多重PCR种特异性检测恶性疟原虫和间日疟原虫方法的建立

目的建立恶性疟原虫和间日疟原虫种特异性检测的多蕈PCR方法,用于疟疾的检测和诊断.方法根据疟原虫18S核糖体小亚基ssRNA的基因序列设计合成8对11条引物,通过对恶性疟、间日疟患者及健康对照者血样的DNA进行扩增,选择出敏感性和特异性最佳的引物用于建立多重PCR方法,并用梯度变化的方法分别对引物浓度、复性温度、延伸温度和循环次数等反应参数进行比较分析,优化PCR反应条件.利用优化后的多重PCR埘采自云南和上海的139份疟疾患者血样和32份非疟疾患者血样进行检测,以镜检方法为金标准,分析多重PCR方法检测患者血样的敏感性和特异性.结果从8对11条引物中优选出2对共3条引物用于建立多重PCR.利用这3条引物进行多重PCR,一次反应即可完成对恶性疟原虫和间日疟原虫的种特异性鉴定.对疟疾和非疟疾患者血样检测结果显示,该方法检测患者血样的敏感性为97.8%,特异性为100%.结论多重PCR方法敏感、特异、可进行批量检测,适用于对人群的疟疾监测和疑似疟疾病例的诊断,并能鉴定恶性疟原虫和间日疟原虫虫种.     

多重PCR快速检测恶性疟和间日疟的研究

目的 建立一种简便快速、能同时检测恶性疟和间日疟的核酸检测方法。方法 针对两种疟原虫18S rRNA基因设计2对(3条引物),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多重PCR。并进行最低检测限确定和临床标本检测,以镜检法为金标准分析灵敏度和特异度等指标。结果 该方法可扩增出431 bp(恶性疟原虫)和341 bp(间日疟原虫)基因片段,最低检测限为10²copies/反应,检测临床标本的结果与镜检法无差别(P＞0.05),敏感度为93.55%,特异度为70.83%,阳性预测值为89.23%,阴性预测值为80.95%。结论 所建立的多重PCR方法可快速检测疟疾感染并鉴别分型,灵敏度高,值得推广。     

A novel real-time PCR assay for the detection of Plasmodium falciparum and Plasmodium vivax malaria in low parasitized individuals

The rapid, accurate diagnosis of Plasmodium spp. is essential for the effective control of malaria, especially in asymptomatic infections. In this study, we developed a sensitive, genus-specific, real-time quantitative PCR assay. It was compared with the microscopic examination of Giemsa-stained blood smears and two different molecular diagnostic techniques: nested PCR and multiplex PCR. For the effective quantitative detection of malaria parasites, all reagents were designed with a lyophilized format in one tube. Plasmodium was detected successfully in all 112 clinically suspected malaria patients, including 32 individuals with low parasitemia (1-100 parasites/μl). The sensitivity threshold was 0.2 parasites/μl and no PCR-positive reaction occurred when malaria parasites were not present. This may be a useful method for detecting malaria parasites in endemic areas.     

Polymerase chain reaction detection of rearranged immunoglobulin heavy chain DNA in plasma samples is useful in the diagnosis of B-cell lymphoma

Using DNA extracted from plasma samples of B-cell non-Hodgkin's lymphoma (B-NHL) patients, we attempted to detect the monoclonal rearrangement of immunoglobulin heavy chain gene by amplifying complementarity-determining region 3 (CDR3) by semi-nested polymerase chain reaction (PCR) method (plasma PCR). In 19 of 37 (51%) cases, clonal DNA was detected. With the same PCR method using DNA extracted from peripheral blood mononuclear cells, clonal DNA was detected in 8 of the 37 cases (22%). These 8 were in advanced stages with bone marrow (BM) invasion mostly. On the other hand, the 19 positive cases by plasma PCR included those in early stages without BM invasion or with normal soluble interleukin-2 receptor (sIL-2R) and lactate dehydrogenase (LDH) values. In 15 healthy volunteers, plasma PCR showed no clonal DNA. In cases in which tumor biopsy was difficult to perform, plasma PCR was helpful for determining whether or not the tumor was B-NHL. Plasma PCR is simple and has high specificity, although its sensitivity is insufficient.     

The indirect hemagglutination test for malaria. Evaluation of antigens prepared from Plasmodium falciparum and Plasmodium vivax.

Soluble antigens were prepared from Plasmodium falciparum and P. vivax and were evaluated in the indirect hemagglutination test. These antigens, attached to aldehyde-fixed type "O" erythrocytes, detected antibodies in more than 91% of infections with the homologous Plasmodium species. Detection rates in infections caused by the heterologous species ranged from 72% to 76%. Positive reactions occurred in less than 2% of sera from persons without malaria infection.     

