Analysis of initial laboratory diagnosis of malaria and its accuracy compared with re-testing from 2013 to 2018 in Yunnan Province, China.

BACKGROUND: According to China's Malaria Eradication Action Plan, malaria cases diagnosed and reported by health authorities at the county level must be further re-confirmed by provincial laboratories. The Yunnan Province Malaria Diagnostic Reference Laboratory (YPMDRL) began the synchronous implementation of microscopic examinations and nested polymerase chain reaction (nested-PCR) testing to re-test the malaria cases initially diagnosed by county-level laboratories and to evaluate the consistency of Plasmodium species identified between by YPMDRL and by the county-level laboratories from 2013 to 2018 in Yunnan Province. METHODS: Data on malaria initial diagnosis completed by county-level laboratories in Yunnan Province were collected weekly from the "China Disease Prevention and Control Information System" from 2013 to 2018. The YPMDRL performed Plasmodium microscopic examination and 18S rRNA gene nested-PCR testing on every malaria case managed by the China Disease Prevention and Control Information System. The re-testing detection results were fed back to the initial diagnosis and reporting unit for revision of malaria case types. RESULTS: A total of 2,869 malaria cases were diagnosed and reported by county-level laboratories in Yunnan Province from 2013 to 2018. The re-testing rate was 95.6% (2,742/2,869), and the re-testing rate increased from 2013 to 2018. Among the re-tested 2,742 cases, 96.7% (2651/2742), 2.2% (59/2742), and 1.1% (32/2742) were doubly examined by microscopy and by nested-PCR, only by microscopy, and only by nested-PCR, respectively. The total Plasmodium species accuracy rate at county-level laboratories was 92.6% (2,543/2,742) reference to the diagnosis by YPMDRL. Among the inconsistent 199 cases, they were identified as including 103 negative cases, 45 falciparum malaria cases, 30 vivax malaria cases, 11 ovale malaria cases, and 10 malariae malaria cases by YPMDRL. From 2013 to 2018, the revised and registered malaria cases by the China Disease Prevention and Control Information System in Yunnan Province was 2,747 cases, including 2,305 vivax malaria cases, 421 falciparum malaria cases, 11 ovale malaria cases, and 10 malariae malaria cases. CONCLUSIONS: The double re-testing strategy by microscopy and by gene testing increases the accuracy of diagnoses malaria in Yunnan Province, and gene testing can reliably differentiate Plasmodium species. The re-testing results provided by YPMDRL are the authoritative basis for revising malaria kind in Yunnan Province.

The spatiotemporal distribution of Japanese Encephalitis cases in Yunnan Province, China, from 2007 to 2017.

BACKGROUND: Japanese encephalitis (JE) is a vector-borne disease with a high prevalence in Yunnan Province, China. However, there has been a lack of a JE epidemic systematic analysis, which is urgently needed to guide control and prevention efforts. METHODS: This study explored and described the spatiotemporal distribution of JE cases observed among two different age groups in Yunnan Province from 2007 to 2017. The epidemiological features and spatial features were analyzed according to basic statistics, ArcGIS software (version 9.3; ESRI, Redlands, CA) and SPSS software (version 20; IBM Corp., Armonk, New York). RESULTS: Overall, the whole province had a high incidence of JE. The annual incidence rates in 2007 and 2017 were 1.668/100,000 and 0.158/100,000, respectively. The annual mortality was under 0.095/100,000 for these years. Although the whole province was in danger of JE, the Diqing autonomous prefecture and the Lijiang autonomous prefecture had no JE cases recorded for over 10 years. The JE cases were reported by hospitals located in 60 counties of 14 municipalities. The top ten areas with the most JE cases were Kunming City, Zhaotong City, Jinghong City, Wenshan City, Mangshi City, Pu'er City, Baoshan City, Dali City, Chuxiong City, and Gejiu City. The incidence declined smoothly, with a peak occurring from June to September, which accounted for 96.1% of the total cases. Children whose age was equal or less than 10 years old (LEQ10) still maintained a high frequency of JEV infection, and a large number of cases were reported in August, despite the Expanded Program on Immunization (EPI), which was established in April 2008. There was no difference in the quantity of cases between the two groups (t = -0.411, P>0.05); additionally, the number of JE cases among patients LEQ10 were significantly greater than those among patients older than 10 years (GTR10). Further analysis using local indicators of spatial association (LISA) revealed that the distribution of JE exhibited a high-high cluster characteristic (Z = 2.06, P<0.05), which showed that Jinghong City, Guangnan County, Yanshan County, Funing County, and Mengzi City were hot spots for the JE epidemic. CONCLUSIONS: Although the EPI was established in 2008 and the incidence of JE declined smoothly in Yunnan Province, there was no difference in the number of cases between the two age groups, which reveals that the EPI has been conducted with a low level success. In the context of limited vaccine supply capacity, we should strengthen the implementation of the children's immunization program before strengthening other immunization programs.

