The Comparison of PCR Kits for the Detection of Erythrocytic Parasites on Filter Paper.

Dried blood spot (DBS) based PCR was considered an inexpensive and feasible method for detecting pathogens in the blood. The DBS carrier filter paper and PCR kits are crucial for accurate diagnosis. We evaluated 4 types of filter papers and 20 PCR kits for DBS samples. The PCR detecting Plasmodium results showed that the minimum detection limit of the 4 filter papers was 1 × 102 parasites/µL, and the positive rates of 20 PCR kits ranged from 0% to 100%. PCR results were satisfactory for detecting Plasmodium falciparum (P. falciparum) and Plasmodium. vivax (P. vivax) in archived DBS samples and Babesia gibsoni (B. gibsoni) in fresh pet DBS samples. Our results provided a useful reference for the detection of blood pathogens with DBS samples and direct PCR, especially for screening the cost-efficacy combination of filter paper and PCR kit in resource-limited areas.

[Quality analysis of malaria blood smear preparations of febrile patients in Chenzhou Prefecture, 2014].

OBJECTIVE: To standardize the malaria smear preparations of febrile patients in Chenzhou Prefecture, Hunan Province, so as to provide the technical support for malaria elimination. METHODS: According to the Technical Solutions to Eliminate Malaria (2011 edition) , the blood smear preparations of febrile patients from each county, included more than 3% negatives and all positives, were reviewed monthly in 2014. The quality of blood smear manufacture, dyeing, cleanliness and results was reviewed by malaria microscopic examination experts. The data were analyzed with the descriptive epidemiological methods. RESULTS: Totally 231 blood smears were reviewed in 2014 with a reviewed rate of 6.91%. The blood smear production qualified rate was 80.52%, the dyeing pass rate was 84.42%, the cleanliness pass rate was 86.58%, and there were no false detections and no leak detections. The highest blood smear production qualified rate, dyeing pass rate and cleanliness pass rate were found in Guiyang County and Linwu County, with all the rates of 100% respectively. The lowest blood smear production qualified rate and cleanliness pass rate were found in Yizhang County, with the rates of 52.94 % and 70.59% respectively. The lowest blood smear dyeing pass rate was found in Yongxing County with the rate of 63.64%. There were statistically significant differences between Guiyang County and Yizhang County in the production qualified rates and cleanliness pass rates (χ2 = 18.60, 9.73, both P < 0.01). There was a statistically significant difference between Guiyang County and Yongxing County in the dyeing pass rates (χ2 = 11.43, P < 0.01). CONCLUSION: Through the review of blood smears, the problems will be timely discovered. Therefore, the blood smear quality should improve, which is helpful for achieving the goal of malaria elimination.

[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.

[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.

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.