins (Shiga toxin 1 and/or Shiga toxin 2) were 100% concordant with conventional PCR based testing results. With more applications of mass spectrometry and whole-genome sequencing in clinical microbiology laboratories, this combined phenotypic and genetic typing platform (MS-H plus WGS-HOT) should be ideal for pathogenic E. coli typing.

Mass Spectrometry-Based Escherichia coli H Antigen/Flagella Typing: Validation and Comparison with Traditional Serotyping.

BACKGROUND: Escherichia coli H antigen typing with antisera, a useful method for flagella clinical identification and classification, is a time-consuming process because of the need to induce flagella growth and the occurrence of undetermined strains. We developed an alternative rapid and analytically sensitive mass spectrometry (MS) method, termed MS-based H antigen typing (MS-H), and applied it at the protein sequence level for H antigen typing. We also performed a comparison with traditional serotyping on reference strains and clinical isolates. METHODS: On the basis of international guidelines, the analytical selectivity and sensitivity, imprecision, correlation, repeatability, and reproducibility of the MS-H platform was evaluated using reference strains. Comparison of MS-H typing and serotyping was performed using 302 clinical isolates from 5 Canadian provinces, and discrepant results between the 2 platforms were resolved through whole genome sequencing. RESULTS: Repeated tests on reference strain EDL933 demonstrated a lower limit of the measuring interval at the subsingle colony (16.97 µg or 1.465 × 10(7) cells) level and close correlation (r(2) > 0.99) between cell culture biomass and sequence coverage. The CV was <10.0% among multiple repeats with 4 reference strains. Intra- and interlaboratory tests demonstrated that the MS-H method was robust and reproducible under various sample preparation and instrumentation conditions. Using discrepancy analysis via whole genome sequencing, performed on isolates with discrepant results, MS-H accurately identified 12.3% more isolates than conventional serotyping. CONCLUSIONS: MS-H typing of E. coli is useful for fast and accurate flagella typing and could be very useful during E. coli outbreaks.

Molecular mutation profile of pfcrt in Plasmodium falciparum isolates imported from Africa in Henan province.

BACKGROUND: Anti-malarial drug resistance is a primary public health problem. Haplotypes of pfcrt gene have been implicated to be molecular markers of chloroquine (CQ) resistance. This study aims to explore the prevalence of polymorphisms in pfcrt in Plasmodium falciparum-infected patients imported from Africa in Henan province. METHODS: Blood samples were collected from 502 patients who were infected with P. falciparum returning from Africa in Henan province during 2012-2015. The single nucleotide polymorphisms in pfcrt (codons 72-76) were assessed by nested PCR with DNA sequencing and restriction digestion, the haplotype prevalences were also determined. RESULTS: Four haplotypes coding 72-76 of pfcrt were found including CVMNK (wild type), CVIET (mutation type), CVIEK (mutation type), and CV M/I N/E/D/K K/T (mixed type), with 61.95 % (311/502), 33.07 % (166/502), 0.20 % (1/502), and 4.78 % (24/502) prevalence, respectively. Except mixed type, CVIET and CVIEK were the largest proportion of the mutant type in West Africa, accounting for 44.83 % (91/203), followed by East Africa (8/21, 38.10 %), North Africa (4/11, 36.36 %), Central Africa (36/135, 26.67 %), and South Africa (28/132, 21.21 %). There was significant difference among the groups (χ(2) = 23.78, P < 0.05). Mixed type was the largest proportion in North Africa (9.09 %), followed by Central Africa (6.67 %), East Africa (4.76 %), South Africa (4.55 %), and West Africa (3.45 %). There was no significant difference among the groups (χ(2) = 2.31, P > 0.05). The position 72 and 73 of pfcrt showed predominance for the wild type with rates of 100 % (502/502). CONCLUSIONS: This study identified four haplotypes of pfcrt in P. falciparum-infected patients imported from Africa in Henan province. The prevalence of mutations in the pfcrt was dropped comparing with other people's researches. It establishes fundamental data for detection of P. falciparum CQR with molecular markers for the imported P. falciparum in China, and it also provides complementary information of CQR for the malaria endemic countries and assesses the evolution of anti-malarial drug resistance.

