Genetic diversity of Plasmodium vivax population before elimination of malaria in Hainan Province, China.

BACKGROUND: Hainan Province is one of the most severe endemic regions with high transmission of Plasmodium falciparum and Plasmodium vivax in China. However, the incidence of P. falciparum and P. vivax has dropped dramatically since 2007 and a national elimination malaria programme (NEMP) was launched after 2010. To better understand the genetic information on P. vivax population before elimination of malaria in Hainan Province, the extent of genetic diversity of P. vivax isolates in Hainan Province was investigated using four polymorphic genetic markers, including P. vivax merozoite surface proteins 1, 3α, and 3ß (pvmsp-1, pvmsp-3α, and pvmsp-3ß) and circumsporozoite protein (pvcsp). METHODS: Isolates of P. vivax (n = 27) from Hainan Province were collected from 2009 to 2010 and pvmsp-1 and pvcsp were analysed by DNA sequencing, respectively. Using polymerase chain reaction-restriction fragment length polymorphism were analysed in pvmsp-3α, and pvmsp-3ß. RESULTS: The DNA sequencing analysis on pvmsp1 revealed that there were three allele types: Salvador-1 (Sal-1), Belem and recombinant (R) types. Among them, Sal-1 type was a dominant strain with eight variant subtypes (88.9%), whereas R- (3.7%) and Belem-type strains (7.4%) had one variant subtypes, respectively. All the isolates carried pvcsp with VK210 type accounting for 85.2% (23/27 isolates) and VK247 type accounting for 14.8% (4/27). Only type A and type B alleles were successfully amplified in pvmsp-3α gene, and a high level of polymorphism was observed in pvmsp-3α. Considering pvmsp-3ß gene, type A was the predominant type in 17 isolates (63%), whereas type B was dominant in only ten isolates (37%). CONCLUSION: The present data indicate that there was high degree of genetic diversity among P. vivax population in Hainan Province of China during the pre-elimination stage of malaria, with 26 unique haplotypes observed among 27 samples.

Sensitivity of Plasmodium falciparum to antimalarial drugs in Hainan Island, China.

Pyronaridine and artesunate have been shown to be effective in falciparum malaria treatment. However, pyronaridine is rarely used in Hainan Island clinically, and artesunate is not widely used as a therapeutic agent. Instead, conventional antimalarial drugs, chloroquine and piperaquine, are used, explaining the emergence of chloroquine-resistant Plasmodium falciparum. In this article, we investigated the sensitivity of P. falciparum to antimalarial drugs used in Hainan Island for rational drug therapy. We performed in vivo (28 days) and in vitro tests to determine the sensitivity of P. falciparum to antimalarial drugs. Total 46 patients with falciparum malaria were treated with dihydroartemisinin/piperaquine phosphate (DUO-COTECXIN) and followed up for 28 day. The cure rate was 97.8%. The mean fever clearance time (22.5 ± 10.6 hr) and the mean parasite clearance time (27.3 ± 12.2 hr) showed no statistical significance with different genders, ages, temperatures, or parasite density (P > 0.05). The resistance rates of chloroquine, piperaquine, pyronarididine, and artesunate detected in vitro were 71.9%, 40.6%, 12.5%, and 0%, respectively (P < 0.0001). The resistance intensities decreased as follows: chloroquine > piperaquine > pyronarididine > artesunate. The inhibitory dose 50 (IC50) was 3.77 × 10(-6) mol/L, 2.09 × 10(-6) mol/L, 0.09 × 10(-6) mol/L, and 0.05 × 10(-6) mol/L, and the mean concentrations for complete inhibition (CIMC) of schizont formation were 5.60 × 10(-6) mol/L, 9.26 × 10(-6) mol/L, 0.55 × 10(-6) mol/L, and 0.07 × 10(-6) mol/L, respectively. Dihydroartemisinin showed a strong therapeutic effect against falciparum malaria with a low toxicity.

Prevention measures and socio-economic development result in a decrease in malaria in Hainan, China.

