Shuisen Zhou

Background: Artemisinin-based combination therapy (ACT) has been recommended as the first-line treatment by the World Health Organization to treat uncomplicated Plasmodium falciparum malaria. However, the emergence and spread of P. falciparum resistant to artemisinins and their partner drugs is a significant risk for the global effort to reduce disease burden facing the world. Currently, dihydroartemisinin-piperaquine (DHA-PPQ) is the most common drug used to treat P. falciparum, but little evidence about the resistance status targeting DHA (ACT drug) and its partner drug (PPQ) has been reported in Shandong Province, China. Methods: A retrospective study was conducted to explore the prevalence and spatial distribution of Pfk13 and Pfcrt polymorphisms (sites of 72–76, and 93–356) among imported P. falciparum isolates between years 2015–2019 in Shandong Province in eastern China. Individual epidemiological information was collected from a web-based reporting system were reviewed and analysed. Results: A total of 425 P. falciparum blood samples in 2015–2019 were included and 7.3% (31/425) carried Pfk13 mutations. Out of the isolates that carried Pfk13 mutations, 54.8% (17/31) were nonsynonymous polymorphisms. The mutant alleles A578S, Q613H, C469C, and S549S in Pfk13 were the more frequently detected allele, the mutation rate was the same as 9.7% (3/31). Another allele Pfk13 C580Y, closely associated with artemisinin (ART) resistance, was found as 3.2% (2/31), which was found in Cambodia. A total of 14 mutant isolates were identified in Western Africa countries (45.2%, 14/31). For the Pfcrt gene, the mutation rate was 18.1% (77/425). T₇₆T₃₅₆ and T₇₆ were more frequent in all 13 different haplotypes with 26.0% (20/77) and 23.4% (18/77). The CVIET and CVIKT mutant at loci 72–76 have exhibited a prevalence of 19.5% (15/77) and 3.9% (3/77), respectively. The CVIET was mainly observed in samples from Congo (26.7%, 4/15) and Mozambique (26.7%, 4/15). No mutations were found at loci 97, 101 and 145. For polymorphisms at locus 356, a total of 24 isolates were identified and mainly from Congo (29.2%, 7/24). Conclusion: These findings indicate a low prevalence of Pfk13 in the African isolates. However, the emergence and increase in the new alleles Pfcrt I356T, reveals a potential risk of drug pressure in PPQ among migrant workers returned from Africa. Therefore, continuous molecular surveillance of Pfcrt mutations and in vitro susceptibility tests related to PPQ are necessary.

Background: Plasmodium vivax is the most widely distributed malaria parasite, and its drug resistance poses unique challenges to malaria elimination. The Greater Mekong Subregion (GMS) is known as the global epicenter of multidrug resistance. Surveillance of molecular markers associated with drug resistance in this area will help to inform drug policy. Methods: Dry blood spots from 58 patients out of 109 with P. vivax infection between 2017, December and 2019, March were obtained from Yingjiang County, Yunnan Province, along the China–Myanmar border. Pvdhfr, Pvdhps, Pvmdr1 and Pvcrt-o were amplified and sequenced to assess gene mutations. The polymorphism and prevalence of these molecular markers were analyzed. Results: Mutations in Pvdhfr at codons 57, 58, 61, 99 and 117 were detected in 27.59, 48.28, 27.59, 32.76 and 48.28% of the isolates, respectively. Single mutant haplotype (I₁₃F₅₇S₅₈T₆₁S₉₉S₁₁₇I₁₇₃) was the most frequent (29.31%, 17/58), followed by double mutant haplotype (20.69%, 12/58). Of three types of tandem repeat variations of Pvdhfr, deletion type was the most common. Pvdhps showed a lower prevalence among mutation genotypes. Single mutant was dominant and accounted for 34.48% (20/58). Prevalence of Pvmdr1 mutations at codons 958 and 1076 were 100.00% and 84.48%, respectively. The proportion of double and single mutant types was 84.48% (49/58) and 15.52% (9/58), respectively. Eleven samples (18.97%, 11/58) showed K10 “AAG” insertion in chloroquine resistance transporter gene Pvcrt-o. Conclusions: There was moderate diversity of molecular patterns of resistance markers of Pvdhfr, Pvdhps, Pvmdr1 and Pvcrt-o in imported P. vivax cases to Yingjiang county in Western Yunnan, along the China–Myanmar border. Prevalence and molecular pattern of candidate drug resistance markers Pvdhfr, Pvdhps, Pvmdr1 and Pvcrt-o were demonstrated in this current study, which would help to update drug policy.

