Genetic polymorphism of circumsporozoite protein of Plasmodium falciparum among Chinese migrant workers returning from Africa to Henan Province.

BACKGROUND: Plasmodium falciparum malaria is recognized as a major global public health problem. The malaria vaccine was important because the case fatality rate of falciparum malaria was high. Plasmodium falciparum circumsporozoite protein (PfCSP) is one of the potential vaccine candidates, but the genetic polymorphism of PfCSP raises concerns regarding the efficacy of the vaccine. This study aimed to investigate the genetic polymorphism of PfCSP and provide data for the improvement of PfCSP-based vaccine (RTS,S malaria vaccine). METHODS: Blood samples were collected from 287 Chinese migrant workers who were infected with P. falciparum and returning from Africa to Henan Province during 2016-2018. The Pfcsp genes were analysed to estimate the genetic diversity of this parasite. RESULTS: The results showed that there were two mutations at the N-terminus of imported Pfcsp in Henan Province, including insertion amino acids (58.71%, 118/201) and A → G (38.81%, 78/201). The number of repeats of tetrapeptide motifs (NANP/NVDP/NPNP/NVDA) in the central repeat region ranged mainly from 39 to 42 (97.51%, 196/201). A total of 14 nonsynonymous amino acid changes were found at the C-terminus. The average nucleotide difference (K) of imported Pfcsp in Henan Province was 5.719, and the haplotype diversity (Hd) was 0.964 ± 0.004. The estimated value of dN-dS was 0.047, indicating that the region may be affected by positive natural selection. The minimum number of recombination events (Rm) of imported Pfcsp in Henan Province was close to that in Africa. The analysis of genetic differentiation showed that there may be moderate differentiation between East Africa and North Africa (Fst = 0.06484), and the levels of differentiation in the other regions were very small (Fst < 0.05). CONCLUSIONS: The N-terminus of Pfcsp was relatively conserved, and the central repeat region and the Th2R and Th3R regions of the C-terminus were highly polymorphic. The gene polymorphism pattern among Chinese migrant workers returning from Africa to Henan Province was consistent with that in Africa. The geographical pattern of population differentiation and the evidence of natural selection and gene recombination suggested that the effect of polymorphism on the efficacy of PfCSP-based vaccines should be considered.

Molecular Surveillance of Drug Resistance of Plasmodium falciparum Isolates Imported from Angola in Henan Province, China.

Angola was the main origin country for the imported malaria in Henan Province, China. Antimalarial drug resistance has posed a threat to the control and elimination of malaria. Several molecular markers were confirmed to be associated with the antimalarial drug resistance, such as pfcrt, pfmdr1, pfdhfr, pfdhps, and K13. This study evaluated the drug resistance of the 180 imported Plasmodium falciparum isolates from Angola via nested PCR using Sanger sequencing. The prevalences of pfcrt C72V73M74N75K76, pfmdr1 N86Y184S1034N1042D1246, pfdhfr A16N51C59S108D139I164, and pfdhps S436A437A476K540A581 were 69.4%, 59.9%, 1.3% and 6.3%, respectively. Three nonsynonymous (A578S, M579I, and Q613E) and one synonymous (R471R) mutation of K13 were found, the prevalences of which were 2.5% and 1.3%, respectively. The single nucleotide polymorphisms (SNPs) in pfcrt, pfmdr1, pfdhfr, and pfdhps were generally shown as multiple mutations. The mutant prevalence of pfcrt reduced gradually, but pfdhfr and pfdhps still showed high mutant prevalence, while pfmdr1 was relatively low. The mutation of the K13 gene was rare. Molecular surveillance of artemisinin (ART) resistance will be used as a tool to evaluate the real-time efficacy of the artemisinin-based combination therapies (ACTs) and the ART resistance situation.

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.

[ Effect of Malaria Prevention and Control in Henan Province from 2010 to 2014].

During 2010-2014 a total of 1 779 malaria cases were reported in Henan Province, including 958 indigenous cases, and 821 imported cases, with 12 deaths. No indigenous malaria cases were reported during 2012-2014, but the number of imported cases showed a trend of increase, rising from 106 cases in 2010 to 216 in 2014. Blood examination was conducted in 5 210 428 patients with fever during 2010-2014. A total of 4 829 anophelines were captured, of which 4 741 were Anopheles sinensis and 88 were Anopheles anthropophagus. A total of 28 067 patients were given pre-transmission medications. In addition, 3 112 staff for Plasmodium microscopical examinations and 783 epidemiological researchers received training in the five years. Seventy-two counties (cities, districts) passed the evaluation for malaria elimination. Future work should focus on the enhancement of inspection and management of imported malaria to prevent its further dissemination and to achieve the final goal of malaria elimination in the province.