BACKGROUND: Historically, the incidence of malaria in the Hainan Province, China has been high. However, since 2001 the malaria incidence in Hainan has decreased due to large-scale, public educational, promotional campaigns and the adoption of preventative measures against malaria following the fast growth of socio-economic development. The present study analysed the correlation between prevention measures and social economic development on the incidence of malaria in Hainan from 2001 to 2013. METHODS: The data of malaria preventative measures and socio-economic development were collected from various cities and counties in Hainan Province from 2001 to 2013 and analysed by the grey correlation analysis system. RESULTS: Seasonal preventive medication and local fiscal revenue increases are significantly related to the reduction of malaria incidence from 2001 to 2013 (R1 = 0.751677; R5 = 0.764795). CONCLUSION: Malaria prevention and control measures and local economic development in Hainan decreased malaria incidence from 2001 to 2013.

Eliminating Plasmodium falciparum in Hainan, China: a study on the use of behavioural change communication intervention to promote malaria prevention in mountain worker populations.

BACKGROUND: In the island of Hainan, the great majority of malaria cases occur in mountain worker populations. Using the behavioral change communication (BCC) strategy, an interventional study was conducted to promote mountain worker malaria prevention at a test site. This study found the methods and measures that are suitable for malaria prevention among mountain worker populations. METHODS: During the Plasmodium falciparum elimination stage in Hainan, a representative sampling method was used to establish testing and control sites in areas of Hainan that were both affected by malaria and had a relatively high density of mountain workers. Two different methods were used: a BCC strategy and a conventional strategy as a control. Before and after the intervention, house visits, core group discussions, and structural surveys were utilized to collect qualitative and quantitative data regarding mountain worker populations (including knowledge, attitudes, and practices [KAPs]; infection status; and serological data), and these data from the testing and control areas were compared to evaluate the effectiveness of BCC strategies in the prevention of malaria. RESULTS: In the BCC malaria prevention strategy testing areas, the accuracy rates of malaria-related KAP were significantly improved among mountain worker populations. The accuracy rates in the 3 aspects of malaria-related KAP increased from 37.73%, 37.00%, and 43.04% to 89.01%, 91.53%, and 92.25%, respectively. The changes in all 3 aspects of KAP were statistically significant (p < 0.01). In the control sites, the changes in the indices were not as marked as in the testing areas, and the change was not statistically significant (p > 0.05). Furthermore, in the testing areas, both the percentage testing positive in the serum malaria indirect fluorescent antibody test (IFAT) and the number of people inflicted decreased more significantly than in the control sites (p < 0.01). CONCLUSION: The use of the BCC strategy significantly improved the ability of mountain workers in Hainan to avoid malarial infection. Educational and promotional materials and measures were developed and selected in the process, and hands-on experience was gained that will help achieve the goal of total malaria elimination in Hainan.

[Adsorption Characteristics of Phosphate on Cerium Modified Water Hyacinth Biochar].

A cerium-modified water hyacinth biochar adsorbent(Ce-BC) was developed through co-precipitation-pyrolysis to remove phosphate from wastewater. The study investigated the effects of Ce-BC dosing, wastewater pH, reaction time, and coexisting competing ions on the adsorption process. The results showed that the best adsorption performance was achieved with a maximum adsorption capacity of 35.00 mg·g-1 at a Ce-BC dosage of 0.4 g·L-1 and an initial pH range of 3-10 in the phosphate solution. The adsorption process of phosphate by the Ce-BC followed the quasi-second-order kinetic model, and a phosphate removal efficiency of 98% within 1 h was achieved. In addition, Ce-BC had a strong anti-competitive anion interference and a good regeneration ability; after four cycles of regeneration, the adsorption efficiency remained above 90%. Characterization using field emission scanning electron microscopy-energy dispersive spectrometry(FESEM-EDS), Fourier-transform infrared spectroscopy(FTIR), X-ray diffraction(XRD), and X-ray photoelectron spectroscopy(XPS) showed that the adsorption mechanism of Ce-BC with respect to phosphate mainly involved ligand exchange and inner sphere complexation. The Ce-BC adsorbent prepared in this study effectively removed and recovered phosphates found in domestic sewage, thereby avoiding the eutrophication of water bodies as well as enabling the recovery and utilization of phosphorus resources.

[Efficacy of dihydroartemisinin-piperaquine and artemether-lumefantrine in the treatment of uncomplicated falciparum malaria in Hainan, China].