Application and Comparison of Laboratory Parameters for Forecasting Severe Hand-Foot-Mouth Disease Using Logistic Regression, Discriminant Analysis and Decision Tree.

BACKGROUND: In recent years, the prevalence of hand-foot-mouth disease (HFMD) in China and some other countries has caused worldwide concern. Mild cases tend to recover within a week, while severe cases may progress rapidly and tend to have bad outcome. Since there is no vaccine for HFMD and anti-inflammatory treatment is not ideal. In this study, we aimed to establish a valid forecasting model for severe HFMD using common laboratory parameters. METHODS: Retrospectively, 77 severe HFMD cases from Zhengzhou Children's hospital in the peaking period between years 2013 to 2015 were collected, with 77 mild HFMD cases in the same area. The study recorded common laboratory parameters to assist in establishment of the severe HFMD model. After screening the important variables using Mann-Whitney U test, the study also matched the logistic regression (LR), discriminant analysis (DA), and decision tree (DT) to make a comparison. RESULTS: Compared with that of the mild group, serum levels of WBC, PLT, PCT, MCV, MCH, LCR, SCR, LCC, GLO, CK-MB, K, S100, and B in the severe group were higher (p < 0.05), while MCR, EOR, BASOR, SCC, MCC, EO, BASO, NA, CL, T, Th, and Th/Ts were lower (p < 0.05). Five indicators including MCR, LCC, Th, CK-MB, and CL were screened out by LR and the same for DA, and five variables including EO, LCC, CL, GLO, and MCC screened out by DT. The area under the curve (AUC) of LR, DA, and DT was 0.805, 0.779 and 0.864, respectively. CONCLUSIONS: The findings were that common laboratory indexes were effectively used to distinguish the mild HFMD cases and severe HFMD cases by LR, DA, and DT, and DT had the best classification effect with an AUC of 0.864.

[Results of Soil-transmitted Nematode Monitoring in Henan Province in 2011-2015].

To understand the endemic situation of soil-transmitted nematodiasis (Ascaris lumbricoides, Trichuris trichiura and Ancylostoma sp.) in Huaiyang County, Henan Province. Over 1 000 fecal samples from inhabitants in Huaiyang County of Henan Province were collected each year during 2011-2015, in which the soil-transmitted nematodes eggs and other intestinal helminth eggs were examined by Kato-Katz technique. The helminth-positive samples were examined by filter paper culture method to identify the species of hookworm. The cellophane swab method was used to detect Enterobius vermicularis eggs in children aged 3～12 years. Soil samples were collected from vegetable field, lavatory, courtyard and kitchen of 10 families randomly selected in each year to examine Ascaris eggs by a modified saturated sodium nitrate floatation method. In 2011-2015, 5 229 people were examined and 54 person infected with intestinal helminths were found. Five intestinal helminthes, A. lumbricoides, T. trichiura, Ancylostoma duodenale, Enterobius vermicularis and Trichostrongylus orientalis were found with 13, 2, 9, 29 and 1 infection person respectively. All showed mild infection and no multiple infections were found. There was no significant difference between the year 2015 which had the highest soil-transmitted nematode infection rate 0.6%（7/1 134） and the year 2013 which had the lowest infection rate 0.3%（3/1 037）（P>0.05）. The infection rate of intestinal helminths was highest in group of <10 years（2.8%, 25/905）, followed by the groups of >70 years（1.6%, 4/256） and 30～40 years（1.2%, 8/671）（P>0.05）. The average infection rate of E. vermicularis was 1.8%（18/993） The infection rate of E. vermicularis was relatively higher in kindergarten kids（1.6%, 6/366） and students（1.3%, 13/1 005） than that in farmers（0.3%, 10/3 782）（P<0.01）. No Ascaris eggs were found in the 200 randomly collected soil samples. The intestinal helminth infection status maintaines at low level in Henan Province during 2011-2015.

[Medical Costs on Malaria in Henan Province and Analysis of the Influential Factors].