[The Role of Medical Institutions in the Control of Imported Malaria Since the Initiation of Malaria Elimination Program in Henan Province].

Objective: To retrospectively overview the diagnosis and treatment of malaria during 2010-2014 in Henan Province and understand the role of medical institutions in imported malaria control. Methods: Information on malaria epidemic situation as well as its diagnosis and treatment during 2010-2014 in Henan Province was collected from the infectious disease surveillance system and information management system for the prevention and treatment of parasitic disease. Descriptive analysis was performed to determine the role of medical institutions in the reporting, diagnosis and treatment of malaria. Results: A total of 821 imported malaria cases were reported during 2010-2014 in Henan Province, among whom 12 died, with a case fatality rate of 1.7%. The 12 deaths were all due to falciparum malaria and from Africa. The number of cases reported by medical institutions and disease control agencies were 432（52.6%） and 389（47.4%）, respectively. Among the 569 imported malaria cases with diagnosis records, 380 were determined to be malaria at first-diagnosis, with a diagnostic accuracy of 66.8%（380/569）. The accuracy of first diagnosis by medical institutions（49.2%, 178/362） was significantly lower than that by disease control agencies（97.6%, 202/207）（χ2=139.147, P<0.01）. The accuracy of first diagnosis by medical institutions at the town-ship level or below, the county level, the city/prefecture level and the provincial level was 14.2%（18/127）, 43.4% （23/53）, 73.6%（67/97） and 76.9%（70/91）, respectively（χ2=112.764, P<0.01）. However, there was no significant difference in the accuracy among disease control agencies of the above levels（χ2=0.380, P＞0.05）. The cases diagnosed by medical institutions and disease control agencies constituted 48.9%（278/569） and 51.1%（291/569）, respectively, with no significant difference（χ2=0.594, P＞0.05）. In addition, the cases diagnosed by medical institutions at the town-ship level or below, the county level, the city/prefecture level and the provincial level constituted 1.2%（7/569）, 3.7%（21/569）, 12.5%（71/569） and 31.5%（179/569）, respectively（χ2=299.143, P<0.01）. Similar results were also obtained for disease control agencies of the above levels（χ2=91.569, P<0.01）. Conclusion: There are considerable differneces of the first diagnosis accuracy and the diagnosis rate among medical institutions of different levels. Medical institutions of lower levels, which establish a diagnosis mainly based on microscopic examination, have lower diagnosis rate than the disease control agencies at same levels.

[Malaria situation in Henan Province in 2013].

In 2013, 197 cases were reported in Henan Province and all of them were imported cases. Among them, 154 (78.2%) were Plasmodium falciparum cases, 18 (9.1%) were P. vivax cases, 2 (1.0%) were P. malariae cases, and 23(11.7%) were P. ovale cases. The ratio of males to females was 97.5 : 1. The average age was (37.8±9.9) years old. 183(92.9%) patients were returned from Africa. Most of the cases were peasants, export labours and workers. Cases were reported every month with 28 cases in May. The median interval from symptom appearing to diagnosis was 4 d, only 17 patients (8.6%) were diagnosed within 24 h. 61 cases (31.0%) were reported by provincial medical institutions. 60 cases (30.5%) were reported by county CDCs. A total of 193 cases recovered with chemotherapy and 4 cases died.

[Assessment of malaria knowledge and capability on Plasmodium detection among professionals from class III malaria-endemic counties of Henan Province in 2012].