OBJECTIVE: To assess the therapeutic efficacy of the combinations dihydroartemisinin-piperaquine and artemether-lumefantrine in treating uncomplicated falciparum malaria cases in an area with high level resistance of Plasmodium falciparum to chloroquine in Hainan Province. METHODS: Patients aged 1 to 60 with uncomplicated P. falciparum infection and parasite density 1,000 to 200,000 parasites/microl were enrolled following an informed consent. Eligible patients were randomly assigned to 2 groups for receiving either a 3-day course of dihydroartemisinin-piperaquine (40/320 mg, DP, group A) or 6-dose course of artemether/lumefantrine (20/120 mg tablets, AL, group B) over three days. They were followed up with clinical and laboratory examinations until day 28 using standard WHO in vivo antimalarial drug test protocol. RESULTS: Altogether 107 eligible patients were enrolled but 106 completed the study. Adequate clinical and parasitological response (ACPR) was observed in 51 (100%) and 55 (100%) cases in groups A and B respectively. The mean time of fever clearance and mean time of asexual parasite clearance were (20.99 +/- 11.38) h and (36.45 +/- 12.60) h in AL and (22.35 +/- 13.26)h and (34.99 +/- 12.28) h in DP, respectively. There was no statistical difference on the mean time of fever clearance and asexual parasite clearance between AL and DP (P > 0.05). None of the participants showed recrudescence and serious adverse effect. CONCLUSION: Both combinations artemether-lumefantrine and dihydroartemisinin-piperaquine show a high cure rate and proper tolerability among the patients with uncomplicated falciparum malaria in Hainan.

[Primary evaluation on the application of nested/multiplex PCR in malaria diagnosis and surveillance].

OBJECTIVE: To compare the usefulness of Tag-primer nested/multiplex PCR (UT-PCR) method with microscopy in malaria diagnosis and surveillance. METHODS: 400 blood smears and blood samples on filter paper were taken from febrile patients which were initially diagnosed as malaria or suspected malaria during surveillance in mixed endemic areas of Plasmodium falciparum (Pf) and P. vivax (Pv) in Hainan and Yunnan provinces and a malaria-controlled area in Guangxi Zhuang Autonomous Region. The initial test results of both UT-PCR and microscopy for the 400 samples were compared under double blind condition. Blood smears with discrepant results between the two methods were retested by an experienced microscopist, and also repeated by UT-PCR for 2-3 times. The sensitivity and specificity of the two methods were evaluated following the Tjitra's method. RESULTS: Among the 400 blood samples, 234 were found plasmodium-positive by microscopy with 125 Pf and 109 Pv; 235 were positive by UT-PCR including 124 Pf, 109 Pv and 2 mixed infection. Altogether, the coincidence between the two methods stood for 92.5 % (370/400), including 154 negatives and 216 positives (Pv 117, Pf 99). 25 samples with discrepancy from the initial detections were retested, which covered 11 microscopy negative and PCR positive, 10 microscopy positive and PCR negative, 3 microscopy Pf positive and PCR Pv positive, 1 microscopy Pv positive but PCR mixed infection. 15 of the 25 samples showed same UT-PCR results, 7 "false positives" and 3 "false negatives" . Therefore, the sensitivity and specificity of UT-PCR was 99.6% and 98.8% respectively. CONCLUSION: As a diagnosis method, UT-PCR is useful for confirmation of malaria diagnosis and differentiation of Plasmodium species, also for improving the effectiveness and quality of malaria surveillance.

[Study on the haplotypes of pfcrt polymorphism from Hainan Province].

OBJECTIVE: To develop a method for comparing the haplotypes of pfcrt polymorphism of Hainan Province with those from other areas of the world. METHODS: Nested PCR was used to amplify the polymorphic region including codon 72 to 76 and 97 of pfcrt gene. The PCR products were digested by ApoI restriction endonuclease to determine the allelic types. According to the allelic types, 6 products from each of mutant type and wild type were sequenced to analyze the haplotypes of pfcrt polymorphism. RESULTS: Bands in size of 195 bp appeared in all 19 samples. After ApoI digestion, 11 samples contained one ApoI site when codon 76 of the pfcrt gene codes for lysine (K76), which were visualized by the presence of 98 bp and 97 bp restriction fragments. The DNA sequencing revealed that 6 samples of chloroquine resistant P. falciparum carried pfcrt alleles encoding an amino acid haplotype of CVIET (residues 72-76), and the haplotype of CVMNK was found in other 6 samples with wild type pfcrt gene. CONCLUSION: Haplotypes of pfcrt polymorphism from Hainan were the same as those from southeast Asia and Africa.

Extensive Resistance of Anopheles sinensis to Insecticides in Malaria-Endemic Areas of Hainan Province, China.

