Classification of induced malaria case in an elimination setting: investigation of transfusion-transmitted malaria cases.

BACKGROUND: Since the National Malaria Elimination Action Plan was launched in China in 2010, local malaria transmission has decreased rapidly. Zero indigenous cases were reported since 2017. However, after 2010, the proportion of imported cases in China increased from 45.7% in 2010 to 99.9% in 2016, and almost all provinces of China have reported imported cases in recent years. Prevention of the reintroduction of malaria into China is crucial for the maintenance of its malaria-free status. Hence, it is of utmost importance to correctly identify the source of malaria infections within the country. CASE INTRODUCTION AND RESPONSE: In 2016 and 2017, three laboratory-confirmed cases of malaria caused by Plasmodium falciparum were identified in patients with no previous travel history to endemic areas were reported in Jiangsu Province, China, where malaria due to P. falciparum was eliminated about 30 years ago. These were diagnosed after 41, 31 and 39 days of seeking treatment, respectively, and all of them had received blood transfusions. Further investigations indicated that two of the cases had received blood from foreign students (from Indonesia and Ghana), and the other had received blood from an individual who had worked in Equatorial Guinea. All three blood donors were traced, and found to be carrying asymptomatic P. falciparum infections by microscopic examination and PCR. Furthermore, five polymorphic microsatellite markers (C1M4, C4M62, C13M13, C14M17, and C13M63) were typed and used to link parasites from the donors with those of the transfusion-receiving patients. CONCLUSIONS: Three transfusion-transmitted malaria cases were identified in China, all of which were due to the transfusion of blood donated by individuals who had contracted malaria outside the country. These cases can provide a reference for those faced with similar challenges in malaria case identification and classification in other regions. In addition, a stricter screening policy including the use of appropriate detection methods for malaria parasites should be developed and adopted for blood donation in regions undergoing malaria elimination.

[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.

[Epidemiological analysis of malaria prevalence in Jiangsu Province in 2014].

OBJECTIVE: To understand the malaria epidemic situation and characteristics in Jiangsu Province in 2014, so as to provide the evidence for formulating and adjusting appropriate strategies and measures for malaria elimination in this province. METHODS: The reported malaria cases from the Internet Reporting System and the epidemiological data of malaria in Jiangsu Province were collected and analyzed. RESULTS: A total of 355 malaria cases were reported in Jiangsu Province in 2014, which was increased by 4.11% comparing to that in 2013 (341 cases), and the malaria incidence was 0.046/10 000. All the 355 cases were imported from other countries, among which, 4 cases (1.13%) were from Southeast Asia; the other 351 cases (98.87%) were from 21 African countries. Though the cases were distributed in all the 13 prefecture-level cities in Jiangsu Province, the number of cases in 5 of them namely Huai' an, Nantong, Lianyungang, Yangzhou and Taizhou accounted for 63.38% (225/ 355). A total of 292 falciparum malaria cases, 4 tertian malaria cases, 10 quartan malaria cases, 46 ovale malaria cases and 3 mixed infection cases were confirmed after re-checked by Jiangsu Provincial Reference Lab of Malaria. The follow-up observation of the cases showed that among the 355 cases, 6 falciparum malaria cases recrudesced, and 4 ovale malaria cases and 1 tertian malaria case recurred. CONCLUSIONS: There have been no local malaria cases reported from Jiangsu Province in the last three years, indicating the object of malaria elimination has been achieved initiatively. However, there are still many imported malaria cases from other countries, with a diverse species of plasmodium. Therefore, the surveillance of the imported malaria, the training for diagnosis and treatment of malaria as well as the health education to the key population should be strengthened.

[Surveillance of malaria in provincial surveillance sites in Jiangsu Province, 2013].