OBJECTIVE: To evaluate the capability of malaria parasite detection among professionals from Class III malaria endemic counties of Henan Province in 2012. METHEDS: The capacity assessment of professionals from the Centres for Disease Control and the medical institutions was done in September to December 2012. The content of the assessment included malaria knowledge (including malaria etiology, clinical manifestation, diagnosis, treatment and epidemiology and so on, 100 scores as full marks and 60 as passing score), making blood slides of Plasmodium (4 slides in 1 hour, including Giemsa staining, 40 scores as full marks and 24 as passing score) and identification of species with microscopy (6 slides, 8 min per slide 60 scores as full marks and 36 as passing score). All the participanats were grouped by gender, age, professional title, level and type of institution. Their scores were statistically analyzed by SSPS 17.0 software. RESULTS: The average total score in 891 participants was 162.1, the highest was 200 (full markers), and the lowest was 96, and 871 (97.8%) participants passed the test (> or = 120 scores). There were no significant differences for the scores of blood slide making among gender, age, professional title and level of institution (P > 0.05). No significant differences in the scores of malaria knowledge and blood slide reading among gender female participants (162.97 +/- 17.64) was higher than that of males (159.01 +/- 20.33) (P < 0.05). The film-reading and total scores of 50-plus age group (34.62 +/- 14.82, 144.62 +/- 20.33) was significantly lower than the other three groups (under age 30 group: 45.75 +/- 13.58 and 162.50 +/- 18.90, age 31-40 group: 46.53 +/- 12.72 and 163.51 +/- 17.77, age 41-50 group: 46.22 +/- 13.38 and 159.80 +/- 17.32) (P < 0.05). The scores of malaria knowledge in 50-plus age group (84.38 +/- 9.41) was lower than that of under age 30 group (89.91 +/- 7.81), age 31-40 group (89.96 +/- 7.74) (P < 0.05). The scores of malaria knowledge (88.33 +/- 8.23, 90.00 +/- 7.76, 92.37 +/- 7.29), film-reading (44.88 +/- 13.62, 46.59 +/- 12.88, 49.57 +/- 11.98) and total scores (159.61 +/- 18.37, 163.81 +/- 18.03, 169.15 +/- 16.38) of primary, intermediate and senior groups was proportional to the level of the titles, and the difference between the groups was statistically significant (P< 0.05). The theory (88.28 +/- 8.30, 90.84 +/- 7.32, 93.54 +/- 6.10), film-reading (44.54 +/- 13.14, 47.69 +/- 13.40, 52.62 +/- 11.04) and total scores (159.48 +/- 18.33, 165.92 +/- 17.31, 171.97 +/- 15.53) of the three institution level groups (township, county and province) were proportional to their level, and the difference between the groups was statistically significant (P < 0.05). There was no significant differences for the scores between the CDCs and hospitals (P < 0.05). CONCLUSION: The capabilities of malaria parasite detection in Class III malaria endemic counties of Henan Province is balanced. It needs to strengthen the skills training for the professionals of the junior, intermediate and primary care units.

Reemergence of Visceral Leishmaniasis in Henan Province, China.

Visceral leishmaniasis (VL) was widely prevalent in Henan Province in the 1950s. Through active efforts by the government, there were no local cases reported from 1984 to 2015. In 2016, local VL cases reoccurred, and there was an increasing trend of VL cases in Henan Province. To provide a scientific control of VL, an investigation was conducted in Henan Province from 2016 to 2021. The data from VL cases were obtained from the Disease Surveillance Reporting System of the Chinese Center for Disease Control and Prevention. The rK39 immunochromatographic test (ICT) and PCR assay were performed among high-risk residents and all dogs in the patients' village. ITS1 was amplified, sequenced, and subjected to phylogenetic analyses. A total of 47 VL cases were reported in Henan Province during 2016-2021. Of the cases, 35 were local, and they were distributed in Zhengzhou, Luoyang, and Anyang. The annual average incidence was 0.008/100,000, showing an upward trend year by year (χ2 = 3.987, p = 0.046). Their ages ranged from 7 months to 71 years, with 44.68% (21/47) in the age group of 0-3 years and 46.81% (22/47) in the age group ≥15 years. The cases occurred throughout the year. The high-risk populations were infants and young children (age ≤3), accounting for 51.06% (24/47), followed by farmers at 36.17% (17/47). The ratio of males to females was 2.13:1. The positive rates of rK39 ICT and PCR were 0.35% (4/1130) and 0.21% (1/468) in the residents. The positive rates of rK39 ICT and PCR were 18.79% (440/2342) and 14.92% (139/929) in the dogs. The ITS1 amplification products in the patients and positive dogs were sequenced. The homology between the target sequence and Leishmania infantum was more than 98%. The phylogenetic analysis indicated that the patients and the positive dogs were infected by the same type of Leishmania, which was consistent with the strains in the hilly endemic areas in China. This paper showed that patients and domestic dogs were infected by the same type of L. infantum and that the positive rate in dogs was relatively high in Henan Province. Because the measures of patient treatment and culling of infected dogs were not effective in reducing VL incidence in Henan Province, it is urgent to develop new approaches for the control of VL, such as wearing insecticide-impregnated collars on dogs, treating the positive dogs, spraying insecticide for sandflies control, and improving residents' self-protection awareness to prevent the further spread of VL in Henan Province.