Semi-nested, multiplex polymerase chain reaction for detection of human malaria parasites and evidence of Plasmodium vivax infection in Equatorial Guinea

A semi-nested, multiplex polymerase chain reaction (PCR) based on the amplification of the sequences of the 18S small subunit ribosomal RNA (ssrRNA) gene was tested in a field trial in Equatorial Guinea (a hyperendemic focus of malaria in west central Africa). The method uses a primary PCR amplification reaction with a universal reverse primer and two forward primers specific for the genus Plasmodium and to mammals (the mammalian-specific primer was included as a positive control to distinguish uninfected cases from inhibition of the PCR). The second amplification is carried out with the same Plasmodium genus-specific forward primer and four specific reverse primers for each human Plasmodium species. The PCR amplified products are differentiated by fragment size after electrophoresis on a 2% agarose gel. Four villages from three regions of the island of Bioko (Equatorial Guinea) and two suspected Plasmodium vivax-P. ovale infections from the hospital of Malabo were tested by microscopy and PCR. The PCR method showed greater sensitivity and specificity than microscopic examination and confirmed the existence of a focus of P. vivax infections in Equatorial Guinea suspected by microscopic examination. It also provided evidence of several mixed infections, mainly P. falciparum and P. malariae, the two predominant species causing malaria in Equatorial Guinea.     

Polymerase chain reaction using noninvasively obtained samples, for the detection of Leishmania infantum DNA in dogs

A polymerase chain reaction (PCR) procedure using noninvasively obtained samples, for the identification of Leishmania infantum in canine tissues, was evaluated and compared with serologic testing and culture. A total of 92% of naturally infected, symptomatic, seropositive dogs were found to be positive by use of DNA from conjunctival swabs. Spleen or lymph node aspirates were found to be positive by PCR in 86% and by culture in 74% of these dogs. The sensitivity and specificity of conjunctival PCR were 92% and 100%, respectively. Experimentally infected dogs were found to be positive by conjunctival PCR already at 45 days of infection (83%) and before seroconversion. PCR using noninvasively obtained conjunctival samples will be useful for epidemiological studies and for direct diagnosis of canine visceral leishmaniasis.     

Paroxysm serum from a case of Plasmodium vivax malaria inhibits the maturation of P. falciparum schizonts in vitro

In concurrent infections in vivo, the blood stages of Plasmodium vivax suppress those of Plasmodium falciparum. To see if the paroxysm (i.e. the periodic febrile episode) of P. vivax infection contributes to this suppression, sera from a P. vivax-infected volunteer were added to cultures of whole blood taken from cases of P. falciparum malaria. The crude 'rate' of schizont generation from the ring forms, measured as the percentage of all asexual parasites that were schizonts after incubation for 24 h, was similar whether the cultures contained serum samples collected during paroxysms or those collected, from the same volunteer, at other times (19.1% v. 18.9%; P=0.842). After a random-effect linear regression was used to adjust for disparities between the P. falciparum isolates, however, the degree of schizont maturation, measured as the mean number of nuclei per schizont, was significantly lower for the cultures with 'paroxysm serum' than for those with 'non-paroxysm serum' (4.8 v. 5.3; P=0.002). The proportion of schizonts considered mature was also significantly lower when 'paroxysm serum' was used (3.7% v. 6.3%: P=0.03). This appears to be the first in-vitro study in which sera collected during a paroxysm of P. vivax have been shown to inhibit the maturation of P. falciparum schizonts. The role of this mechanism in intra- and inter-specific competition is discussed.     

Plasmodium falciparum: detection and strain identification of Indian isolates by polymerase chain reaction

The polymerase chain reaction (PCR) was employed for detection and strain identification of P. falciparum in a comparative field study of Indian isolates. The primers were selected from highly conserved regions flanking the variable, tandemly repeated regions of highly polymorphic cell surface antigens, major merozoite surface antigen-1 (MSP-1), major surface antigen-2 (MSP-2), circumsporozoite surface antigen (CSP) and ring-infected erythrocyte surface antigen (RESA). Out of the 52 microscopically positive P. falciparum infected field samples, 47 samples were positive by PCR. Variation in the size of the amplified products was observed using MSP-1, MSP-2 specific primers respectively in different field isolates of P. falciparum, but CSP and RESA did not exhibit any variation in size of the amplified product. The multiplex PCR results demonstrated that amplified products from these surface antigens vary in size and there is a specific pattern for each strain and this could be utilized to identify a particular field isolate. One P. falciparum infected field sample detected by the above PCR method was found to be a mixed infection by two different strains. Five microscopically positive P. vivax infeced samples were also analyzed by PCR method using P. falciparum cell surface antigen (MSP-2) specific primers. PCR results showed one P. vivax infected sample was positive when P. falciparum specific primers were used, this could be due to inaccurate and reduced limit of detection of Plasmodial species by microscopic examination.     