OBJECTIVE: To understand the epidemic situation and influencing factors of malaria in Jiangsu Province and grasp its epidemic regularity and trend. METHODS: According to the malaria prevalence in Jiangsu Province, 6 counties (city, district) including Yixing, Suining, Wujin, Hai'an, Ganyu and Xuyi were selected as provincial surveillance sites to survey malaria epidemic conditions. The basic information, blood test results of fever patients, case investigation, information of malaria patients, monitoring data of investigation and disposition of the malaria focus were collected and analyzed. RESULTS: In 2013, the blood tests of 66 723 fever patients were performed, the average blood smear checking rate was 1.10%, and the average positive rate was 0.08% (52 plasmodium positive individuals) in the 6 areas. For these 52 plasmodium positive individuals, the blood retests and case investigations were completed within 3 days after these cases were reported by the network system, and the investigation confirmed that they were foreign imported malaria cases. The malaria focus investigation and disposition were finished within 1 week and the data were reported by the Parasitic Diseases Information System. Four of 52 cases were recrudescence during the follow-up. Among the 52 cases, 20 people went abroad themselves and 4 were labors of private enterprises, 21 people came back without the accompanied. CONCLUSIONS: With the development of the malaria elimination program in Jiangsu Province, the eliminating malaria "targeted 1-3-7" working pattern has been comprehensively implemented. The personnel monitoring for labors who returned from overseas working will be a key in the future.

[Epidemiological analysis of malaria prevalence in Jiangsu Province in 2012].

OBJECTIVE: To understand the malaria epidemic situation and characteristics in Jiangsu Province in 2012, so as to provide the evidence for formulating and adjusting effective malaria elimination strategies and measures. METHODS: The reported malaria cases from the Internet Reporting System and the epidemiological data of malaria in Jiangsu Province were collected and analyzed. RESULTS: A total of 198 malaria cases were reported in Jiangsu Province in 2012 with the incidence of 0.026/10 000, which decreased by 47.06% compared with that in 2011(374 cases). A total of 198 malaria cases were reported from 13 prefectures of Jiangsu and the cases were mainly distributed in Yangzhou (34 cases), Nantong (31 cases), Nanjing (22 cases), Taizhou (21 cases), Xuzhou (17 cases) and Huaian (17 cases), which accounted for 71.72% (142/198) among the total cases of the province. There were no local malaria cases reported from Jiangsu in 2012, and the imported malaria cases from other countries decreased by 45.15% compared with that in 2011. CONCLUSIONS: For the first time, there are no local malaria cases reported from Jiangsu in 2012. However, the imported case distribution is further expanded and the infected plasmodium parasites are more diverse. Imported malaria from other countries remains the key for malaria control in Jiangsu Province.

[Epidemiological analysis of malaria prevalence in Jiangsu Province in 2013].

OBJECTIVE: To understand the malaria epidemic situation and characteristics in Jiangsu Province in 2013, so as to provide the evidence for formulating and adjusting effective malaria elimination strategies and measures. METHODS: The reported malaria cases from the Internet Reporting System and the epidemiological data of malaria in Jiangsu Province were collected and analyzed. RESULTS: A total of 341 malaria cases were reported in Jiangsu Province in 2013 with the incidence of 0.050/10 000, which increased by 72.22% compared with that in 2012 (198 cases). All the cases were imported from other countries including one infected by blood transfusion resulted from imported infection. The cases were mainly distributed in Lianyungang City (15.84%, 54 cases), Nantong City (14.08%, 48 cases), Yangzhou City (14.08%, 48 cases), Huaian City (11.44%, 39 cases) and Yancheng City (8.50%, 29 cases). All the cases were confirmed in Jiangsu Provincial Reference Laboratory and there were 286 cases of Plasmodium falciparum, 8 cases of P. vivax, 9 cases of P. malariae, 30 cases of P. ovale and 8 cases of mixed infections. CONCLUSIONS: There were no local malaria cases reported from Jiangsu Province in the last two years which reflected effective achievements of malaria elimination. However, the situation of imported malaria is more serious and the species of infected plasmodium are more diverse. Imported malaria from other countries remains the key of malaria control in Jiangsu Province.

[Field application of multiplex PCR to distinguish Plasmodium vivax and Plasmodium falciparum].

