SYBR Green实时PCR检测恶性疟原虫和间日疟原虫的临床检验技术研究

目的建立鉴别恶性疟和间日疟的SYBR Green实时PCR检测方法。方法针对恶性疟原虫和间日疟原虫18S rRNA基因设计引物,优化引物浓度与退火温度,建立可扩增出两种疟原虫基因片段的SYBR Green实时PCR,并进行54例临床标本检测,其中恶性疟32例、间日疟22例。以镜检法为金标准分析敏感度和特异度等指标。结果该方法可扩增出恶性疟原虫和间日疟原虫18S rRNA基因片段,并能检出混合感染,特异性好。该方法检测恶性疟原虫或间日疟原虫的敏感度为97.30%(36/37),特异度为5.88%(1/17);检测恶性疟原虫,敏感度为87.50%(21/24),特异度为63.33%(19/30);检测间日疟原虫,敏感度为69.23%(9/13),特异度为68.29%(28/41)。结论所建立的SYBR Green实时PCR方法能较准确地诊断疟疾并鉴别虫种,敏感度高,在混合感染的诊断方面具有优越性。     

套式/多重PCR方法应用于疟疾诊断与监测的初步评价

目的 与镜检法比较评价标签引物-套式/多重PCR（UT-PCR）在疟疾监测中的应用价值。 方法 在海南、云南省恶性疟和间日疟混合流行区和广西疟疾控制区的疟疾监测中,采集初诊为疟疾或疑似疟疾的发热患者的血片与滤纸血样400份,在双盲条件下比较镜检法与UT-PCR的初检结果,对结果不一致的血片再次镜检复查,同时对其滤纸血样重复PCR 2～3次;评估UT-PCR与镜检法的敏感性和特异性。 结果 400例发热患者血样中,镜检法初检检出疟原虫阳性234例,其中恶性疟125例,间日疟109例;UT-PCR检出疟原虫阳性235例,其中恶性疟124例,间日疟109例;恶性疟和间日疟混合感染2例。两法初检结果一致的血样占92.5%（370/400）,其中阴性154例,阳性216例（间日疟117例,恶性疟99例）。复查25份初检结果不一致的血样,包括镜检阴性PCR阳性11例,镜检阳性PCR阴性10例,镜检为恶性疟PCR为间日疟3例,镜检为间日疟而PCR为混合感染1例,其中15份与UT-PCR的初检结果一致,7份“假阳性”原因不明,仅3份为PCR的假阴性。根据复查结果评估PCR的敏感性为99.6%,特异性为98.8%。 结论 采用更敏感的UT?鄄PCR疟疾诊断方法有助于解决疟疾诊断与鉴别诊断中的疑难问题,提高疟疾监测的质量和效率。     

云南省瑞丽市景颇族、汉族和傣族的疟疾发病观察

瑞丽市位于云南省西南部,是一个多民族混合居住的边境地区。人口8万余人。为了解不同民族的疟疾发病情况,于1994—1996年进行了调查。方法与结果瑞丽市卫生防疫站对就诊的当地景颇族、汉族和傣族“四热病人”均采耳垂血检查;各乡卫生院、卫生所的血检标本均送市卫生防疫站鉴定后进行分类统计。结果:景颇族1471例,检出疟原虫阳性166例,阳性率为11.3%。其中间日疟85例,恶性疟80例,混合感染1例;傣族2573例,检出疟原虫阳性189例,阳性率为7.3%。其中间日疟98例,恶性疟89例,混合感染2例;汉族共血检7119例,检出疟原虫阳性196例,阳性率为2.8%。其中…     

PCR检测疟疾混合感染的现场应用研究

目的　评价PCR检测技术在间日疟与恶性疟混合感染区诊断疟疾的现场应用价值。　方法　采集海南省疟疾混合感染流行区 3 0 4份滤纸干血滴样本 ,根据红内期疟原虫SSUrRNA基因序列 ,设计合成 3条引物 ,采用PCR技术在同一反应体系中扩增出间日疟原虫和恶性疟原虫不同的DNA片段 ,检测现场所采样本中的间日疟原虫或恶性疟原虫DNA ;同时与镜检法进行比较。　结果　在 3 0 4份样本中 ,PCR法阳性 15份 ,其中间日疟 7份 ,恶性疟 8份 ;镜检法阳性11份 ,其中间日疟 6份 ,恶性疟 5份。镜检阳性的样本PCR均为阳性 ;镜检阴性而PCR阳性的 4份样本 ,其扩增产物经限制性酶切鉴定 ,证实为间日疟原虫或恶性疟原虫DNA。　结论　此PCR检测体系灵敏、特异 ,对诊断或鉴别诊断间日疟原虫和恶性疟原虫混合感染具有实用价值。     