Thrombocytopenia in Plasmodium falciparum,Plasmodium vivax and mixed infection malaria: A study from Bikaner (Northwestern India)

The occurrence, relation and magnitude of thrombocytopenia in different species of malaria are not clearly defined. This study included 1,064 patients admitted with malaria to study thrombocytopenia (platelet count <150,000 /cumm) in Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) mono infection and mixed infection (Pf?+?Pv). The species diagnosis was done by peripheral blood film (PBF) and rapid diagnostic test (RDT). Validation by polymerase chain reaction (PCR) was done only in patients with severe thrombocytopenia (platelet count <20,000 /cumm). The breakup of patients was 525 (49.34%) Pf, 460 (43.23%) Pv and 79 (7.42%) mixed malaria (Pf?+?Pv). Thrombocytopenia was observed in 24.6% (262/1064) patients. The risk was greatest in the mixed infections in comparison to monoinfection individually (43.04% [34/79]; mixed vs Pv monoinfection: Odds Ratio [OR]?=?1.675 [95% Confidence Interval (CI) 1.029–2.726], p?<?0.0366; mixed vs Pf monoinfection: OR=3.911 [95% CI 2.367–6.463], p?<?0.0001). Pv monoinfection (31.09% [143/460]) had greater risk compared to Pf monoinfection (16.19% [85/525]; OR?=?2.335 [95% CI 1.722–3.167], p?<?0.0001). The occurrence of severe thrombocytopenia was also higher in Pv monoinfection (18.18% [26/143]) in comparison to either Pf monoinfection (10.59% [9/85], OR?=?1.877 (95% CI 0.834–4.223)) or mixed infection (11.76% [4/34]; OR?=?1.667 (95% CI 0.540–5.142) but this association was statistically not significant. Six patients (3 Pv, 2?Pf and 1 mixed) developed severe epistaxis requiring platelet transfusion. There was no relation between parasite density and platelet count as many patients with severe thrombocytopenia had parasite density similar to patients without thrombocytopenia. We found that the association of thrombocytopenia was statistically more significant with P. vivax monoinfection as compared to P. falciparum.     

Thrombocytopenia in Plasmodium falciparum, Plasmodium vivax and mixed infection malaria: a study from Bikaner (Northwestern India)

The occurrence, relation and magnitude of thrombocytopenia in different species of malaria are not clearly defined. This study included 1,064 patients admitted with malaria to study thrombocytopenia (platelet count <150,000 /cumm) in Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) mono infection and mixed infection (Pf?+?Pv). The species diagnosis was done by peripheral blood film (PBF) and rapid diagnostic test (RDT). Validation by polymerase chain reaction (PCR) was done only in patients with severe thrombocytopenia (platelet count <20,000 /cumm). The breakup of patients was 525 (49.34%) Pf, 460 (43.23%) Pv and 79 (7.42%) mixed malaria (Pf?+?Pv). Thrombocytopenia was observed in 24.6% (262/1064) patients. The risk was greatest in the mixed infections in comparison to monoinfection individually (43.04% [34/79]; mixed vs Pv monoinfection: Odds Ratio [OR]?=?1.675 [95% Confidence Interval (CI) 1.029-2.726], p?相似文献   

Infectious reservoir of Plasmodium vivax and Plasmodium falciparum malaria in an endemic region of Sri Lanka

The infectious reservoir of Plasmodium vivax and P. falciparum in a malaria endemic region in Sri Lanka was defined in a population of 3,625 by directly feeding mosquitoes on a sample of infected individuals during a period of 17 months. The malaria case incidence in this population was concurrently monitored. P. vivax gametocyte densities were highest in the youngest age groups, and decreased steadily with increasing age. However, the infectivity per gametocyte appeared to be lower in the younger age groups than in the older ones. There was no significant correlation between the age of patients and their gametocyte densities for P. falciparum, to which this population was only recently exposed, nor was there a discernible trend in the infectivity per gametocyte in different age groups. The average infectivity of patients was lowest in the youngest (0-5 years) and the oldest (greater than 50) age groups. The contribution made by P. vivax patients in the different age groups to the reservoir of infection was estimated. Patients in the 6-25 year age groups made the largest contribution to the reservoir, followed by those in the 26-50 year age group. Patients in the youngest and the oldest age groups contributed least to the infectious reservoir. When population sizes in the different age groups were taken into consideration, the age groups between 6 and 50 years contributed almost equally to approximately 87% of the infectious reservoir. The reservoir of P. falciparum malaria was very small, being confined to 9% of the patients, and this appears to be a characteristic of epidemic malaria, as was the case with P. falciparum.     