Anopheles sinensis is one of the major malaria vectors and among the dominant species in Hainan Province, China. The resistance of An. sinensis to insecticides is an important threat to malaria control. However, few reports on insecticide resistance of An. sinensis were reported in this area. Eight districts in Hainan Province were selected as the study areas. Insecticide susceptibility bioassays were tested on wild-caught female mosquitoes of An. sinensis to 4% dichlorodiphenyltrichloroethane (DDT), 0.05% deltamethrin, and 5% malathion by using the World Health Organization standard resistance tube assay procedure. All the tested An. sinensis mosquitoes demonstrated resistance to 4% DDT, with less than 72% mortality in the standard assay. The populations from Baisha and Qiongzhong demonstrated possible resistance to 0.05% deltamethrin, with 94-95% mortality, whereas the populations from other districts demonstrated resistance to 0.05% deltamethrin in the standard assay. The populations from Baisha, Qiongzhong, and Dongfang demonstrated susceptibility to 5% malathion, but the populations from other districts demonstrated resistance. These results facilitate the improvement of effective control strategies for malaria vector mosquitoes in Hainan.

A Cost-Effectiveness Analysis of Plasmodium falciparum Malaria Elimination in Hainan Province, 2002-2012.

In Hainan Province, China, great achievements in elimination of falciparum malaria have been made since 2010. There have been no locally acquired falciparum malaria cases since that time. The cost-effectiveness of elimination of falciparum malaria has been analyzed in Hainan Province. There were 4,422 falciparum malaria cases reported from 2002 to 2012, more cases occurred in males than in females. From 2002 to 2012, a total of 98.5 disability-adjusted life years (DALYs) were reported because of falciparum malaria. Populations in the age ranges of 15-25 and 30-44 years had higher incidences and DALYs than other age groups. From 2002 to 2012, malaria-related costs for salaries of staff, funds from the provincial government, national government, and the GFATM were US$3.02, US$2.24, US$1.44, and US$5.08 million, respectively. An estimated 9,504 falciparum malaria cases were averted during the period 2003-2012. The estimated cost per falciparum malaria case averted was US$116.5. The falciparum malaria elimination program in Hainan was highly effective and successful. However, funding for maintenance is still needed because of imported cases.

[Analysis of malaria epidemic situation in Hainan Province, 2010].

A total of 78 malaria cases were reported in 2010 and the incidence of malaria in this year was 9 per million in Hainan Province. The positive rate of blood tests in floating population was higher than that in local residents, and the antibody-positive rate of residents was higher than that of students, and all the reported malaria patients received the correct treatment. The rate of the therapy for vivax malaria relapse was 96.56% and the rate of prevention medicine in the high-risk groups was 94.15%. Totally 4 086 households were sprayed with insecticides and 26 843 persons were protected. Meanwhile, 153 116 LLINs were used and 250 851 persons were protected. In conclusion, the measures of malaria elimination is effective, however, we should strengthen malaria epidemic reports, case verification, treatment of epidemic foci, malaria prevention on floating population, malaria surveillance and so on.

[Relationship between falciparum malaria incidence and the number of Anopheles dirus in Hainan from 2001 to 2008].

OBJECTIVE: To analyze the relationship between falciparum malaria incidence and the number of Anopheles dirus in Hainan Province from 2001 to 2008, so as to provide the evidence for malaria control and prevention in Hainan Province. METHODS: The falciparum malaria incidence and the numbers of Anopheles dirus in 2001 - 2008 were calculated according to the data from Wuzhishan City and Qiongzhong County in Hainan Province. The Cochran-Armitage trend test was employed to explore the trend of falciparum malaria incidence. And Spearman rank correlation analysis was applied to analyze the relationship between the falciparum malaria incidence and the numbers of Anopheles dirus in Hainan from 2001 to 2008. RESULTS: A fluctuating but distinctly declining temporal trend of falciparum malaria incidence was identified in these two areas (Z(Wuzhishan) = -6.16, P < 0.01; Z(Qionghong) = -7.17, P < 0.01). The falciparum malaria incidences were not correlated with the numbers of Anopheles dirus in these two areas (r(wuzhishan) = 0.36, P > 0.05; r(Qiongzhong) = 0.67, P > 0.05). CONCLUSION: The control and prevention of falciparum malaria should be implemented from many aspects, as well as Anopheles dirus control, environment management, susceptible people protection and so on.