[Polymorphism analysis of the block 5 region in merozoite surface protein-1 gene of imported and local Plasmodium vivax in Yunnan Province].

Objective: To analyze the sequence of Plasmodium vivax merozoite surface protein-1(PvMSP-1) and allele polymorphism in imported and local vivax malaria parasites in Yunnan Province. Methods: Blood samples on filter paper were collected from imported and local vivax malaria cases in Yunnan Province during August 2012 and September 2015 and information of epidemiological history was recorded. Plasmodium DNA was extracted by a DNA extraction kit, and the block 5 region in PvMSP-1 gene was amplified by PCR. The PCR products were sequenced and blasted with reference sequences M75674, AAN86237, M60807, ABJ53045, AAN86238 and BAA18977. The sequence polymorphism in block 5 region of PvMSP-1 was analyzed with MEGA 5.04 and Arlequin3.5.1 softwares. The conserved sites, genetic distances among sequences and Shannon-wiener index among alleles were calculated. The clustering tree was drawn according to the genetic distances between the amino-acid sequences. Results: A total of 847 blood samples were collected from the malaria cases, comprising of 61 samples from local cases, 66 from imported cases from Africa, and 720 from Myanmar. The block 5 region in PvMSP-1 was successfully amplified in 278 samples, and sequencing was successfully made in 206 of them. The peptide coded by the block 5 region had a length of 193 to 222 aa. The amino acid sequence alignment showed that in 206 samples the proportion of genotypes of Sal-1, Belem and Recombine was 59.2%(122/206),23.3%(48/206) and 17.5%(36/206), respectively. The proportion of Sal-1 genotype in imported cases from Myanmar and Africa and in local cases was 58.8%(104/177),73.3%(11/15) and 50%(7/14), respectively. The genotypes Sal-1, Belem and Recombine had 51, 9 and 6 different alleles. The 66 alleles had a Shannon Wiener index (H') of 0.955 and an expected heterozygosis (He) of 0.567. The 206 DNA sequences had a 665-bp homologous locus, comprising of 75 conserved sites (11.3%,75/665) and 590 variable sites (88.7%, 590/665). The genetic distances between sequences were all less than 0.4. The clustering analysis showed that the 206 sequences were clustered into two categories with three branches. The homology of Recombine with Belem genotype was 91%-92%, higher than with Sal-1 genotype (82%-83%). Conclusion: The block 5 region in PvMSP-1 gene from local and imported Plasmodium vivax in Yunnan Province has varied forms of alleles, and the Sal-1 genotype is predominant among the three genotypes.

[Polymorphism of 72-76 Coding Sequence within Exon 2 Region of Pfcrt in Yunnan Province].

