Hongwei Zhang

Background: Malaria, which is transmitted by Anopheles mosquitoes (Diptera: Culicidae), remains a critical global public health problem. Vector control interventions, particularly insecticide-based strategies, are pivotal for malaria control and elimination, as the efficacy of these interventions is heavily dependent on the high susceptibility of Anopheles mosquitoes to insecticides. However, insecticide resistance in mosquito vectors poses a considerable threat to the sustainability of these control efforts. Notably, no synthesis data on insecticide resistance have been reported in China in recent decades. Methods: This systematic review and meta-analysis aimed to assess the mortality rates and frequency of knockdown resistance (kdr) and acetylcholinesterase-1 (ace-1) mutations in Anopheles mosquitoes. The Chinese National Knowledge Infrastructure, Wan Fang, PubMed, Embase, and Web of Science databases were searched from 2000 to 2024 to identify relevant articles. Meta-analysis was performed using R and Stata software. Results: Thirty articles reporting 30,065 An. sinensis were included. The pooled mortality rate for insecticide resistance was 61% [95% confidence interval (CI): 53–68]. The mortality rates of various insecticides were as follows: dichlorodiphenyltrichloroethane (DDT), 49%; deltamethrin, 47%; malathion, 81%; propoxur, 69%; permethrin, 61%; beta-cyfluthrin, 28%; fenitrothion, 82%; beta-cypermethrin, 48%; cyfluthrin, 59%; and lambda-cyhalothrin, 56%. Moreover, the frequency of knockdown resistance (kdr) to insecticides was 36%, whereas the frequency of acetylcholinesterase-1 (ace-1) resistance was 78%. Kdr genotype analysis revealed that 13% of the reported mosquitoes were homozygote resistant, 13% were heterozygote resistant, and 74% were zygote susceptible. Ace-1 genotype analysis revealed that 42% of the reported mosquitoes were homozygote resistant, 25% were heterozygote resistant, and 33% were zygote susceptible. Conclusions: Of An. sinensis, 36% had kdr mutations, and 78% had ace-1 mutations. These vectors were resistant to pyrethroid, organochlorine, carbamate, and organophosphate insecticides. To prevent the development of resistance to alternative insecticides, it is critical to target Anopheles mosquitoes with novel chemical insecticides or biocontrol approaches. Graphical

Sulfadoxine-pyrimethamine (SP) is a key drug recommended by the World Health Organization for the chemoprevention of malaria. However, the strategy is affected by the parasite resistance to SP. This study evaluated Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes, associated with SP resistance, from 508 P. falciparum isolates imported from West African countries to Henan Province, China, during 2012–2022. High mutant prevalence of the genes Pfdhfr (94.7%) and Pfdhps (96.8%) was observed. The mutants Pfdhfr N51I, C59R, S108N, and Pfdhps A437G were at high frequency in all countries analyzed. The overall prevalence of the mutant Pfdhps K540E was low (3.4%), but with a high frequency in Liberia (24.3%). The frequency of mutants Pfdhps I431V, A581G, and A613S was 11.7%, 9.8%, and 16.2%, respectively, all of which had the highest mutant prevalence in Nigeria. The mutant Pfdhps A581G and A613S were identified in the absence of K540E. The partially resistant haplotype (I₅₁R₅₉N₁₀₈ - G₄₃₇) was the most common (72.6%), and the fully resistant haplotype (I₅₁R₅₉N₁₀₈ - G₄₃₇E₅₄₀) had a low prevalence of 3.4% and mainly occurred in Liberia. No super resistant haplotype was identified. The mutant Pfdhps I431V and the octuple mutant haplotype I₅₁R₅₉N₁₀₈ - V₄₃₁A₄₃₆G₄₃₇G₅₈₁S₆₁₃ deserve more attention. In areas of high SP resistance, the intervention still reduces low birthweight and maternal anaemia. SP should continue to be used in areas of high SP resistance until more effective alternatives for malaria chemoprevention are found. It is important to continuously monitor the molecular markers associated with SP resistance to better implement intermittent preventive treatment policies in pregnancy (IPTp) and infants (IPTi).

What is already known about this topic? China’s “1-3-7” approach outlines specific targets to guide and monitor the processes of case reporting, investigation, and response. However, few studies have examined the time intervals preceding the initial step, and the timeline from the arrival of imported malaria cases in China to their diagnosis has been largely overlooked. What is added by this report? The study demonstrated that the median duration from arrival in China to the onset of symptoms for P. ovale was 78 days, with 71.59% of imported cases manifesting symptoms after more than one month. For P. vivax, the median interval was 42 days, with 55.91% exceeding one month. Additionally, the median time from symptom onset to malaria treatment in China between 2014 and 2021 was 2 days, with an interquartile range (IQR) of 1–4 days. What are the implications for public health practice? This study represents the initial effort to delineate the chronology of imported malaria cases, from their arrival in China to their subsequent treatment. The results underscore the importance of providing malaria health education to populations arriving from overseas. Furthermore, enhancing physician training is crucial for improving the diagnosis of malaria.

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.

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. Graphical

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.

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.

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.