OBJECTIVE: To explore the application value of multiplex PCR in the diagnosis of malaria in field. METHODS: The plasmodium genus-specific primer, Plasmodium vivax, P. falciparum species-specific primers were synthesis based on the specific target segments of small subunit of 18 S rRNA ribosomal. The multiplex PCR system was optimized, and a PCR diagnostic method of malaria was established based on the genomic specific DNA fragment of P. vivax, and P. falciparum was amplified in the same PCR reaction system. The sensitivity, specificity, and the value of field application of the multiplex PCR were investigated. RESULTS: The sizes of amplification products of multiplex PCR amplifying genomic DNA of P. vivax and P. falciparum were 833 bp and 1 451 bp, respectively, and the amplification did not take place with the samples DNA of P. berghei, P. cynomolgus and healthy human blood. The sensitivities of multiplex PCR to detect P. vivax and P.falciparum were 1.1 x 10(-6) and 5.6 x 10(-7) parasitemia, respectively. Compared with the microscopic examination, the positive rate of multiplex PCR to detect 119 cases of field samples was 54%, missed diagnosis rate was 0.8%, and the misdiagnosis rate was naught, while the positive rate of the microscopic examination was 53%, its misdiagnosis rate and missed diagnosis rate were 1.7% and 3.4%, respectively. The compliance rate between the multiplex PCR and microscopic examination was 94%. CONCLUSION: The multiplex PCR for detecting malaria is simple, rapid, specific, sensitive, etc., which is suitable for the differential diagnosis of suspected cases, and molecular epidemiology investigation.

[Detection of Plasmodium sporozoites in mosquitoes by using fluorescent quantitative PCR].

OBJECTIVE: To establish a fluorescent quantitative PCR (FQ-PCR) method for quantitative detection and species identification of Plasmodium sporozoites in Anopheles mosquitoes. METHODS: One pair of human Plasmodium genus-specific primers based on 18S rRNA genes were used and the reaction system and reaction condition of FQ-PCR were optimized by using the mixture of Plasmodium 18S rRNA gene recombinant plasmids and Anopheles DNA as a template. The specificity was verified by using four Plasmodium spp. 18S rRNA gene plasmid DNA as well as mosquito DNA and the Plasmodium species was identified according to the value of melting temperature (Tm). The standard curve was made by using P. vivax 18S rRNA gene recombinant plasmids which were serially diluted by negative Anopheles DNA as a template. The sensitivity was analysed by using plasmid DNA and laboratory infected sporozoite positive mosquito DNA, respectively. The different parts and different amounts of Anopheles DNA were added into the reaction system to investigate the influence of Anopheles DNA on the assessment. RESULTS: There was no specific amplification for mosquito DNA and human blood DNA. There was specific amplification for Plasmodium 18S RNA gene recombinant plasmids and the Tm(s) of P. malariae, P. falciparum, P. ovale and P. vivax were 71.0, 72.7, 73.9 degrees C and 75.9 degrees C, respectively, which were easy to be identified. The standard curve indicated a good linear relationship between the cycle threshold (Ct) and template concentration (r = -0.99). The sensitivity was 50 copies of plasmid DNA or one sporozoite positive mosquito DNA diluted by 32 times of mosquito DNA. Anopheles DNA could inhibite the FQ-PCR reaction. The Ct value of amplification showed a good reproducibility both within the same experiment and among different experiments. CONCLUSION: The novel SYBR Green I based FQ-PCR method developed in this study shows a high sensitivity and specificity and it can be used for quantitative detection and species identification of sporozoites in mosquitoes.

[Establishment of early warning system of malaria epidemic situation in Jiangsu Province I impact of temperature on development of Plasmodium vivax in Anopheles sinensis in Jiangsu Province].