Objective: To understand the endemic situation of chloroquine-resistant falciparum malaria in Yunnan Province by analyzing the polymorphism of the 72-76 amino-acid coding sequence within exon 2 region of Plasmodium falciparum chloroquine resistant transporter (Pfcrt) gene （referred to as the 72-76 coding region） in malaria patients. Methods: The filter paper blood samples and relative information of falciparum malaria cases were collected in 13 prefectures of Yunnan Province （excluding Diqing, Wenshan, Zhaotong prefectures） from August 2012 to September 2015. The source of infection was determined by epidemiological investigation and the place of case discovery was confirmed according to the endemic registration in the Infectious Diseases Reporting Manage System, Chinese Center for Disease Control and Prevention. The exon2 region of Pfcrt gene was amplified by nested PCR and sequenced. The polymorphism of the 72-76 coding region was analyzed with MEGA 5.04. The variable sites and genetic distance between sequences were calculated. The constituent ratio of the polymorphism in sub-populations was analyzed with IBM SPSS Statistics 21 software. Results: Two hundred and thirty-two blood samples were collected in the period and source of infection included Yunnan of China, Africa and Myanma. Nested-PCR resulted in positive products in 210 samples. Sequence analysis showed the presence of chloroquine-sensitive genotype（CVMNK）（15.2%, 32/210） and mutated chloroquine-resistant genotype（CVIET, SVMNT and CVMNT）（76.2%, 160/210; 6.7%, 14/210; 1.9%, 4/210） 72-76 coding regions. The proportion of the CVMNK type was 100%（32/32） in cases with the range of 19-55 years, 46.9% （15/32） in farmers, and 59.4% （19/32） in patients with infection source in Southeast Asia, all significantly higher than those of other cases in the same groups（0; 31.3%, 10/32; and 37.5%, 12/32 respectively, χ2=13.674, 8.478, 6.292, P<0.05）. The proportion of the CVIET and SVMNT genotypes in patients with infection source in Myanma and Cambodia was 81.3%（130/160） and 78.6%（11/14） respectively, significantly higher than those in patients with infection source in Yunnan Province（6.3%, 10/160; 21.4%, 3/14）（χ2=6.519 and 6.620, P<0.05）. In samples with Africa infection source, the proportion of CVIET was 12.5%（20/160）, with no detection of SVMNT. There was a 145 bp homologous locus among the 210 exon2 regions, of which the conservative sites accounted for 95.2%（138/145） and variable sites for 4.8%（7/145）. The genetic distance between the 210 sequences ranged 0.000-0.036（0.012±0.005）. The genetic distances from genotypes CVIET, SVMNT and CVMNT to the chloroquine-sensitive genotype CVMNK were（0.029±0.015）, （0.021±0.013） and （0.014±0.001） respectively. 178 cases with chloroquine-resistant P. falciparum distributed in all the 13 prefectures. Among them, the regions with top detection rate of chloroquine-resistant genotypes were Dehong（51.7%, 92/178）, Baoshan（24.7%, 44/178） and Lincang（5.6%, 10/178） bordering on Myanmar and Kunming (4.5%, 8/178). Conclusion: There are three chloroquine-resistant genotypes of the 72-76 coding region in falciparum malaria cases in Yunnan Province, which distribute in 81.3%（13/16） of prefectures in the Province.

[Polymorphism Analysis of Plasmodium falciparum K13 Gene Kelch Domain Associated with Resistance to Artemisinin in Yunnan Province].

Objective: To investigate the polymorphism of Plasmodium falciparum K13 gene kelch domain region and provide basis for understanding the artemisinin resistance of falciparum malaria in Yunnan Province. Methods: The filter blood samples and relative information of falciparum malaria cases were collected in 16 prefectures of Yunnan Province from January 2013 to December 2015. The source of infection was determined by epidemiological investigation and the place of case discovery was confirmed according to the China Information System for Disease Control and Prevention Epidemic Registration. The K13 gene kelch domain region was amplified by nested PCR, sequenced, and blasted against the reference strain 3D7（PF3D7_1343700）. The K13 gene kelch domain region polymorphism was analyzed with Mega 5.04. The variable sites and genetic distance between sequences were analyzed. The constituent ratio of amino acid mutation sites was calculated and analyzed with χ2 test. Results: A total of 202 blood samples were collected from 2013 to 2015, comprising 190 from imported cases, 12 from local cases in Yunnan Province. The constitutent ratio of infection cases were 30.7% （62/202）, 34.2% （69/202） and 35.1% （71/202） respectively, increased year by year. The K13 gene kelch domain was successfully amplified from 192 samples and 190 were successfully sequenced, detecting missense mutation of K13 gene in 66 samples, the mutation rate was 34.7% （66/190）. The detection rate of K13 gene mutation was 40.9% （27/66）, 37.9% （25/66） and 21.2% （14/66） respectively, decreased year by year. In this study, ten types of mutations were detected, which were F446I, A578S, N458Y, P574L, A676D, G449A, C469Y, V494I, E556D and S16L. The highest mutation rate occurred in F446I which was 72.7% （48/66）. The proportion of F446I mutation type was 58.3% （28/48） in an age-range of 18-56 years, 70.8% （34/48） in farmers, and 91.7% （44/48） in patients with infection source in Southeast Asia, all significantly higher than that of other groups with the same characteristics （41.7%, 20/48; 29.2%, 14/48; and 8.3%, 4/48, respectively）（χ2=4.633, 5.556 and 5.152, both P＜0.05）. There was a 248 bp homologous sequence in the 190 sequences, composed of 235 conservative sites （94.8%）, 13 variable sites （5.2%）, 5 parsim-info sites （2.0%）, and 8 singleton sites （3.2%）. The genetic distance among the 190 sequences ranged 0.000-0.036, with an average of 0.001±0.001. Conclusion: There are 10 types of mutations in the K13 kelch domain in Yunnan Province, the predominant mutation type was F446I.