Storage duration and polymerase chain reaction detection of Plasmodium falciparum from blood spots on filter paper

To evaluate the effect of long-term storage of sample filters on the sensitivity of polymerase chain reaction (PCR) detection of malaria, 252 blood spots from patients with microscopically confirmed Plasmodium falciparum malaria were analyzed and stratified by storage duration. The spots were collected between 1996 and 2000 on filter paper and stored at room temperature. A Chelex-based method was used to extract the DNA. Unexpectedly, after the first purification, the sensitivity of the PCR from recently stored samples was low and showed progressively increased with time storage (P = 0.003, by chi-square test for linear trend). This suggested that PCR inhibitors were easier to dissolve from the more recent blood spots (< 4 years old) than from blood spots > or = 4 years old, thus leading to a time-dependent increase in PCR sensitivity. However, if DNA was purified again (when the first PCR result was negative), the cumulative sensitivity was not influenced by storage duration. This indicated that length of storage is not a critical issue providing purification is sufficient.     

Efficacy of monthly tafenoquine for prophylaxis of Plasmodium vivax and multidrug-resistant P. falciparum malaria

We assessed monthly doses of tafenoquine for preventing Plasmodium vivax and multidrug-resistant P. falciparum malaria. In a randomized, double-blind, placebo-controlled study, 205 Thai soldiers received either a loading dose of tafenoquine 400 mg (base) daily for 3 days, followed by single monthly 400-mg doses (n = 104), or placebo (n = 101), for up to 5 consecutive months. In volunteers completing follow-up (96 tafenoquine and 91 placebo recipients), there were 22 P. vivax, 8 P. falciparum, and 1 mixed infection. All infections except 1 P. vivax occurred in placebo recipients, giving tafenoquine a protective efficacy of 97% for all malaria (95% confidence interval [CI], 82%-99%), 96% for P. vivax malaria (95% CI, 76%-99%), and 100% for P. falciparum malaria (95% CI, 60%-100%). Monthly tafenoquine was safe, well tolerated, and highly effective in preventing P. vivax and multidrug-resistant P. falciparum malaria in Thai soldiers during 6 months of prophylaxis.     

Polymerase chain reaction-based diagnosis of Mediterranean spotted fever in serum and tissue samples

A nested polymerase chain reaction (PCR) assay has been developed and used in the diagnosis of fatal and benign cases of Mediterranean spotted fever (MSF). The test was based on specific primers derived from a Rickettsia conorii 17-kD protein gene. A positive signal was obtained from spotted fever group (SFG) and typhus group (TG) rickettsiae. Discrimination between SFG and TG rickettsiae was based on a restriction fragment length polymorphism test. Other gram-negative bacterial species tested did not generate a signal, attesting for the specificity of the assay. The SFG-specific DNA fragment was detected in four of 29 acute-phase sera from serologically confirmed patients with MSF, while acute-phase sera from 25 patients without MSF were PCR negative. Acute-phase sera samples (five of five) and tissue autopsies (six of seven) from fatal suspected cases of MSF were PCR positive. The results demonstrate that sera and tissue samples are suitable specimens for the nested PCR tests, especially in fatal cases.     

Relative utility of dipsticks for diagnosis of malaria in mesoendemic area for Plasmodium falciparum and P. vivax in northeastern India

For diagnosis of malaria, popular brands of rapid test kits collectively termed as "dipsticks" were subject to field evaluation in northeastern India for their comparative sensitivity and specificity vis-à-vis conventional microscopic results. Dipsticks based on Plasmodium falciparum-specific histidine-rich protein (Pf HRP-2) antigen capture assay revealed 100% sensitivity and high specificity (94-100%); thus, they were concluded to be reliable tools for confirmed diagnosis of malarial infection. However, an advanced version of the same kit, having incorporated additional pan-malarial monoclonal antibody, was found to be less sensitive (71%) for non-falciparum infections. Besides, Pf HRP-2-based kits continued to show positive results up to day 7, even after clearance of parasitemia on account of persistent antigenemia. This very limitation seemed to have been overcome by parasite-specific lactate dehydrogenase (pLDH) enzyme-based kit. This kit was observed to have high sensitivity (81-89%) and specificity (100%) for both falciparum and non-falciparum malaria, but cannot distinguish mono-infection from mixed infections. It is concluded that the rational use of these kits would accord health benefits in terms of early detection and prompt treatment, reduce drug pressure, and possibly delay the emergence and spread of multi-drug-resistant strains of malarial parasites.     

间日疟、恶性疟混合感染1例治疗报告

2009年龙岩市报告1例间日疟、恶性疟混合感染病例,患者在国外感染、发病,曾接受过治疗,回国后再次发病。在国内经3个疗程青蒿琥酯(1 800 mg)和4个疗程的氯伯8 d疗法(氯喹4 800 mg、伯氨喹720 mg)治疗后痊愈。