Background: Resistance to anti-malarial drugs hinders malaria elimination. Monitoring the molecular markers of drug resistance helps improve malaria treatment policies. This study aimed to assess the distribution of molecular markers of imported Plasmodium falciparum infections. Methods: In total, 485 P. falciparum cases imported from Africa, Southeast Asia, and Oceania into Zhejiang province, China, from 2016 to 2018 were investigated. Most were imported from Africa, and only a few cases originated in Asia and Oceania. Blood samples were collected from each patient. Plasmodium falciparum chloroquine resistance transporter (Pfcrt) at residues 72–76 and Kelch13-propeller (k13) were determined by nested PCR and DNA sequence. Results: Wild-type Pfcrt at residues 72–76 was predominant (72.61%), but mutant and mixed alleles were also detected, of which CVIET (22.72%) was the most common. Mutant Pfcrt haplotypes were more frequent in patients from West Africa (26.92%), North Africa (25%), and Central Africa (21.93%). The number of cases of P. falciparum infections was small in Southeast Asia and Oceania, and these cases involved Pfcrt mutant type. For the k13 propeller gene, 26 samples presented 19 different point mutations, including eight nonsynonymous mutations (P441S, D464E, K503E, R561H, A578S, R622I, V650F, N694K). In addition, R561H, one of the validated SNPs in k13, was detected in one patient from Myanmar and one patient from Rwanda. A578S, although common in Africa, was found in only one patient from Cameroon. R622I was detected in one sample from Mozambique and one sample from Somalia. The genetic diversity of k13 was low in most regions of Africa and purifying selection was suggested by Tajima’s D test. Conclusions: The frequency and spatial distributions of Pfcrt and k13 mutations associated with drug resistance were determined. Wild-type Pfcrt was dominant in Africa. Among k13 mutations correlated with delayed parasite clearance, only the R561H mutation was found in one case from Rwanda in Africa. Both Pfcrt and k13 mutations were detected in patients from Southeast Asia and Oceania. These findings provide insights into the molecular epidemiological profile of drug resistance markers in the study region.

World Malaria Day, established by the World Health Assembly at its 60 th session in May 2007, is commemorated each year on April 25. According to the World Health Organization's (WHO) World Malaria Report 2019, an estimated 228 million cases of malaria (95% confidence interval [CI]: 206-258 million) occurred worldwide and most of the cases (213 million, 93%) were in the WHO African Region in 2018. There were an estimated 405,000 deaths from malaria, of which 272,000 (67%) were children aged under 5 years old. There were no global gains in reducing new infections over the period of 2014 through 2018, and nearly as many people died of malaria in 2018 as the year before (1). On World Malaria Day 2020, the WHO joins the RBM Partnership to End Malaria in promoting "Zero Malaria Starts with Me", a grassroots campaign that aims to keep malaria high on the political agenda, mobilize additional resources, and empower communities to take ownership of malaria prevention and care (2). During the COVID-19 pandemic, the malaria community must remain committed to supporting the prevention of malaria infection, illness, and death through preventive and case management services, while maintaining a safe environment for patients, clients, and staff. Deaths due to malaria and its comorbidities (anemia, undernutrition, etc.) must continue to be prevented. The National Health Commission of China has announced China's 2020 National Malaria Day theme: "Eliminating malaria and containing COVID-19: co-mitigation of imported cases and re-establishment" (3).

Background: In order to meet the requirement of malaria elimination (ME), three courses of the External Competency Assessment of Malaria Microscopists (ECAMM) were conducted during 2017–2018 in China by facilitators designated by the World Health Organization (WHO-ECAMM). A training course with a model copied from the WHO-ECAMM course was also held a week ahead of ECAMM in March 2018. Thirty-six participants completed these courses and obtained different results. Methods: The slide structures, agendas, score calculations, and the levels of certifications of the four courses strictly adhered to the WHO guidelines. All the data were collected in Excel 2016 and analysed in Graphpad Prism5 or SPSS 23. Significant differences were evaluated in Graphpad Prism5 by two-tailed paired t tests between the pre-assessment and final-assessment for each of the four courses, as well as one-way ANOVAs with Kruskal–Wallis tests and Dunn’s post hoc tests among the final assessments of the four courses. Correlations between participants’ competency results and their ages, years working on malaria, and numbers of malaria cases reported in their provinces were evaluated by bivariate correlations (two-tailed) and linear regression (excluding cases pairwise) in SPSS 23. The Pearson correlation coefficients (r values), P values (two tailed), adjusted R square (Adjusted R²), standardized coefficients (β) and Sig. P values were recorded. The percentages of participants who gave the right answer to each slide (PPS) in the final assessments of the three WHO-ECAMM courses were calculated. Correlation analysis between PPS and parasitaemia (100–2000 parasites/μL) of Plasmodium falciparum slides used in species identification and parasite counting, were also evaluated via bivariate correlations (two-tailed) tests. Results: Among the 36 participants, 16 participants were certificated as Level 1 (two from NRL), 10 were certified as Level 2 (one from NRL). Within the same course, participants had improved their average scores from pre-assessments to final assessments. The numbers of malaria cases reported in participants’ provinces were strongly correlated to their species identification (SI) scores; r = 0.45, P = 0.040, n = 21; r = 0.57, P = 0.001, n = 32; r = 0.56, P = 0.007). The parasitaemia of P. falciparum within 100–2000 parasites/μL was correlated significantly (r = 0.44, P = 0.008, n = 36) with the PPS of all counting slides but not with slides for identification (r = − 0.018, P = 0.93, n = 30). Conclusions: The analysis and comparison of participants’ competency results not only verified that the model of the WHO-ECAMM course had strong power in improving and assessing microscopists’ competencies but also reflected the correlation between decreased numbers of indigenous malaria cases and microscopists’ competencies in certain areas in China.