PCR检测疟疾混合感染的现场应用研究

目的 评价PCR检测技术在间日疟与恶性疟混合感染区诊断疟疾的现场应用价值。方法 采集海南省疟疾混合感染流行区304份滤纸干血滴样本，根据红内期疟原虫SSUrRNA基因序列，设计合成3条引物．采用PCR技术在同一反应体系中扩增出间日疟原虫和恶性疟原虫不同的DNA片段，检测现场所采样本中的间日疟原虫或恶性疟原虫DNA；同时与镜检法进行比较。结果 在304份样本中，PCR法阳性15份，其中间日疟7份．恶性疟8份；镜检法阳性11份，其中间日疟6份，恶性疟5份。镜检阳性的样本PCR均为阳性；镜检阴性而PCR阳性的4份样本，其扩增产物经限制性酶切鉴定，证实为间日疟原虫或恶性疟原虫DNA。结论 此PCR检测体系灵敏、特异．对诊断或鉴别诊断间日疟原虫和恶性疟原虫混合感染具有实用价值。     

PCR鉴别诊断间日疟及恶性疟的研究

目的 :在同一反应体系中建立能鉴别诊断间日疟与恶性疟的 PCR检测方法。方法 :根据红内期疟原虫 SSUr RNA编码基因序列 ,设计合成 3个引物 ,采用聚合酶链反应技术 ,在同一反应体系中 ,间日疟原虫和恶性疟原虫分别预期被扩增出 34 1bp和 4 31bp的 DNA条带。结果 :间日疟原虫和恶性疟原虫模板在同一反应体系中分别被扩增出预期大小的 DNA片段 ,并经限制性酶切证实 ;杜氏利什曼原虫、弓形虫、健康人血及空白对照均无特异性扩增条带 ;以该检测体系至少可检出原虫血症为 2 .56× 10 ^-7间日疟原虫感染和 1.0 8× 10 ^-5恶性疟原虫感染 ;69份镜检阳性的间日疟和 2份恶性疟患者血样 PCR检测均为阳性 ,1例发热待查患者PCR诊断为间日疟 ,与镜检复查结果一致 ,所有疟疾阳性标本中未检出混合感染 ,2 0份健康人血 PCR均为阴性。结论 :该检测体系灵敏、特异 ,对于诊断间日疟和恶性疟以及鉴别间日疟原虫和恶性疟原虫混合感染具有一定的应用价值。     

2017年江苏省疟疾诊断参比实验室样本检测结果分析

目的 分析2017年江苏省疟疾诊断参比实验室样本检测结果,为巩固江苏省疟疾诊断水平提供科学依据。方法 由江苏省疟疾诊断参比实验室收集2017年疟疾网报病例诊断结果和样本;对每份病例样本分别采用镜检、核酸检测复核和疟疾快速诊断试纸条（RDT）检测;比较分析不同地区和不同疟原虫虫种样本检测结果符合情况。结果 2017年江苏省共有疟疾网报病例242例,经复核确定疟疾病例共239例,其中恶性疟163例、间日疟21例、三日疟11例、卵形疟43例、恶性疟和卵形疟原虫混合感染1例。13个设区市疟疾网报病例的诊断符合率均＞80%,总符合率为88.8%;恶性疟原虫、间日疟原虫、三日疟原虫、卵形疟原虫检测符合率分别为98.8%、57.1%、63.6%、81.4%,RDT对4种疟原虫感染检出率分别为95.7%、85.0%、63.6%、79.1%。结论 2017年江苏省疟疾网报病例诊断质量总体较高,对非恶性疟人体疟原虫的虫种鉴别能力有待提高,RDT对非恶性疟人体疟原虫感染检测效果不理想。在当前消除疟疾阶段,应加强和保持各部门的疟疾诊断能力。     

体外培养的食蟹猴疟原虫用于间日疟的间接荧光抗体实验

培养的恶性疟原虫作为抗原用于间接荧光抗体实验(IFA)检出感染恶性疟患者的血清疟疾抗体灵敏度较高,但检出感染间日疟后血清中的抗体则灵敏度低或不理想。早巳证实与间日疟原虫相对应的食蟹猴疟原虫可用于IFA测试感染间日疟后的血清抗体,本文报道了用培养的食蟹猴疟原虫作抗原用于IFA测试间日疟病人血清中的抗体。 抗原片的制作:液氮保存已培养70天的食蟹猴疟原虫(PcC)复苏后再培养28天,按照Sulzer等的方法制成抗原片。将培养原虫去上清液后,用     

