逆转录聚合酶链反应用于检测恶性疟原虫的初步研究

目的 建立逆转录聚合酶链反应系统,用于检测恶性疟原虫感染。方法 以恶性疟原虫小亚单位核糖体核糖核酸基因为靶片段,设计一对特异引物,分别采用RT－PCR技术和PCR技术进行检测恶性疟原虫血样并比较两才检测的敏感性。结果 从培养的恶性疟原血样中扩增出359bp特定扩增带,而间日疟和健康人血样均未见扩增带。经限制性内切酶分析证实扩增产物为目的片段。检测系统初步显示可检出恶性疟血样中0．34个疟原虫所含的RNA模板。以RNA为检测对象的RT－PCR肉眼可见条带的检测水平较以DNA为检测对象的PCR扩增高。结论 RT－PCR检测恶性疟原虫方法具有敏感性高、特异性强的特点,有一定的应用价值。     

逆转录聚合酶链反应检测疟原虫混合感染的研究

〔目的〕建立在同一反应体系中能同时检测和鉴定恶性疟、间日疟的一步逆转录聚合酶链反应（RT-PCR）检测方法,并用于国境口岸归国劳务人员的疟疾检测。〔方法〕以疟原虫小亚单位核糖体核糖核酸基因为靶片段,设计恶性疟原虫和间日疟原虫的上游通用引物和各自的下游特异性引物,在同一反应体系中同时扩增恶性疟原虫和间日疟原虫的特异片段,对疟疾患者的PCR产物进行序列测定和分析。〔结果〕用建立的RT-PCR检测方法对广东口岸回国劳务人员中发现的2名疟疾患者的不同采样时间的全血进行检测,2006年10月和2007年1月采集的样本被分别扩增出预期大小为360bp和450bp特异扩增带,推测2名患者为恶性疟原虫和间日疟原虫的混合感染,其在入境时处于恶性疟的发作期和间日疟的潜伏期,均属于典型的集体输入性疟疾案例。〔结论〕RT-PCR检测疟原虫方法具有敏感性高、特异性强的特点,对国境口岸疟疾诊断、镜检质量控制和流行病学研究具有较大的实用价值。     

套式PCR检测恶性疟原虫与间日疟原虫方法的建立及其应用研究

目的建立简便、灵敏、低成本的恶性疟原虫与间日疟原虫套式PCR检测方法,并探讨应用于间日疟原虫实验室检测的效果。方法制备抗凝静脉血的干滤纸血滴标本,采用干滤纸5%Chelex-100煮沸法提取疟原虫DNA,并进行套式PCR反应;通过比较PCR检测结果与疟原虫镜检结果,评价套式PCR检测恶性疟原虫与间日疟原虫方法的效果。结果在42份样本中,有34份血样PCR检测结果与镜检结果一致,占81%;5份镜检结果无法判断的血样,PCR检测为阳性或阴性;2份镜检结果为阴性的血样,套式PCR检测为阳性。结论套式PCR检测恶性疟原虫与间日疟原虫结果可靠,比镜检方法灵敏,有望在疟疾的实验室诊断中得到应用。     

分型基因芯片检测疟原虫的研制及应用

目的 研制快速检测间日疟原虫和恶性疟原虫等并可进行分型的基因芯片。方法 筛选疟原虫高度保守而且具有种属特异性的瓣RNA基因片断为探针，制备疟原虫诊断和分型的基因芯片；检测时提取标本中的疟原虫核酸，用不对称PCR技术进行扩增和荧光标记，然后与检测芯片杂交，进行扫描和分析。结果 建立的疟原虫检测芯片能特异和敏感的对间日疟原虫和恶性疟原虫诊断及分型。结论 成功制备用于疟原虫快速侦检和分型的基因芯片，对疟疾的预防和控制具有重要意义。     

湖北省首例输入性疟疾混合感染的实验室诊断分析

目的应用多重巢式PCR技术对一例输入性疟疾混合感染病例进行诊断和鉴定。方法采集镜检初诊为输入性卵形疟患者血样,进行疟疾快速诊断试剂检测、显微镜镜检复核、多重巢式PCR检测及测序分析。结果该患者经快速诊断试剂检测结果为非恶性疟原虫阳性;镜检复核查见寄生于红细胞内的疟原虫环状体、大滋养体和配子体,根据形态鉴定为卵形疟;多重巢式PCR产物经琼脂糖凝胶电泳,在760bp和205bp处有特异性扩增条带,测序后经Blast比对,分别与Gen Bank数据库中卵形疟原虫变异亚种和恶性疟原虫部分序列的一致性为99.00%。结论该患者经多重巢式PCR和序列分析确诊为湖北省首例输入性卵形疟原虫变异亚种与恶性疟原虫混合感染病例。     