Objective: To analyze the costs for the diagnosis and treatment of malaria in Henan Province and the influential factors. Methods: Malaria cases diagnosed in and reported by medical institutions in Henan Province from November 2013 to October 2014 were selected. General information and clinical information of those with further microscopic confirmation were also collected. The rank sum test for two or more independent samples and stepwise regression analysis were used to investigate the influential factors for medical costs. Results: A total of 218 malaria cases were finally included, of whom 73.4% were from rural areas. On average, the medical costs for patients from rural areas and cities/towns were 1 503 yuan and 4 833 yuan respectively. The average medical cost per patient with first-visit in rural hospitals was 2 600 yuan, and that with first-visit in provincial hospitals was 7 800 yuan. The average medical costs for patients diagnosed in county/city-level hospitals and provincial hospitals were 1 022.5 yuan and 6 170 yuan, respectively. There was null cost for patients diagnosed at the first-visit, while for those diagnosed after 3 or more visits the average cost per patient was 5 621 yuan. Factors significantly associated with medical costs were the current living locality of patients, the hospital level of first-visit, the hospital level of diagnosis and the number of visits before diagnosis（P＜0.05）. Stepwise regression analysis showed that the hospital level of first-visit was the most important influential factor for medical cost, followed by the hospital level of diagnosis and the number of visits before diagnosis. The higher hospital levels of first-visit and diagnosis, the higher cost. The same applied to the number of visits before diagnosis. Conclusion: There is an considerable correlation between medical cost and health seeking behavior in malaria patients.

[Analysis of Pfcrt Gene Polymorphism in Imported Falciparum Malaria Cases in Henan Province in 2015].

Objective: To analyze Plasmodium falciparum chloroquine resistant transporter (Pfcrt) gene polymorphism in imported falciparum malaria cases in Henan Province in 2015. Methods: Blood samples were collected from 132 cases of imported falciparum malaria in Henan Province in 2015. DNA was extracted from the samples, and the Pfcrt was amplified by nested PCR using specific primers. The PCR products were digested by restriction endonuclease enzyme Apol I and sequenced. Pfcrt gene polymorphism and distribution were analyzed. Results: Most of the 132 cases of imported malaria were young male adults returning from the Africa, with the highest percentage in those from West Africa（38.6%, 51/132）, then Central Africa（26.5%, 35/132）, South Africa（25.0%, 33/132）, East Africa（8.3%, 11/132）, and North Africa（1.5%, 2/132）. The nested PCR yielded a 145-bp product for each sample, and 66.7%（88/132） of the products were completely digested by Apol I, resulting in two fragments of 114 bp and 31 bp; 32.6%（43/132） could not been digested and only a single fragment of 145 bp was shown; and 0.8%（1/132） were incompletely digested, yielding three fragments of 145 bp, 114 bp and 31 bp. By blasting against chloroquine sensitive strain 3D7, we found mutations of Pfcrt at sites correspondig to residues 74, 75 and 76 from ATG, AAT and AAA to ATT, GAA and ACA （i.e. M74I, N75E and K76T） in 43 of the 132 blood samples, and mixed type mutations into ATG/T, A/GAA/T and AA/CA at sites correspondig to residues 74, 75 and 76（CVM/I, N/E/D/K, T/K） in one blood sample. The other 88 blood samples showed a wild type with no mutation (CVMNK). Mutations occurred mainly in cases from West Africa（41.2%, 21/51）, then East Africa（36.4%, 4/11）, South Africa（30.3%, 10/33）, and Central Africa（22.9%, 8/27）（χ2=4.07, P＞0.05）. The 2 cases from the North Africa both had wild type Pfcrt; the one with mixed type mutation was from West Africa. Conclusion: Three haplotypes of Pfcrt have been found, including wild type (CVMNK), mutation type (CVIET) and mixed type (CVM/I, N/E/D/K, K/T) in the imported malaria cases. The wild type occupies the highest proportion （66.7%）, while the mutation type possesses a high proportion of 41.2% in cases from West Africa.

