| Malaria is one of the most devastating infectious tropical diseases.According to WHO report 2015, it was estimated that there were214 million cases of malaria globally, leading to 438,000 deaths. Plasmodium ovale is one of four major Plasmodium species caused human malaria. It mainly distributed in tropical Africa and Southeastern Asia.The prevalence rate of Plasmodium ovale ranged from3% to10.5% in Africa and 1.6% to 6.1% in Southeastern Asia. Infection with Plasmodium ovale is generally mild and easily curable.Severe cases have rarely been described,but could occur nonetheless. Throughout the past decades, ovale malaria was seldom reported in China. In recent years, the imported ovale malaria cases dramatically increased in China.There are still many problems in the detection of Plasmodium ovale.Microscopic diagnosis of P.ovale by blood smear examination is time-and-effort consuming, and identification from P.vivax is very difficult.The requirements of equipment and technology limit the utility of nucleic acid detection in routine clinical practice and the field clinics. RDTs are simple and rapid to use, easy to interpret results and minimal operator training. But so far there are no P.ovale specific RDTs available. Some reports mentioned that RDTs are poor to detect Plasmodium ovale.But the performance of RDTs for ovale malaria diagnosis has not been systematically evaluated, and the main factors causing misdiagnosis are never been investigated.In this study, we evaluated four common RDTs for detecting Plasmodium ovale and analyzed the influence of parasitaemia, sub-species, concentration and polymorphism of the targeted antigens on the performance. It will provide evidence for different levels of medical institutions in their choice of suitable RDTs for ovale malaria and provide scientific basis for development of next generation malaria RDTs.This study includes the following three parts:PartⅠEvaluation of four RDTs for detecting P. ovaleObjectiveTo assess the performance of four common RDTs for the detection of P. ovale.Methods Four RDTs(Wondfo, Care Start, SD and Binax NOW) were performed to test 100 blood samples from ovale malaria patients,according to the manufacturers’ instructions.The detection rates were compared among different RDTs, the result of different RDTs combination was analyzed as well.Results1.The detection rate of Wondfo, Care Start, SD and Binax NOW were70%, 55%,18%, and37%respectively,the difference was significant(P<0.05). The detection rate of SD was lower than the other three RDTs(P < 0.008, adjusting test standard).Binax NOW(Pan-Aldolase based) was able to detect seven cases which were all misdiagnosed by the three Pan-p LDH based RDTs.2.If combine two of different RDTs, the detection rate of(Wondfo+Binax NOW)was highest(78%),and(SD+Binax NOW) was lowest(40%). The detection rates of combination(Wondfo +Care Start),(Wondfo+SD) and(Wondfo+Binax NOW) have no advantage comparing with Wondfo alone(P > 0.05).The combination of(Care Start+SD) and(Care Start+Binax NOW)were no superior to Care Start either(P>0.05). No difference was observed between(SD+Binax NOW)and Binax NOW(P >0.05).Conclusion1. Overall, all four RDTs were not as good as expected to detect P. ovale. While Wondfo performed slightly better than others, SD showed the worst performance.2.Best test performance of combination RDTs to detect P. ovale was achieved by(Wondfo+Binax NOW),(SD+Binax NOW) showed the worst performance.The performance was no improved if combinding 2 RDTs.But combination Pan-p LDH based RDTs and Pan-Aldolase based RDTs would slightly increase the detection rate of P. ovale.PartⅡ Effects of parasitaemia and concentration of the targeted antigens on the performance of RDTsObjectiveTo analyze the influence of parasitaemia and concentration of the targeted antigens on the performance of RDTs for detecting P. ovale.Methods1.Corresponding blood smears of 100 blood samples were independently read by two qualified microscopists. Parasite density was determined by thick blood smears counting method.According to the level of parasitaemia,all samples were divided into3 groups:low parasitaemia ≤ 500parasites/μL,medium parasitaemia between501parasites/μL and 5,000 parasites/μL,high parasitaemia ≥ 5,001parasites/μL.Then the detection rate of the four RDTs was compared between different groups.2.The concentration of p LDH in 100 blood samples was measured by QuantimalTMp LDH Malaria CELISA.All samples were divided into 3 groups:low concentration of p LDH(A values≤0.100),medium concentration of p LDH(A values between 0.101 and 0.500), high concentration of p LDH(A values≥0.501).Then the detection rate of three p LDH-based RDTs was compared between the groups of different pLDH concentration.Results1.The detection rates of Wondfo, Care Start, SD and Binax NOW