基因扩增检测四种感染人的疟原虫种类、数量的方法

目的建立对四种感染人的疟原虫种类、数量进行基因检测的方法。方法根据恶性疟、三日疟、卵形疟、间日疟的18SrRNA基因序列,设计属、种特异性引物和TaqMan探针,用荧光定量扩增反应确定标本中的基因拷贝数,用电泳区别各种疟原虫,并对2份疟疾血样进行检测。结果建立的检测方法对疟疾血样进行检测,荧光定量PCR具有良好的反应性,通过电泳能区分检测标本中的疟原虫株。结论建立的方法能够特异而灵敏地检测出标本中疟原虫的种类、数量,适合疟疾防治检测的需要。     

交叉引物等温扩增技术在恶性疟疾快速检测中的应用

目的 建立一种快速、敏感、特异检测恶性疟原虫的交叉引物等温扩增(cross priming amplification,CPA)技术.方法 根据疟原虫的18S rRNA基因保守序列,设计CPA引物和探针.分别用CPA法、吉氏染色镜检法检测78份发热患者血样,验证CPA法特异性和灵敏性.结果 采用CPA法对不同病原体核酸的检测结果显示,38份含有恶性疟原虫血样中检出37例阳性,其他病原体检测结果均为阴性,证明其特异性良好;该方法检测的灵敏度可达102拷贝/μl;与镜检法比较,其检测的灵敏度为97.37％,特异性为100％,准确度为98.72％.结论 用于检测恶性疟原虫的CPA检测体系具有简便、快速、灵敏、特异的优点,可用于临床、预防和出入境口岸现场疟疾的快速检测.     

1例输入性卵形疟原虫实验室检测

目的对1例输人性疑似疟疾患者的血样进行实验室确诊。方法首先制备血涂片,吉姆萨染色后镜检疟原虫;其次对血样进行RDT检测;最后利用疟原虫属特异性（通用型）和4种疟原虫种特异性的巢式PCR和多重PCR检测方法,对该血样进行分子生物学检测及分型。结合分子生物学检测结果,再对血片进行镜检复核。结果初次镜检结果为间日疟原虫,RDT结果为阴性。巢式PCR检测结果,仅扩增出预期大小约800bp的卵形疟条带;多重PCR（Pf／Pv）检测无特异性条带产生。重新对薄血膜复核镜检,改判为卵形疟原虫。结论综合巢式PCR、多重PCR、RDT和镜检等检测结果,确诊该患者为卵形疟原虫感染。     

2017年锦州市输入性病例疟原虫18S rDNA 基因种类鉴定及序列分析

目的研究锦州市输入性病例疟原虫18S rDNA的基因差异和种系发育。方法收集2017年2例输入性疟疾患者血样,提取血样中的疟原虫DNA,采用巢式PCR对其18S rDNA基因进行扩增、测序分析,并与GenBank中疟原虫相应序列进行比较分析,利用邻接法(neighbor joining,NJ)构建系统进化树。结果 2例患者PCR电泳结果获得片段均为205 bp,是恶性疟原虫感染。从系统进化树中显示,2个检测基因片段与恶性疟原虫形成一个分支,与GenBank中来自喀麦隆(KC428741、KC428742)、巴西(KC906718)和马来西亚(HQ283221)等7个分离株聚集在一起,具有很高的相似性(98.5%～99.0%),与来自印度(HM014353)的相似性较高(98.0%～98.5%),与其他间日疟原虫、三日虐原虫、卵型疟原虫、诺氏疟原虫基因序列相似性较低(36.4%～64.2%)。结论 2例输入性疟疾患者感染的疟原虫18S rDNA基因变异较小,基因型别较为稳定,均无明显种内差异。     

多重qPCR检测疟原虫方法的建立及应用评价

目的 建立一种能检测4种疟原虫多重qPCR(multiplex quantitative real-time polymerase chain reaction)方法，并对其进行临床应用评价。方法 以4种疟原虫18S rRNA基因作为靶序列，在保守区域设计双侧扩增引物，根据4种疟原虫特异性区域分别设计荧光探针，建立多重qPCR检测方法，并测试其特异性、敏感性，同时应用该方法对疟疾或疑似疟疾的临床血样进行检测。结果 建立检测4种疟原虫多重qPCR方法具有良好的特异性与敏感性，检测下限可达10拷贝/μl。该方法对浙江省2020年—2021年115份疟疾或疑似疟疾的临床血样进行检测，结果与疟疾分子诊断参考方法巢式PCR一致，临床特异度与敏感度均为100%。结论 本研究所建立的检测4种疟原虫多重qPCR方法简单快速、特异性好、敏感性高，可应用于疟疾临床病原诊断和参比实验室样本复核。     