两种检测方法在间日疟和卵形疟诊断中的应用分析

目的分析疟原虫镜检和巢式PCR检测间日疟和卵形疟效果。方法收集2012～2016年输入性间日疟和卵形疟病例血样,巢式PCR检测疟原虫ssRNA基因,并与显微镜检结果进行比对。结果显微镜检和巢式PCR检测71份血样,间日疟、卵形疟、混合感染分别占74.6%、25.4%、0%和63.4%、29.6%、7%,符合率为81.7%;亚洲23份血样,镜检与巢式PCR均为间日疟,符合率为100%,巢式PCR检测非洲血样48份,间日疟、卵形疟和混合感染分别占45.8%、43.8%和10.4%,镜检与巢式PCR符合率为72.9%;巢式PCR检测26份卵形疟,经典curtisi、变种wallikeri和混合感染分别占80.8%、11.5%和7.7%,检出变种wallikeri总数占卵形疟19.2%。结论巢式PCR检测疟原虫虫种鉴别优于传统镜检法,可提高疟疾病例诊断水平。     

福建省3例输入性卵形疟的实验诊断分析

目的 随着输入性疟疾的增加,通过镜检结合PCR方法,提高检出率。方法 福建省2013年成立基因诊断参比实验室,用PCR方法回顾性筛查过往病例时发现有3例卵形疟原虫误诊为间日疟。结果 镜检结合PCR扩增和序列分析发现FJ1、FJ2为卵形疟亚种curtisi和恶性疟混合感染,FJ3为单一卵形疟亚种curtisi感染。结论 福建省以往卵形疟报道很少,需要加强诊断培训,镜检结合PCR检查提高检出率。     

High prevalence of malaria infection in Amazonas State, Venezuela

This study was carried out to determine the incidence of malaria in an endemic region of Amazonas State, Venezuela. For this, 200 random samples were collected from symptomatic and asymptomatic individuals from San Fernando de Atabapo and Santa Barbara. Epidemiological factors were related to malaria infection, which was diagnosed by microscopy observation and amplification of the 18S rDNA sequence by PCR. Malaria prevalence in these populations was 28.5%, whilst P. vivax and P. falciparum prevalences were 12 and 17%, respectively. No infection by P. malariae was found. A mixed infection was found on an asymptomatic individual. Prevalence patterns differed between age groups depending on the Plasmodium species. We found that 34.8% of the P. vivax and 15.2% of the P. falciparum infections were asymptomatic. The use of nets was helpful to prevent P. vivax infection, but did not protect against P. falciparum infection. The results suggest the presence of more than one mosquito vector in the area, displaying a differential pattern of infection for each Plasmodium species. There appear to be risk factors associated with malaria infections in some individuals. The population based approach and PCR diagnosis improved the accuracy of the statistical analysis in the study.     

Application of real-time polymerase chain reaction (PCR) analysis for detection and discrimination of malaria parasite species in Thai patients

A TaqMan real-time PCR system was used to detect and discriminate the 4 species of human malaria parasites in clinical blood samples. A 150-base pair (bp) region of the small subunit ribosomal RNA (SSU rRNA) gene of each malaria parasite, including species-specific sequences to be detected by TaqMan probe, was used as a target for PCR analysis. The PCR method used universal primers and species-specific TaqMan probes for Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. The detection threshold for the method, as determined with serial dilution of cultured P. falciparum-infected erythrocytes, was 5 parasite-infected erythrocytes per reaction. Fifty blood samples of falciparum malaria and a second set of 50 samples of vivax malaria, diagnosed by microscopic examination at the Hospital for Tropical Diseases, Mahidol University, Thailand, were analyzed by real-time PCR. In the 50 samples of microscopically-diagnosed falciparum malaria, 40 were regarded as P. falciparum single infection, 7 were P. falciparum and P. vivax mixed infections, and 3 were P. vivax single infection by real-time PCR. In the second set of 50 samples of microscopically diagnosed vivax malaria, all were considered P. vivax single infection by PCR. Neither P. ovale nor P. malariae infection was identified in the 100 blood samples. Real-time PCR analysis was shown to be more sensitive and accurate than routine diagnostic methods. Application and extension of the PCR method reported here will provide a powerful tool for further studies of malaria.     