[Quality Analysis of Laboratory Malaria Diagnosis in Yunnan Province during 2012-2014].

OBJECTIVE: To assess the quality of microscopy-based malaria diagnosis in Yunnan Province from August 2012 to October 2014, and analyze the relevant factors. METHODS: Blood samples were collected from patients diagnosed as malaria by microscopy in county-level laboratories of Yunnan Province. The blood smears and blood filter paper samples were prepared and submitted to the provincial malaria diagnosis reference laboratory for further confirmation by both microscopy and the genetic approach. Coincidence rates for species identification between county and provincial laboratories were analyzed using the SPSS 21.0 software. RESULTS: From August 2012 to October 2014, 1 400 malaria cases were diagnosed with microscopy in 72 counties of Yunnan Province. Among them, the cases of falciparum malaria, vivax malaria, and unclassified malaria accounted for 18.4% (252/1,400), 79.3% (1,105/1,400) and 3.1% (43/1,400), respectively. The percentage of unclassified malaria cases reached a peak in 2012 (3.5%, 9/257). The coincidence rate for species identification with microscopy between county-level and provincial-level laboratories was 70.1% (845/1,216) in 2012, being the lowest during 2012-2014, and the coincidence rate for diagnosis of positive infection was 77.6% (943/1,216). Similarly, the coincidence rates for species identification and for positive infection between county-level laboratories using microscopy and the provincial-level laboratory using the genetic approach were 81.3% (150/185) and 85.0% (157/185) respectively in 2012, being also the lowest during 2012-2014. In the provincial laboratory, the inconsistency rate for species identification between microscopy and the genetic approach was 8.7% (97/1 120), predominately the infection-negative results by microscopy versus falciparum malaria, vivax malaria or mixed infection revealed by the genetic approach (57.7%, 56/97). The sampling coverage rate in counties was the lowest in November 2012 (46.9%, 82/175). The blood smear preparation scored 69.8, 70.4 and 78.8 (P < 0.05) in 2012, 2013 and 2014, respectively. CONCLUSION: The quality of laboratory malaria diagnosis has been significantly improved in most counties of Yunnan Province since 2013.

[Serological survey of Toxoplasma gondii infection in some areas of Pu' er City].

OBJECTIVE: To understand the status of Toxoplasma gondii infection in the population of Pu'er City, so as to provide the evidence for formulating the strategy of toxoplasmosis control. METHODS: The population from Jingdong, Jinggu, and Menglian counties in Pu' er City was surveyed; IgG of T. gondii in serum was detected by ELISA. RESULTS: Totally 906 resident serum samples were detected and the IgG positive rate was 24.2%. The positive rates were higher in the aged groups of 30-39 years and 60-69 years, and the difference among different aged groups was significant (Χ2 = 17.77, P < 0.01). There were no significant differences between different sexualities, and among different educational levels and living habits (P > 0.05). The positive rates were 26.6% (194/730), 15.5% (22/142), and 8.8% (3/34) in farmers, students and other occupations, respectively, and there was a significant difference among them (Χ2 = 12.51, P < 0.01). The positive rates were 23.3% (198/849)and 36.8% (21/57) in the farmers who had the habit of rearing pigs in pens and the farmers who had the habit of free ranging pigs, respectively, and there was a significant difference between them (Χ2 = 5.33, P < 0.05). CONCLUSION: The IgG positive rate of T. gondii is very high in Pu'er City, and therefore, the health education for toxoplasmosis control should be strengthened.