Rapid, Sensitive, and Specific Escherichia coli H Antigen Typing by Matrix-Assisted Laser Desorption Ionization-Time of Flight-Based Peptide Mass Fingerprinting.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has gained popularity in recent years for rapid bacterial identification, mostly at the genus or species level. In this study, a rapid method to identify the Escherichia coli flagellar antigen (H antigen) at the subspecies level was developed using a MALDI-TOF MS platform with high specificity and sensitivity. Flagella were trapped on a filter membrane, and on-filter trypsin digestion was performed. The tryptic digests of each flagellin then were collected and analyzed by MALDI-TOF MS through peptide mass fingerprinting. Sixty-one reference strains containing all 53 H types and 85 clinical strains were tested and compared to serotyping designations. Whole-genome sequencing was used to resolve conflicting results between the two methods. It was found that DHB (2,5-dihydroxybenzoic acid) worked better than CHCA (α-cyano-4-hydroxycinnamic acid) as the matrix for MALDI-TOF MS, with higher confidence during protein identification. After method optimization, reference strains representing all 53 E. coli H types were identified correctly by MALDI-TOF MS. A custom E. coli flagellar/H antigen database was crucial for clearly identifying the E. coli H antigens. Of 85 clinical isolates tested by MALDI-TOF MS-H, 75 identified MS-H types (88.2%) matched results obtained from traditional serotyping. Among 10 isolates where the results of MALDI-TOF MS-H and serotyping did not agree, 60% of H types characterized by whole-genome sequencing agreed with those identified by MALDI-TOF MS-H, compared to only 20% by serotyping. This MALDI-TOF MS-H platform can be used for rapid and cost-effective E. coli H antigen identification, especially during E. coli outbreaks.

Application of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for the detection of SFTSV-specific human IgG and IgM antibodies by indirect ELISA.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease that was first reported in China in 2011. It is caused by SFTS virus (SFTSV) which is a member of the Phlebovirus genus in the Bunyaviridae family. SFTSV has been classified as a BSL3 pathogen. There is a need to develop safe and affordable serodiagnostic methods for proper clinical management of infected patients. METHODS: The full length nucleocapsid (N) gene of SFTSV Yamaguchi strain was amplified by RT-PCR and cloned to an expression vector pQE30. The recombinant (r) SFTSV-N protein was expressed by using Escherichia coli (E. coli) expression system and purified under native conditions. rSFTSV-N protein based indirect IgG and IgM enzyme linked immunosorbent assay (ELISA) systems were established to detect specific human IgG and IgM antibodies, respectively. One hundred fifteen serum samples from clinically suspected-SFTS patients were used to evaluate the newly established systems and the results were compared with the total antibody detecting sandwich ELISA system. RESULTS: The native form of recombinant (r) SFTSV-N protein was expressed and purified. Application of the rSFTSV-N protein based indirect IgG ELISA to the 115 serum samples showed results that perfectly matched those of the total antibody sandwich ELISA with a sensitivity and specificity of 100 %. The rSFTSV-N protein based indirect IgM ELISA missed 8 positive samples that were detected by the total antibody sandwich ELISA. The sensitivity and specificity of rSFTSV-N-IgM capture ELISA were 90.59 and 100 %, respectively. CONCLUSIONS: The rSFTSV-N protein is highly immunoreactive and a good target for use as an assay antigen in laboratory diagnosis. Its preparation is simpler in comparison with that used for the total antibody sandwich system. Our rSFTSV-N protein-based IgG and IgM ELISA systems have the advantage of distinguishing two types of antibodies and require small volume of serum sample only. They are safe to use for diagnosis of SFTS virus infection and especially fit in large-scale epidemiological investigations.

Knockdown resistance of Anopheles sinensis in Henan province, China.