The detection of Plasmodium falciparum and P. vivax in DNA-extracted blood samples using polymerase chain reaction

Seventeen pairs of published primer sets were compared for their relative sensitivity to detect malaria DNA extracted from blood samples, which were obtained from Pakistani patients suffering from malaria. The primer sets investigated consisted of: (i) 9 pairs of direct primers and 3 sets of nested primers for detecting Plasmodium falciparum, (ii) 2 pairs of direct primers and 2 sets of nested primers for detecting P. vivax, and (iii) 1 set of multiplex primers for detecting both P. falciparum and P. vivax, simultaneously. After a miniscreen of 9 DNA-extracted blood samples using the 17 primer sets stated above, 5 primer sets were short-listed (based on their superior sensitivity) and used for a maxi-screen of DNA extracted from 126 microscopy-positive blood samples from Pakistan, with the following results. (i) For the detection of P. falciparum, the direct primer pair 'PF1 + PF2' gave a sensitivity of 95% and the nested primer set 'RIT405 + RIT406/RIT371 + RIT372' gave a sensitivity of 97%. (ii) For the detection of P. vivax, the direct primer pair 'Forward + Reverse' and the nested primer set 'PLF + UNR/PLF + VIR' both gave a sensitivity of 94%. (iii) The nested multiplex primer set 'rPLU5 + rPLU6/rFAL1 + rFAL2 + rVIV1 + rVIV2' gave a sensitivity of 97% and 96% for P. falciparum and P. vivax, respectively. It was concluded that the nested multiplex primer set was the most optimal primer set to use for the detection of malaria DNA extracted from blood samples. Furthermore, the nested multiplex primer set has the advantage of simultaneously detecting and differentiating between P. vivax and P. falciparum.     

Optimization of a semi-nested multiplex PCR to identify Plasmodium parasites in wild-caught Anopheles in Bolivia, and its application to field epidemiological studies

Without an adequate DNA extraction protocol, the identification of Plasmodium species in whole mosquitoes by PCR is difficult because of the presence of reaction inhibitors from the insects. In this study, eight DNA extraction protocols were tested, from which a chelex-based protocol was selected. Then a semi-nested multiplex PCR technique that detects and distinguishes among the four human Plasmodium species in single mosquitoes and in pools of up to 100 mosquitoes was optimized. The technique was used to detect P. vivax in wild-caught Anopheles pseudopunctipennis from a village in the Andean valleys of Bolivia in May 2003. The prevalence of infection was 0.9%. This is the first direct evidence of P. vivax transmission by this vector in this country. The extraction and PCR technique presented here can be useful to: (1) estimate Plasmodium prevalence in Anopheles populations in low prevalence areas where large numbers of individual mosquitoes would need to be processed to obtain a reliable estimate; (2) incriminate Anopheles species as malaria vectors; (3) identify all the circulating Plasmodium species in vectors from an area; (4) detect mixed infections in mosquitoes; and (5) detect mosquitoes with low-level parasite infections.     

Demonstration by the polymerase chain reaction of mixed Plasmodium falciparum and P. vivax infections undetected by conventional microscopy.

Mixed malaria infections (Plasmodium falciparum and P. vivax) are suspected to occur at a greater frequency than is detected by conventional light microscopy. To determine this frequency we carried out a prospective 'blinded' comparison of diagnosis by conventional light microscopy and enzymatic amplification of the circumsporozoite gene extracted from dried spotted blood samples. Patients were previously healthy, active duty Thai soldiers assigned to a malaria risk area presenting with malaria. Microscopy (oil immersion objective at 1000 x magnification) involved examination of Giemsa-stained thick and thin blood films by an experienced microscopist. Whole blood samples (25 microliters) dried on filter paper were used for species-specific parasite deoxyribonucleic acid (DNA) amplification by the polymerase chain reaction (PCR) and hybridization with radiolabelled P. falciparum and P. vivax probes. Of 137 consecutive cases of malaria studied, 9% (3/32) of microscopically diagnosed P. falciparum infections and 5% (5/104) of microscopically diagnosed P. vivax infections were found to be mixed by the PCR/DNA probe systems, while 1 case was diagnosed as mixed by both microscopy and PCR. The possibility that malaria patients may have undetected mixed infections should be kept in mind because of the specific therapy required both for P. falciparum and for radical cure of P. vivax.     