Evaluation of the Binax NOW ICT test versus polymerase chain reaction and microscopy for the detection of malaria in returned travelers

Microscopic detection of Plasmodium species has been the reference standard for the diagnosis of malaria for more than a century. However, maintaining a sufficient level of expertise in microscopic diagnosis can be challenging, particularly in non-endemic countries. The objective of this study was to compare a new rapid malaria diagnostic device (NOW ICT Malaria Test; Binax, Inc., Portland, ME) to polymerase chain reaction (PCR) and expert microscopy for the diagnosis of malaria in 256 febrile returned travelers. Compared with PCR, the NOW ICT test showed a sensitivity of 94% for the detection of P. falciparum malaria (96% for pure P. falciparum infection) and 84% for non-P. falciparum infections (87% for pure P. vivax infections and 62% for pure P. ovale and P. malariae infections), with an overall specificity of 99%. The Binax NOW ICT may represent a useful adjunct for the diagnosis of P. falciparum and P. vivax malaria in febrile returned travelers.     

Post-arrival screening for malaria in asymptomatic refugees using real-time PCR

Malaria is a significant health risk to refugee populations originating from endemic areas, but there is little consensus on screening and/or treatment approaches for malaria in this population. Furthermore, detection of malaria in semi-immune asymptomatic refugees is limited by the sensitivity of diagnostic tests used for screening. We determined the prevalence of malaria by microscopy and real-time polymerase chain reaction (PCR) in a consecutive population of 324 asymptomatic refugees examined in Edmonton, Canada, during 2009-2010. Although all thick and thin blood smear results were negative, 10 subjects (3.1%) tested PCR positive for Plasmodium DNA. Interestingly, 6 of 10 PCR positive subjects are at risk of malaria relapse by P. vivax or P. ovale infections. These results suggest that appropriate guidelines for malaria screening should consider the risk of relapsing infections, and they highlight the potential usefulness of real-time PCR in the diagnosis of asymptomatic malaria.     

Performance of the OptiMAL assay for detection and identification of malaria infections in asymptomatic residents of Irian Jaya, Indonesia

The OptiMAL assay, a new immunochromatographic "dipstick" test for malaria based on detection of Plasmodium lactate dehydrogenase (pLDH), is purported to detect infections of approximately 200 parasites/microL of blood and to differentiate between Plasmodium falciparum and non-P. falciparum. We evaluated OptiMAL performance by comparing the test strip interpretations of two independent readers with consensus results obtained independently by expert malaria microscopists. Unbiased measures of sensitivity were derived by applying the OptiMAL test for detection and differentiation of light, asymptomatic infections by P. falciparum and Plasmodium vivax. OptiMAL readings were separated in time to determine whether the reaction signal was stable. Microscopy identified infections in 225 of 505 individuals screened; those with P. falciparum (n = 170) averaged 354 asexual forms/microL and P. vivax/Plasmodium malariae (n = 112) averaged 216 asexual forms/microL of blood. Concordance between OptiMAL and microscopy was 81% and 78% by the two independent readings. The assay's sensitivity for detection of any malaria species was 60.4% and 70.2% respectively and specificity was 97% and 89%. Most cases identified by microscopy as P. falciparum were graded as negative or non-falciparum by both OptiMAL readers. OptiMAL false negatives as well as misidentifications were related to low parasitemias (< 500/microL). The OptiMAL assay demonstrated 88-92% sensitivity for detecting infections of 500-1,000 parasites/microL, a range covering the mean parasitemia of primary symptomatic P. falciparum infections in malaria-na?ve Indonesian transmigrants. This device was markedly less sensitive than expert microscopy for discriminating between malaria species and is presently unsuited for use as an epidemiological screening tool. The OptiMAL assay is not approved for diagnostic use but is commercially available for research purposes only.     

Clinical features of children hospitalized with malaria--a study from Bikaner, northwest India