BACKGROUND: Vivax malaria was historically epidemic in Henan Province of China and Anopheles sinensis was the main vectors and poor farming communities bare the greatest burden of disease. Knockdown resistance in An. sinensis is one of the mechanisms of resistance against pyrethroids. In the present study, the frequency of mutations from An. sinensis was examined in Henan province, China. METHODS: Anopheles was collected from Kaifeng, Tongbai, Tanghe, Pingqiao, Shihe, and Yongcheng counties of Henan province in 2013. Molecular identification of Anopheles species was conducted by polymerase chain reaction (PCR) amplifying the internal transcribed spacer 2 (ITS2). Part of the IIS6 domain of the para-type sodium channel protein gene was polymerase chain reaction-amplified and directly sequenced. Frequency and geographic difference of kdr gene mutant types were analysed. RESULTS: 208 Anopheles were received molecular identification, of which 169 (81.25%) were An. sinensis, 25 (12.02%) were Anopheles yatsushiroensis, and 12 (5.77%) were Anopheles lesteri. A 325 bp fragment of the para-type sodium channel gene including position 1014 was successfully sequenced from 139 Anopheles, of which 125 (89.93%) were An. sinensis, 12 (8.63%) were An. yatsushiroensis, 2 (1.44%) were An. lesteri. The molecular analyses revealed that mutations existed at codon 1014 in An. sinensis but not in An. yatsushiroensis and An. lesteri. Frequency of kdr mutation was 73.60% (92/125) from population of An. sinensis in Henan province, of which L1014F (TTT + TTC) allele frequencies accounted for 46.40% (58/125), and was higher than that of L1014C(TGT) which accounted for 27.20% (34/125) ( χ2 = 55.423, P < 0.001). The frequency of kdr mutation in Kaifeng county was 100% (49/49), and was higher than that of 37.93% (11/29) in Tongbai, 54.55% (6/11) in Pingqiao, 50.00% (3/3) in Shihe, and 62.50% (10/16) in Yongcheng county, respectively (χ2 = 39.538, P < 0.001; χ2 = 24.298, P < 0.001; χ2 = 25.913, P < 0.001; χ2 = 20.244, P < 0.001). While 92.86% (13/14) frequency of kdr mutation was found in Tanghe county, which was higher than that in Tongbai county (χ2 = 11.550, P = 0.0018). CONCLUSIONS: A high frequency of kdr gene mutations from population of An. sinensis in Henan province was found.

[Discovery and whole genome sequences analysis of a dengue virus type 1 strain isolated in Henan province].

OBJECTIVE: To diagnose imported dengue fever case from Henan province, and to sequence and analyze the characteristics of whole genome sequence, and to explore the possible viral origin source. METHODS: A suspected dengue fever case was reported in Yuzhou city, Henan province. The patient returned from foshan, Guangdong province on September 19, 2014, after the epidemiological investigation and serum specimen collected, which dengue fever case was diagnosed in the laboratory, then it was inoculated on Vero cells. Whole genome sequence was amplified by several pairs primers and characterized using biologic software. RESULTS: The imported case was diagnosed as dengue virus 1 serotype infection. Dengue 1 strain was isolated using Vero cells successfully. Whole genome was 10,670 nt, which belonged to dengue virus 1 serotype V genotype and didn't found any recombination event. The phylogenetic analysis demonstrated that the strain was closed to Indian starins isolated in 2008-2011, and the homology of nucleotide sequence was between 98.2%-99.4%. CONCLUSION: It was the first time to discover imported dengue 1 serotype case in Henan province. However, according to the patient has been to Guangdong province before onset, it inferred that the Indian strain had been imported to Guangdong province before this case in Henan province.

[Tracing Investigation of One Vivax Malaria Case by Detecting the Gene Encoding Circumsporozoite Protein in Henan].

A vivax malaria case in Henan Province was diagnosed as an indigenous case firstly in June 2013, and replased in April 2014. The clinical data of this case were collected and the epidemiological investigation was conducted. The blood samples were examined by Giemsa-stained blood smear, rapid diagnostic test strip (RDT) and nested PCR. This patient stayed at Myanmar for about one week in May 2013, had the symptoms of chills, fever and sweating in June, and was diagnosed as vivax malaria. After treated with artesunate, the symptoms disappeared. The CSP sequence was amplified from the blood samples of the first and second attack, and there was no difference in the central repeat domain of CSP gene. The identity of our two CSP gene sequences to that of Myanmar isolates (GenBank accessssion No. ABS95455, ABS95456) was 95.1% and 100%, while their nucleotide sequence was with 88.8% and 67.1% identity with that of Henan isolates (accessssion No. KP888996, KP889000), respectively. This patient is therefore confirmed as an imported relapse case of Plasmodium vivax infection.