Insecticide resistance of Anopheles sinensis after elimination of malaria in Henan Province, China.

BACKGROUND: Historically, malaria due to Plasmodium vivax has been epidemic in Henan Province, China, with Anopheles sinensis as the main vector. The most effective measures to prevent malaria transmission are based on vector control through the use of insecticides. However, insecticides exert a strong selective pressure on mosquito populations for insecticide resistance. The aim of this study was to investigate the susceptibility profile and population genetic characteristics of An. sinensis to provide basic data and scientific guidance for the study of resistance mechanisms and the control of An. sinensis in Henan Province. METHODS: Adult Anopheles mosquitoes were collected at sites near local farmers' sheepfolds, pigsties and/or cowsheds located in Pingqiao, Xiangfu, Xiangcheng and Tanghe counties/districts of Henan Province during July-September 2021 for insecticide susceptibility testing. Molecular identification of collected mosquitoes as belonging to genus Anopheles was by PCR, and the frequencies of mutations in the knockdown resistance gene (kdr) and acetylcholinesterase-1 gene (ace-1) were detected using gene amplification. The mitochondrial DNA cytochrome oxidase subunit I (COI) gene was amplified in deltamethrin-resistant and deltamethrin-sensitive mosquitoes to analyze the genetic evolutionary relationship. RESULTS: A total of 1409 Anopheles mosquitoes were identified by molecular identification, of which 1334 (94.68%) were An. sinensis, 28 (1.99%) were An. yatsushiroensis, 43 (3.05%) were An. anthropophagus and four (0.28%) were An. belenrae. The 24-h mortality rates of An. sinensis in Pingqiao, Tanghe, Xiangcheng and Xiangfu counties/districts exposed to deltamethrin were 85.85%, 25.38%, 29.73% and 7.66%, respectively; to beta-cyfluthrin, 36.24%, 70.91%, 34.33% and 3.28%, respectively; to propoxur, 68.39%, 80.60%, 37.62% and 9.29%, respectively; and to malathion, 97.43%, 97.67%, 99.21% and 64.23%, respectively. One mutation, G119S, was detected in the ace-1 gene. The frequencies of the main genotypes were 84.21% of specimens collected in Xiangfu (G/S), 90.63% of speciments collected in Xiangcheng (G/G) and 2.44% of speciments collected in Tanghe (S/S). Significantly higher G119S allele frequencies were observed in both propoxur- and malathion-resistant mosquitoes than in their sensitive counterparts in the Tanghe population (P < 0.05). Three mutations, L1014F (41.38%), L1014C (9.15%) and L1014W (0.12%), were detected in the kdr gene. The genotypes with the highest frequency in the populations of An. sinensis in Xiangfu and Tanghe were the mutant TTT (F/F) and wild-type TTG (L/L), at 67.86% (57/84) and 74.29% (52/70), respectively. In Pingqiao and Xiangfu, higher frequencies of the L1014F allele and lower frequencies of the L1014C allele were observed in mosquitoes resistant for beta-cyfluthrin than in those which were sensitive for this insecticide (P < 0.05). The results of Tajima's D and of Fu and Li's D and F were not significantly negative (P > 0.10), and each haplotype was interlaced and did not form two distinct branches. CONCLUSIONS: High resistance to pyrethroids and propoxur was observed at four sites, but the resistance to malathion varied according to the location. Anopheles belenrae and the L1014W (TGG) mutation in An. sinensis were first discovered in Henan Province. The deltamethrin-resistant and deltamethrin-sensitive mosquito populations showed no genetic differentiation. The generation of resistance might be the result of the combination of multiple factors.

One Health approach to improve the malaria elimination programme in Henan Province.