Severe Plasmodium vivax malaria in adults has been reported from Bikaner (northwestern India) but the reports on children are scanty. This prospective study was done on 303 admitted children of malaria. The diagnosis was done by peripheral blood smear and rapid diagnostic test. Further confirmation of severe P. vivax monoinfection was done by polymerase chain reaction (PCR). The proportion of P. falciparum, P. vivax, and mixed (P. falciparum and P. vivax) infection was 61.01%, 33.99%, and 4.95%, respectively. Severe disease was present in 49.5% (150/303) children with malaria, with the risk greatest among P. vivax monoinfection (63.1% [65/103]) compared with P. falciparum, either alone (42.7% [79/185]; odds ratio [OR] = 2.3 [95% confidence interval (CI) = 1.40-3.76], P = 0.001) or mixed infections (40% [6/15]; OR = 2.57 [95% CI = 0.88-7.48]). In children < 5 years of age, the proportion of severe malaria attributable to P. vivax rose to 67.4% (31/46) compared with 30.4% (14/46) of P. falciparum (OR = 4.7 [95% CI = 2.6-8.6], P < 0.0001) and 2.2% (1/46) of mixed infection (OR = 92 [95% CI = 24.6-339.9], P < 0.0001). The proportion of patients having severe manifestations, which included severe anemia, thrombocytopenia, cerebral malaria, acute respiratory distress syndrome, hepatic dysfunction, renal dysfunction, abnormal bleeding was significantly high in association with P. vivax monoinfection in 0-5 year age group, while the same was significantly high in association with P. falciparum monoinfection in 5-10 year age group. Similarly P. vivax monoinfection had greatest propensity to cause multiorgan dysfunction in 0-5 year age group (34.1% [17/41], P < 0.0001) in comparison to P. falciparum monoinfection, which had similar propensity in 5-10 year age group (36.8% [35/95], P = 0.039). Plasmodium vivax monoinfection was almost equally serious to cause significant mortality in comparison to P. falciparum (case fatality rate of severe P. vivax was 3.9% versus 3.2% of severe P. falciparum malaria; P = 1.0). This study reaffirms the evidence of severe P. vivax malaria in children in Bikaner.     

Performance appraisal of rapid on-site malaria diagnosis (ICT malaria Pf/Pv test) in relation to human resources at village level in Myanmar.

Logistic, economic and technical factors limit rapid access to microscopic confirmation of symptomatic diagnosis of malaria in many rural areas in endemic countries such as Myanmar. A study was conducted to evaluate a rapid on-site immunochromatographic test (ICT Malaria Pf/Pv) for detection of Plasmodium falciparum and P. vivax in two villages in the Taikkyi region of Myanmar. The ICT Malaria tests were performed by a volunteer health worker (VHW) in Yae-Aye-San village and by a professionally trained midwife (MW) in Kankone village. A total of 1000 symptomatic patients participated in the study by providing blood samples for an ICT test and for microscopy. The ICT performance indices, relative to microscopy, were better for the trained MW compared with the less experienced VHW. For P. falciparum and/or P. vivax infections, the sensitivities were 82.7% for the VHW compared with 93.7% for the MW. For P. falciparum infections, the sensitivities were 82.2% for the VHW and 91.3% for the MW, while the corresponding values for P. vivax infections were 66.7 and 79%, respectively. Although the test kit appeared to perform better in more experienced hands, this study questions whether this difference is related to the use of the ICT Malaria Pf/Pv test kit, or related to other factors such as differences in the quality of blood slides prepared by the VHW and MW for microscopic examination. Overall, the results suggest that a rapid diagnostic assay such as the ICT Malaria Pf/Pv test kit can be used in rural settings by relatively inexperienced persons, such as VHWs, with a reasonable degree of sensitivity, thus providing on-site confirmation of symptomatic diagnosis of malaria.     

Endemic malaria in the Peruvian Amazon region of Iquitos

A cross-sectional study was conducted in the Peruvian Amazon to test the hypothesis that a reservoir of asymptomatic malaria parasitemic patients would form the basis for continuing malaria endemicity in the region. Active surveillance yielded a Plasmodium spp. slide-positive prevalence of 4.2% (43 of 1,023) and a polymerase chain reaction (PCR)-positive prevalence of 17.6% (144 of 819). Plasmodium vivax prevalence was 2.9% and 14.2% while Plasmodium falciparum prevalence was 1.3% and 2.6% by microscopy and PCR, respectively. Approximately two-thirds of slide-positive and one-fourth of PCR-positive people were symptomatic. Anemia was associated with slide positivity (P < 0.001) and PCR positivity for P. falciparum (P = 0.003). Sensitivity of field microscopy and agreement between field and reference laboratory microscopists were low, arguing for using PCR for epidemiologic investigation and malaria control. While these data confirm recent findings from the Brazilian Amazon suggesting that sufficient numbers of asymptomatic malaria parasitemic patients are present to form a persistent reservoir for continuous reinfection within the Peruvian Amazon region, these results also indicate that clinical immunity in human populations can be driven in malaria-endemic regions that do not have high intensity malaria transmission.