OBJECTIVE: To observe the developmental threshold temperature and the effective accumulated temperature of Plasmodium vivax sporozoites in Anopheles sinensis in Jiangsu Province, and to analyze the impact of temperature on the development of Plasmodium vivax. METHODS: The Anopheles sinensis mosquitoes were maintained under different temperatures of 16 +/- 0.5, 19 +/- 0.5, 22 +/- 0.5, 25 +/- 0.5, 28 +/- 0.5 degrees C and 31 +/- 0.5 degrees C in incubators after membrane feeding with Plasmodium vivax infected blood at laboratory. The salivary glands of Anopheles sinensis were dissected to confirm the development of sporozoite under different temperatures, and the developmental threshold temperature and the effective accumulated temperature of Plasmodium vivax sporozoites in Anopheles sinensis were calculated. The theoretical number of generations of Plasmodium vivax in Anopheles sinensis per year was calculated based on the average monthly temperatures of 13 municipalities in Jiangsu Province. RESULTS: The average developmental threshold temperature of Plasmodium vivax in Anopheles sinensis in Jiangsu Province was 15.31 degrees C, the average effective accumulated temperature was 109.81 day-degrees, and the optimal temperature for the proliferation of Plasmodium vivax was 25-28 degrees C. The theoretical generation number of Plasmodium vivax in sinensis in south Jiangsu was 1-3 generations being more than that in the north of the province. CONCLUSIONS: Temperature is one of the important influencing factors for the development of Plasmodium. The chance for Anopheles sinensis to transmit malaria increases under the temperature of 25-28 degrees C. However, other factors should be considered in the establishment of early warning system of malaria.

[Survey on malaria vectors in Jiangsu Province, 2005-2009].

OBJECTIVE: To understand the species, density and seasonal variation of malaria vectors in Jiangsu Province, so as to provide scientific evidence for malaria control. METHODS: From 2005 to 2009, 5 towns in Jiangsu Province were selected as surveillance sites, and the species of malaria vectors and their density and seasonal variation were studied by the outdoor and indoor trapping methods. The data of malaria cases were analyzed by the circular distribution method. RESULTS: Only Anopheles sinensis was captured in the 5 surveillance sites from 2005 to 2009, and its density peak was mainly appeared in the first half of July. The peak incidence of malaria was on 16th August, the distribution of cases was accordant with the seasonal variation of vectors. CONCLUSION: The surveillance and control of vectors should still be strengthened in the malaria control, so as to prevent the epidemic from rebounding as the increase of the density of Anopheles sinensis.

Adaptation of a visualized loop-mediated isothermal amplification technique for field detection of Plasmodium vivax infection.

BACKGROUND: Loop-mediated isothermal amplification (LAMP) is a high performance method for detecting DNA and holds promise for use in the molecular detection of infectious pathogens, including Plasmodium spp. However, in most malaria-endemic areas, which are often resource-limited, current LAMP methods are not feasible for diagnosis due to difficulties in accurately interpreting results with problems of sensitive visualization of amplified products, and the risk of contamination resulting from the high quantity of amplified DNA produced. In this study, we establish a novel visualized LAMP method in a closed-tube system, and validate it for the diagnosis of malaria under simulated field conditions. METHODS: A visualized LAMP method was established by the addition of a microcrystalline wax-dye capsule containing the highly sensitive DNA fluorescence dye SYBR Green I to a normal LAMP reaction prior to the initiation of the reaction. A total of 89 blood samples were collected on filter paper and processed using a simple boiling method for DNA extraction, and then tested by the visualized LAMP method for Plasmodium vivax infection. RESULTS: The wax capsule remained intact during isothermal amplification, and released the DNA dye to the reaction mixture only when the temperature was raised to the melting point following amplification. Soon after cooling down, the solidified wax sealed the reaction mix at the bottom of the tube, thus minimizing the risk of aerosol contamination. Compared to microscopy, the sensitivity and specificity of LAMP were 98.3% (95% confidence interval (CI): 91.1-99.7%) and 100% (95% CI: 88.3-100%), and were in close agreement with a nested polymerase chain reaction method. CONCLUSIONS: This novel, cheap and quick visualized LAMP method is feasible for malaria diagnosis in resource-limited field settings.