One Health is a collaborative, multi-sectoral, trans-disciplinary approach with the goal of achieving optimal health outcomes by recognizing the interconnection between people, animals, plants, and the environment and determining how this relates to the control of infectious diseases such as malaria, schistosomiasis and so on. Malarias caused by Plasmodium that commonly infects female Anopheles mosquitoes, which feed on human blood and act as a disease vector. It has been a worldwide important public health problem from ancient times. Also, malaria is one of the infectious diseases with the longest epidemic time and the most serious harm in the history of Henan Province, China. During the past decades, the multi-sectoral, cross-regional, and multi-disciplinary One Health approach contributed to a significant reduction in malaria incidence, resulting in initiation of the Henan Malaria Elimination Action Plan. Herein, we reviewed the history of the fight against malaria in Henan Province. A full picture of malaria epidemics, prevention, and control strategies were showed, with the objective that it will help stakeholders, and policy-makers to take informed decisions on public health issues and intervention designs on malaria control towards elimination in the similar areas.

Surveillance of Antimalarial Drug-Resistance Genes in Imported Plasmodium falciparum Isolates From Nigeria in Henan, China, 2012-2019.

Malaria remains a major public health issue in Nigeria, and Nigeria is one of the main sources of imported malaria in China. Antimalarial drug resistance is a significant obstacle to the control and prevention of malaria globally. The molecular markers associated with antimalarial drug resistance can provide early warnings about the emergence of resistance. The prevalence of antimalarial drug resistant genes and mutants, including PfK13, Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps, was evaluated among the imported Plasmodium falciparum isolates from Nigeria in Henan, China, from 2012 to 2019. Among the 167 imported P. falciparum isolates, the wild-type frequency of PfK13, Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps was 98.7, 63.9, 34.8, 3.1, and 3.1%, respectively. The mutation of PfK13 was rare, with just two nonsynonymous (S693F and Q613H) and two synonymous mutations (C469C and G496G) identified from four isolates. The prevalence of Pfcrt mutation at codon 74-76 decreased year-by-year, while the prevalence of pfmdr1 86Y also decreased significantly with time. The prevalence of Pfdhfr and Pfdhps mutants was high. Combined mutations of Pfdhfr and Pfdhps had a high prevalence of the quadruple mutant I51R59N108-G437 (39.0%), followed by the octal mutant I51R59N108-V431A436G437G581S613 (17.0%). These molecular findings update the known data on antimalarial drug-resistance genes and provide supplemental information for Nigeria.

The "1-3-7" Approach to Malaria Surveillance and Response - Henan Province, China, 2012-2018.

What is already known about this topic? The "1-3-7" approach to malaria surveillance and response was a key measure for malaria elimination in China and was first introduced into the World Health Organization (WHO) as an international guideline for malaria surveillance and response in 2018. What is added by this report? The "1-3-7" approach was well implemented in Henan Province from 2012-2018. Over this study period, a total of 1,294 malaria cases were detected and reported, and all cases were diagnosed and reported within 1 day with 99.23% (1,284/1,294) of cases were investigated within 3 days. In addition, 93.7% (1,212/1,294) of foci were investigated and vector control was implemented within 7 days at all residual non-active foci to prevent further spread. What are the implications for public health practice? The "1-3-7" controlling pattern would be an effective and approachable method for implementation especially in malaria-eliminating countries and regions, but the interval from symptom onset to diagnosis cannot be ignored. Thus, the roles and responsibilities that all actors involved in the health sector must be specified too.

Characterization of Plasmodium ovale spp. imported from Africa to Henan Province, China.

As indigenous malaria has decreased over recent decades, the increasing number of imported malaria cases has provided a new challenge for China. The proportion of imported cases due to Plasmodium ovale has increased during this time, and the difference between P. ovale curtisi and P. ovale wallikeri is of importance. To better understand P. ovale epidemiology and the differences between the two subspecies, information on imported malaria in Henan Province was collected during 2010-2017. We carried out a descriptive study to analyze the prevalence, proportion, distribution, and origin of P. o. curtisi and P. o. wallikeri. It showed that imported P. ovale spp. accounts for a large proportion of total malaria cases in Henan Province, even more than that of P. vivax. This suggests that the proportion of P. ovale cases is underestimated in Africa. Among these cases, the latency period of P. o. curtisi was significantly longer than that of P. o. wallikeri. More attention should be paid to imported ovale malaria to avoid the reintroduction of these two subspecies into China.