Persistent Epstein-Barr viral reactivation in young African children with a history of severe Plasmodium falciparum malaria

Epstein-Barr virus (EBV) and Plasmodium falciparum have overlapping distributions and are thought to have causal interactions, particularly with regard to the aetiology of endemic Burkitt's lymphoma. Using real-time PCR, we quantified and compared EBV DNA levels in the blood before and after antimalarial treatment of age- and gender-matched groups of Gabonese children who presented with either mild or severe P. falciparum malaria. Following treatment, the prevalence of EBV DNA declined in the mild malaria group but increased in the severe malaria group, and a significantly higher proportion of the latter had EBV DNA detectable in their blood when they were healthy and parasite free (67% vs. 39%; P=0.013). High EBV DNA loads were associated with more malaria attacks and with elevated plasma concentrations of both TNF-alpha and IL-12p40. Significantly more under 5 year olds had EBV DNA, highlighting the strong age dependence of the interaction between the two pathogens. These findings confirm that EBV is reactivated during acute P. falciparum malaria but, importantly, also reveal that: (i) EBV activity persists at a higher frequency in children with a history of severe malaria; and (ii) higher peripheral blood EBV DNA loads are associated with susceptibility to more frequent P. falciparum episodes and with altered cytokine activity.     

多重半套式聚合酶链反应在检测脑脊液病原菌中的应用

目的：建立多重半套式聚合酶链反应（PCR）快速检测脑脊液标本中常见的病原菌。方法：通过对病原菌16S rRNA基因保守区和变异区的序列分析，设计通过引物及革兰阴性菌、革兰阳性菌的特异性引物，分别作为外、内侧引物，对脑脊液标本中不同细菌的DNA进行多重半套式PCR扩增；同时与常规细菌培养法作比较，并检测了该方法的敏感性。结果：外侧扩增后革兰阳性菌、革兰阴性菌经扩增后均有长约1032bp的片段产生；内侧扩增两种细菌除1032bp的产物外，革兰阳性菌另有一336bp的特异性产物，革兰阴性菌另有一127bp的特异性产物。该方法最低可检测出8cfu/ml的大肠埃希菌；62份脑脊液标本扩增结果与培养法相比，敏感性、特异性、阳性预测值、阴性预没值分别为93．8％、5．7％、88．2％、97．8％。结论：多重半套式PCR方法能特异、敏感、快速地检测出脑脊液感染的常见病原菌。     

The Rate of Plasmodium vivax Infectivity within Gloucose-6-Phosphate Dehydrogenase (G6PD) Deficient Individuals in Hormozgan Province,Iran

Background

One of the most important enzymatic disorders that interact with malaria is deficiency of G6PD (Gloucose-6-phosphate dehydrogenase). This enzyme protects red blood cells from hydrogen peroxide and other oxidative damages. Distribution of this enzyme deficiency usually accompanies with low level distribution of malaria disease in most malarious areas. So this hypothesis may be considered that the G6PD deficiency could be protective against malaria.

Methods

Totally 160 samples were taken from vivax malaria infected and non-infected individuals. Preparing blood smears and quantitative test for G6PD deficiency were employed for all of the samples. To ensure accuracy of the malaria in negative samples besides using microscopical examination, semi-nested multiplex PCR was also performed for the two groups.

Results

In microscopical examination 36 and 124 samples were vivax malaria positive and negative respectively. Out of 36 P.vivax positive cases 3 (8.3%) cases were detected to be G6PD deficient versus 30 (24.2%) cases out of 124 P. vivax negative cases. The results showed a significant differentiation between P. vivax positive and P. vivax negative cases in the rate of G6PD deficiency (3/36 in positive cases versus 30/124 in negative cases) (P<0.05).

Conclusion

vivax malaria positive individuals with G6PD deficiency showed too mild symptoms of Malaria or even asymptomatic.     

