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.

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.

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.

Efficient Face Detection in the Fisheye Image Domain.

Significant progress has been made for face detection from normal images in recent years; however, accurate and fast face detection from fisheye images remains a challenging issue because of serious fisheye distortion in the peripheral region of the image. To improve face detection accuracy, we propose a light-weight location-aware network to distinguish the peripheral region from the central region in the feature learning stage. To match the face detector, the shape and scale of the anchor (bounding box) is made location dependent. The overall face detection system performs directly in the fisheye image domain without rectification and calibration and hence is agnostic of the fisheye projection parameters. Experiments on Wider-360 and real-world fisheye images using a single CPU core indeed show that our method is superior to the state-of-the-art real-time face detector RFB Net.

A case of group infections with Paraginimus species in Henan, Central China.

In July of 2012, mass infections with Paragonimus species were detected in the Henan province sickening 11 of 51 people. In May 2011, these individuals had participated in an excursion during which freshwater crabs were caught and served after being toasted. Before the group infections with Paraginimus species was confirmed, 5 of the 11 patients had been misdiagnosed as tuberculosis (TB) and treated with an anti-TB drug regimen for six months. The most common and typical manifestations were eosinophilia (11/11, 100%) and pulmonary manifestations including, among others, stethalgia and cough (7/11 63.6%). Sero-examination revealed that all 11 patients were seropositive for Paragonimus species. Surprisingly, in our case, one patient presented with hemoptysis and eggs in respiratory secretions, and this is the first time P. skrjabini eggs are detected in the sputum of a patient from the Henan province. Paragonimus metacercariae were collected from 6 of 11 (54.5%) crabs caught at the infection site and were identified as Paraginiumus skrjabini by morphological and molecular examinations. Epidemiological and laboratory evidence confirmed that this is a case of group infection with P. skrjabini. As one of the most neglected tropical diseases (NTD), paragonimiasis should be differentiated diagnosed from TB to avoid the delay of treatment. To our knowledge, this is the second report of a case of group infections with Paraginimus species in Henan, Central China. The first case was reported in 1995. As a kind of food-borne parasitic disease, paragonimiasis should be included in the public health education agenda.

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.

Prolonged parasite clearance in a Chinese splenectomized patient with falciparum malaria imported from Nigeria.

BACKGROUND: The spleen plays a pivotal role in the rapid clearance of parasitized red blood cells in patients with falciparum malaria after artemisinin treatment. Prolonged parasite clearance can be found in patients who have had a splenectomy, or those with hemoglobin abnormalities and/or reduced immunity, which are all distinguishable from artemisinin resistance. This paper reports on a case of prolonged parasite clearance in a Chinese splenectomized patient with falciparum malaria imported from Nigeria. CASE PRESENTATION: A 35-year-old Chinese male suffered 2 days of febrile illness after returning to Zhumadian city of Henan province from Nigeria on October 1, 2014. The main symptoms were febrile, including the highest axillary temperature of 40 °C, headache, and chills. A peripheral blood smear showed parasitemia (53 913 asexual parasites/µl) of Plasmodium falciparum. The patient had not used any chemoprophylaxis against malaria in Nigeria when he worked there as a construction worker between 2009 and 2014. The patient had three episodes of malaria in Nigeria and had a splenectomy due to a traffic accident 8 years ago from the time he was admitted to hospital. The patient was orally administrated a total of 320 mg/2.56 g dihydroartemisinin-piperaquine for 2 days and intravenously administrated a total of 3 000 mg artesunate for 18 days. The axillary temperature of the patient ranged between 37.0 and 37.7 °C from Day 0 to Day 3, and blood microscopy revealed falciparum malaria parasitemia (26 674 asexual parasites/µl) on Day 3. The patient was afebrile on Day 4, falciparum malaria parasitemia was continuously present and then gradually decreased on the next days, and was negative on Day 21. The patient was cured and left hospital on Day 24 after no plasmodium falciparum was found in the blood on Day 21 to Day 23. No mutation was found in the K13 propeller gene when compared with the PF3D7_1343700 K13 propeller gene reference sequence. CONCLUSIONS: This is the first reported case in China of prolonged parasite clearance in a splenectomized patient with imported falciparum malaria. Artemisinin resistance should be distinguished when prolonged parasite clearance is found in a malaria patient who has had splenectomy.

[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.

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.

[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.

[ 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.

[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.

[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.

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.

First imported relapse case of Plasmodium vivax malaria and analysis of its origin by CSP sequencing in Henan Province, China.

In recent years, there has been a substantial increase of imported Plasmodium vivax incidence in Henan Province. As China is in a pre-elimination phase, the surveillance of imported malaria is essential, but there is no good way to distinguish imported cases from indigenous cases. This paper reports a case of a 39-year-old man who acquired P. vivax while staying in Indonesia for one month in 2013, and relapsed in Henan, China in 2014. This was diagnosed as vivax malaria based on rapid diagnostic test, Giemsa-stained peripheral blood smear and Plasmodium species-specific nested PCR. The genetic sequence for the circumsporozoite protein genes was analysed and the genetic variations were compared with a previously constructed database of Chinese isolates. The results from the circumsporozoite protein (CSP) gene sequence analysis centered on the repeat patterns showed that the imported cases had completely different sequences from any subtypes from Chinese isolates, but well matched with the countries travelled by the patient. The imported vivax cases were able to clearly distinguish from the indigenous vivax cases by detecting the CSP gene and were able to confim its origin by genotyping.

[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.

[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.

[Analysis of malaria epidemiological characteristics in Henan Province from 2005 to 2013].

OBJECTIVE: To analyze the epidemiological characteristics of malaria in Henan Province, and provide the basis for adjusting and formulating measures of malaria elimination timely. METHODS: The data of malaria cases during 2005 and 2013 in Henan Province was collected and analyzed using Microsoft Office Excel 2003 and SPSS14.0. RESULTS: Henan Province reported 17,803 malaria cases in 2005-2013, annual incidence was 0.2/100,000 to 5.2/100,000 with an average of 2.0/100,000. The cases were mainly distributed in Shangqiu (9079), Nanyang (4923), Xinyang (1449), Zhumadian (653), and Zhoukou (564), with more young male adults. The highest-risk population was farmers. More cases concentrated in August and September before 2010, but no obvious seasonal peak were seen after 2011. Vivax malaria occupied 95.4% (16,331/17,126) before 2010, but falciparum malaria was the major one after 2011. The laboratory confirmed cases during 2005-2013 took 54.6%. The median interval from symptom appearance to diagnosis was 4 d, and there was a significant difference among the years (χ2=437.2, P<0.01). The number of imported cases increased year by year, 26 cases in 2008 increased 146 cases in 2011. The reported malaria cases were imported cases during 2012 to 2013, of which 79.6% returned from the Africa, and 10.0% were from the Southeast Asia. CONCLUSION: Malaria is still an important part of public health in Henan Province, and appropriate control measures and effective tools should be strengthened for eliminating the disease.