were27.3%,36.4%,0%and 9.1% respectively for low parasitaemia group;75.0%,45.6%,5.3%and 17.5%respectively for medium parasitaemia group; 75.4%,78.1%,46.9%and 81.3%respectively for high parasitaemia group.The detection rate of Wondfo at low parasitaemia was lower than medium or high parasitaemia(P<0.017).The detection rates of Care Start,SD and Binax NOW at low or medium parasitaemia were lower than high parasitaemia( P<0.017).The detection rates of SD at different different levels of parasitaemia were lower than 50%.2.The detection rates of Wondfo, Care Start and SDwas 6.7%,16.7% and 0%respectively at low p LDH concentration group(A values≤0.100); 95.1%,58.5% and4.9% respectively at medium p LDH concentration group(A values between 0.101 and0.500); 100%,89.7%and 55.2%respectively at medium p LDH concentration group(Avalues ≥ 0.501).The detection rate of Wondfo and Care Startat low p LDH concentration group was lower than medium or high p LDH concentration group(P<0.017).The detection rate of SD at low or medium p LDH concentration group was lower than the high p LDH concentration group(P<0.017).Conclusion1.The performances of RDTs for detecting P. ovale were related to parasitaemia.All four RDTs performed poor at low parasitaemia(≤500parasites/μL).Wondfo, Care Start and Binax NOW performed better when parasitaemia was greater than 5,000parasites/μL,but a certain number of samples were under detected.2.The performances of RDTs for detecting P. ovale were closely related to the concentration of targeted antigens. All three Pan-p LDH based RDTs showed poor performance at low concentration of p LDH.The detection rates significantly improved with the increase of concentration of p LDH in samples. But the detection rate of SDfor samples for high p LDH concentration samples of was still not good.PartⅢ Effects of subtypes and polymorphism of the targeted antigens on the performance of RDTsObjective To analyze the influence of sub-species and polymorphism of the targeted antigens on the performance of RDTs for detecting P.ovale.Methods1.Discrimination of P. o.curtisi and P. o.wallikeri in 100 P. ovale samples was performed by Taq Man Real-time PCR assay.The detection rates of four RDTs for both P. o.curtisi and P. o.wallikeri were compared.2.The primers were designed and synthesized respectively for amplification of Po-LDH and Po-Aldolase gene.PCR products were directly sequenced or cloned and then sequenced.Then the sequences of Po-LDH and Po-Aldolase were analyzed to analysis the impact on the detection rates of four RDTs.Results1. Among 100 P. ovale samples, 52 were determined as P. o. curtisi and 48 as P. o. wallikeri.The detection rates of Wondfo,Care Start,SD and Binax NOW was 73.1%,36.5%,0% and 32.7% respectively for P. o. curtisi;the detection rates was66.7%,75%,37% and 41.7% respectively for P. o.wallikeri.There was no significant difference in the detection rates of 2 sub-species with Wondfo and Binax NOW(P>0.05), but Care Start and SD is better to detect P. o. wallikeri than P. o. curtisi(P<0.05).2.Sequence analysis of LDH in 100 P. ovale samples indicated that the nucleotide sequences of LDH in each sub-species were highly conserved.The nucleotide sequences of LDH in the same sub-species were completely consistent.The gene homology of LDH between 2 sub-species was 97%,there were24 single nucleotide polymorphism(SNP). The homology of LDH amino acid sequences between 2 sub-species was 99%,only 3 amino acids were different.3.PCR successfully amplified Po-Aldolase gene products in 77 samples from 100 P. ovale samples.The sequence of Po-Aldolase gene were divided into 2sub-species,34 were determined as P. o. curtisi and 33 as P. o. wallikeri.The nucleotide sequences of LDH in each sub-species were highly conserved.The gene homology of Aldolase in P. o. curtisi was 100%.There was only one SNP(synonymous mutation) in one isolate among all P. o. wallikeri samples.The gene homology of Aldolase between 2 sub-species was 97%. There were 17SNPsbetween2sub-species. The homology of Aldolase amino acid sequences between 2 types was99.6%,only one amino acids was different.Conclusion1.Wondfo and Binax NOW showed similar performance in detecting 2sub-species.Care Start performed slightly better in detecting P. o. wallikeri. SD misdiagnosed all P. o. curtisi samples and showed poor performance in detecting P. o.wallikeri samples as well.2. Three non- synonymous mutations of Po-LDH between 2 sub-species was probably the reason for misdiagnosis of P. o.curtisi with SD. The homology of Po-LDH and Po-Aldolase gene were very high between 2 sub-species, and high conserved within the sub-species.The polymorphism of the targeted antigens would not explain the poor performance of the other three RDTs for detecting P. ovale samples. |
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