Alternative polymerase chain reaction method to identify Plasmodium species in human blood samples: the semi-nested multiplex malaria PCR (SnM-PCR)

A simplified protocol for the identification of Plasmodium species by semi-nested multiplex polymerase chain reaction (SnM-PCR) in human blood samples is compared with microscopical examination of thin and thick blood films in 2 field trials in C?te d'Ivoire and Cameroon. Also, dried blood spots or liquid blood collected from Dutch soldiers returning from Goma, Zaire (n = 141), Angola (n = 40), and from Marechaussee (Dutch border police) returning from various parts of the world (n = 161) were examined, together with miscellaneous other material obtained from laboratories and hospitals. The method is based on features of the small subunit nuclear ribosomal ribonucleic acid (RNA) gene (ssrDNA), a multicopy gene which possesses both highly conserved domains and domains characteristic for each of the 4 human malaria parasites. The first reaction of the SnM-PCR includes a universal reverse primer with 2 forward primers specific for Plasmodium and mammals, respectively. The mammalian-specific primer was included as a positive control to distinguish uninfected cases from simple PCR failures. The second PCR reaction includes a Plasmodium-specific forward primer plus species-specific reverse primers for P. vivax, P. ovale, P. falciparum and P. malariae. The technique worked better with samples collected in the field as dried blood spots on filter paper and heparinized blood rather than with frozen pelleted blood; it was more sensitive and more specific than the standard microscopical examination.     

鉴别脑膜炎奈瑟菌A、B、C、Y、W135群的多重聚合酶链反应诊断方法

目的建立一种能鉴别脑膜炎奈瑟菌（Nm）A、B、C、Y、W1355个血清群的多重聚合酶链反应（PCR）方法.方法首先PCR扩增Nm crgA基因,确定Nm感染;再通过多重PCR扩增荚膜表达基因orf-2和siaD基因,根据特异条带区分不同群Nm.结果多重PCR检测61株Nm与4株非Nm,能准确地鉴定并可将Nm分成5个血清群;检测4例流行性脑脊髓膜炎患者的脑脊液,2例出现A群400 bp的特异条带,而细菌培养和胶乳凝集方法检测均为阴性;检测18份流行性乙型脑炎患者标本,与胶乳凝集试验结果一致.结论多重PCR诊断方法能正确鉴别不同群Nm,对流行性脑脊髓膜炎的临床诊断与流行病学调查有重要意义.     

Application of Multiplex PCR for Detection and Differentiation of Entamoeba histolytica,Entamoeba dispar and Entamoeba moshkovskii

Background

Entamoeba moshkovskii and E. dispar are impossible to differentiate microscopically from the pathogenic species E. histolytica. Multiplex polymerase chain reaction (Multiplex PCR) is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment.

Methods

For detection and differentiation of the three-microscopy indistinguishable Entamoeba species in human, multiplex PCR assay using different DNA extraction methods was studied. A conserved forward primer was derived from the middle of the small-subunit rRNA gene, and reverse primers were designed from signature sequences specific to each of these three Entamoeba species.

Results

A 166-bp PCR product with E. histolytica DNA, a 580-bp product with E. moshkovskii DNA and a 752-bp product with E. dispar DNA were generated in a single-round and multiplex PCR reaction.

Conclusion

We recommend this PCR assay as an accurate, rapid, and effective diagnostic method for the detection and discrimination of these three Entamoeba species in both routine diagnosis of amoebiasis and epidemiological surveys.     

Differences in automated depolarization patterns of Plasmodium falciparum and P. vivax malaria infections defined by the Cell-Dyn CD4000 haematology analyser

Of 1014 samples submitted for full blood count analysis and malaria screening, 854 were designated malaria-negative by blood film microscopy, 79 were unequivocally identified as Plasmodium vivax and 81 as P. falciparum. All samples were additionally analysed with the Abbott Cell-Dyn CD4000 haematology instrument, and leucocyte differential plots of 90 degrees polarized vs. 90 degrees depolarized (NEU-EOS plot) and 90 degrees depolarized vs. 0 degree light (EOS I plot) scatter were specifically examined for abnormal depolarization patterns. Depolarization pattern types were correlated with microscopy (species) results, and these correlations were consolidated by polymerase chain reaction analysis. All 854 microscopically-designated malaria-negative samples showed a type 1 (normal) CD4000 depolarization pattern. Abnormal pattern types 2, 3a and 3b were entirely restricted to one of the two malaria categories. Plasmodium falciparum malaria showed two CD4000 pattern types only; a 'normal' type 1 pattern was seen in 36/75 (48%) cases and the remaining 39 cases were all abnormal pattern type 3a. In contrast, most (79/85) P. vivax malaria cases showed a distinctive clustered EOS I population (types 2 and 3b patterns) that was not seen with P. falciparum. Automated depolarization analysis provides an effective means of detecting malaria-associated haemozoin, and the patterns of intracellular haemozoin further appear to provide species differentiation between P. falciparum and P. vivax.