| Objective Now China is entering the stage of malaria elimination, Malaria is in low prevalence in China. However, there still exists some submicroscopic malaria patients, who can be the reservoirs of infection. Considering this main problem, In this study, we aim to search a sensitive method detecting submicroscopic malaria patients through comparing the results of the three PCR-based methods such as Multiple PCR, Nested PCR, Real-time PCR, and the main work consists of the definition of the submicroscopic malaria, comparing of the three methods, detection on the field samples. What’s more, on this base, we can also understand the situation and prevalence of the submicroscopic malaria patients. This study can enrich the technological method and theoretic evidence for discovery and management about the source of infection and diagnostic reference laboratory network construction.Methods This study consists of two parts:Compare the three PCR methods in the laboratory and the detection and verification of the submicroscopic malaria. On the first part, it would explore the method of Plasmodium falciparum (FCCI/HN) cultured and synchronized, microscopic detection and three PCR-based methods. Through dilution and detection of the cultured P.f and P.v collected from field, determine the limit of the submicroscopic malaria.Continue diluting the submicroscopic P.f and P.v, and use the three methods(multiple PCR, Nested PCR, Real-time PCR) to detect each density ladder, and compare the limit and detection rate of them; On the second part, on the laboratory base, we would use the three methods to detect147samples collected on field from the patients who had a fever as malaria but was negative diagnosed by microscopist, so as to verify the sensitivity of the three methods and evaluate the proportion of the submicroscopic malaria.(1)P.f was cultured and synchronized to proportion of the ring stage above to99%in vitro, and collect the fresh blood sample of vivax malaria on the field. Both the P.f and P.v were all be tested by microscopist. The thick smear would use about4-4.5μl blood, and the thin smear would need1～1.5μl blood; and then use Giemsa stain to stain the blood smear, the diagnosis of the blood film should by2qualified microscopists who were certified by WHO as the level above Ⅲ; what’s more, both were counted the density, counting the density of the P.f by thin film blood counting method (the density of RBC were counted as4.5×106/μl blood); counting the density of the P.vby thick film blood counting method (the density of WBC was counted by full-automatic blood analyzer).(2) Both the two samples were divided into three sets and then each was diluted by healthy blood of O type as the ladder:1:40,1:80,1:160,1:320......1:40960. All the diluted samples were tested by2microscopists (qualified as the level above Ⅲ by WHO) with Giemsa-stained thick smears and confirm the threshold for the submicroscopic malaria.(3) The sample with submicroscopic malaria was diluted by2-fold as (1:2,1:22,1:23,1:24......), all the samples would be tested by the three different molecular biological methods including multiple PCR, nested PCR and real-time PCR and determine the limit of each method(LOD). Compare the LOD and sensitivity of the three methods.(4) Use the QIAamp DNA mini kit (250) to extract DNA of the147field samples from who had a fever as malaria but was detected negative by microscopist, and the DNA of all the samples was detected by all the above three molecular biological methods.Count the rate of the three molecular biological methods, use the’chi-square test to compare the detection rate of the three methods, so as to evaluate the laboratory result.Result (1)P.f The threshold for the Submicroscopic P.fmalaria is21.92/μl. The LOD of multiple PCR, nested PCR and real-time PCR are0.1713/μl,0.0856/μl, and0.1713/μl, respectively. When the density of P.fis0.1713/μl:the detectable rate of the three methods is66.7%(6/9),100%(9/9),100%(9/9), the difference is significant statistically(P<0.05); When the density of P.fis0.0856/μl:the detectable rate of the three methods is0%(0/9),66.7%(6/9),55.6%(5/9), the difference is significant statistically (P<0.05); When the density of P.f/is0.0428/μl:the detectable rate of the three methods is0%(0/9),33.3%(3/9),44.4(4/9), the difference is significant statistically (P<0.05); When the density of P.fis0.0214/μl:the detectable rate of the three methods is0%(0/9),11.1%(1/9),11.1%(1/9), the difference is not significant statistically (P>0.05); When the density of P.f/is0.0107/μl: the detectable rate of the three methods is0%(0/9).(2) P.v The threshold for the Submicroscopic P.v malaria was11.05/μl. The LOD of multiple PCR, nested PCR and real-time PCR are0.3455/μl,0.0864/μl and03455/μl, respectively. When the density of P.v is0.1728/μl:the detectable rate of the three methods is0%(0/9),100%(9/9),11.1%(1/9), the difference is significant statistically (P<0.05); When the density of P.v is0.0864/μl:the detectable rate of the three methods is11.1%(1/9),77.8%(7/9),0%(0/9), the difference is significant statistically (P<0.05); When the density of P.v is0.0432/μl:the detectable rate of the three methods is0%(0/9),66.7%(6/9),0%(0/9), the difference is significant statistically (P<0.05); When the density of P.v is0.0216/μl:the detectable rate of the three methods is0%(0/9),11.1%(1/9),0%(0/9), the difference is not significant statistically (P<0.05).(3) In the147cases detected as negative by microscopists, the positive number was detected by the three methods as follows:3,13, and8, respectively. The positive rate of detection by the three methods is2.0%(3/147),8.8%(13/147),5.4%(8/147),(/P<0.05); the rate of submicroscopic malaria in Teng Chong County, Yunnan Province was estimated as8.8%,95%CI:(4.2%,13.4%).Conclusion l.As the result, the submicroscopic P.fis21.92/μl, the submicroscopic P.vis11.05/μl. All the LOD of the three methods is lower than microscopic method, so all suit for detecting the submicroscopic malaria, among them, the LOD of the nested PCR is lowest, the sensitivity of it is best (the LOD of it is for P.f0.0856/μl, for P.v0.0864/μl).2. The study on the field demonstrated that there is a high proportion (8.8%) of submicroscopic malaria patients in Tengchong County, Yunnan Province. |
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