Detection of four Plasmodium species by genus- and species-specific loop-mediated isothermal amplification for clinical diagnosis

Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the clinical detection of four species of human malaria parasites: Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. We evaluated the sensitivity and specificity of LAMP in comparison with the results of microscopic examination and nested PCR. LAMP showed a detection limit (analytical sensitivity) of 10 copies of the target 18S rRNA genes for P. malariae and P. ovale and 100 copies for the genus Plasmodium, P. falciparum, and P. vivax. LAMP detected malaria parasites in 67 of 68 microscopically positive blood samples (sensitivity, 98.5%) and 3 of 53 microscopically negative samples (specificity, 94.3%), in good agreement with the results of nested PCR. The LAMP reactions yielded results within about 26 min, on average, for detection of the genus Plasmodium, 32 min for P. falciparum, 31 min for P. vivax, 35 min for P. malariae, and 36 min for P. ovale. Accordingly, in comparison to the results obtained by microscopy, LAMP had a similar sensitivity and a greater specificity and LAMP yielded results similar to those of nested PCR in a shorter turnaround time. Because it can be performed with a simple technology, i.e., with heat-treated blood as the template, reaction in a water bath, and inspection of the results by the naked eye because of the use of a fluorescent dye, LAMP may provide a simple and reliable test for routine screening for malaria parasites in both clinical laboratories and malaria clinics in areas where malaria is endemic.     

套式PCR检测滤纸干血滴中间日疟原虫

从滤纸干血滴上用Ｃｈｅｌｅｘ处理洗脱下的疟原虫ＤＮＡ,经套式ＰＣＲ扩增间日疟虫ＳＳＵｒＲＮＡ基因特异性１２１ｂｐ片段,分析该方法的敏感性和特异性。３７例血栓检测结果一部阳性,当原虫密度低全２５个原虫／ｕＬ血时仍可成功检测到该特异条带,且其它三种人疟原虫（恶性疟虫,三日疟原虫和卵疟原虫）血样均为阴性。提示滤纸干血滴与ＰＣＲ扩增技术相结合,是疟疾诊断或流行病学调查的实用工具。     

检测滤纸干血滴中间日疟原虫(英文)

从滤纸干血滴上用Chelex处理洗脱下的疟原虫DNA,经套式PCR扩增间日疟原虫SSUrRNA基因特异性121bp片段,分析该方法的敏感性和特异性。37例血样检测结果全部阳性,当原虫密度低至25个原虫/uL血时仍可成功检测到该特异条带,且其它三种人疟原虫(恶性疟原虫,三日疟原虫和卵形疟原虫)血样均为阴性。提示滤纸干血滴与PCR扩增技术相结合,是疟疾诊断或流行病学调查的实用工具。     

Detection and species determination of malaria parasites by PCR: comparison with microscopy and with ParaSight-F and ICT malaria Pf tests in a clinical environment

A rapid procedure for the diagnosis of malaria infections directly from dried blood spots by PCR amplification was evaluated with samples from 52 patients. Plasmodium infections were identified with a genus-specific primer set, and species differentiation between Plasmodium falciparum and Plasmodium vivax was analyzed by multiplex PCR. The PCR test with any of the three primer sets was able to detect as few as four parasites per microliter by gel electrophoresis or by nonisotopic paper hybridization chromatography. The diagnoses obtained by PCR correlated closely with those obtained by Giemsa staining except for two samples observed to have mixed P. falciparum-P. vivax infections. These were initially missed by microscopic analysis. In comparison with antigen-capture assays for P. falciparum, the PCR assays were able to detect three infections that were missed by the ParaSight-F test. The PCR test was negative for nine ParaSight-F-positive samples and one ICT Malaria Pf-positive sample, and these were confirmed to be false-positive results. The PCR thus gave no false-negative or false-positive results. Patients undergoing antimalarial therapy were also monitored by the PCR assay. Four of seven patients who were PCR positive for P. vivax at the time of discharge were later readmitted to the hospital with a recurrence of P. vivax infection. We would like to propose that PCR is a sensitive and easy method that can serve as a useful addition to microscopy for the diagnosis and the clinical monitoring of treatment of malaria.     

Usefulness of Seminested Multiplex PCR in Surveillance of Imported Malaria in Spain

The use of a new PCR-based method for the diagnosis of malaria in the Spanish Malaria Reference Laboratory has promoted an increase in confirmed cases of malaria. From August 1997 to July 1998, a total of 192 whole-blood samples and 71 serum samples from 168 patients were received from the hospitals of the Spanish National Health System. Most of the patients came from west-central African countries (85%). This molecular method showed more sensitivity and specificity than microscopy, detecting 12.4% more positive samples than microscopy and 13% of mixed infections undetectable by Giemsa stain. Plasmodium falciparum was the main species detected, with 68% of the total positive malaria cases, followed by Plasmodium malariae (29%), Plasmodium vivax (14%), and Plasmodium ovale (7%), including mixed infections in all cases. This report consists of the first wide, centralized survey of malaria surveillance in Spain. The reference laboratory conducted the analysis of all imported cases in order to detect trends in acquisition. The use of a seminested multiplex PCR permitted confirmation of the origins of the infections and the Plasmodium species involved and confirmation of the effectiveness of drug treatments. This PCR also allowed the detection of the presence in Spain of primaquine-tolerant P. vivax strains from west-central Africa, as well as the detection of a P. falciparum infection induced by transfusion.     

Detection of four Plasmodium species in blood from humans by 18S rRNA gene subunit-based and species-specific real-time PCR assays

There have been reports of increasing numbers of cases of malaria among migrants and travelers. Although microscopic examination of blood smears remains the "gold standard" in diagnosis, this method suffers from insufficient sensitivity and requires considerable expertise. To improve diagnosis, a multiplex real-time PCR was developed. One set of generic primers targeting a highly conserved region of the 18S rRNA gene of the genus Plasmodium was designed; the primer set was polymorphic enough internally to design four species-specific probes for P. falciparum, P. vivax, P. malarie, and P. ovale. Real-time PCR with species-specific probes detected one plasmid copy of P. falciparum, P. vivax, P. malariae, and P. ovale specifically. The same sensitivity was achieved for all species with real-time PCR with the 18S screening probe. Ninety-seven blood samples were investigated. For 66 of them (60 patients), microscopy and real-time PCR results were compared and had a crude agreement of 86% for the detection of plasmodia. Discordant results were reevaluated with clinical, molecular, and sequencing data to resolve them. All nine discordances between 18S screening PCR and microscopy were resolved in favor of the molecular method, as were eight of nine discordances at the species level for the species-specific PCR among the 31 samples positive by both methods. The other 31 blood samples were tested to monitor the antimalaria treatment in seven patients. The number of parasites measured by real-time PCR fell rapidly for six out of seven patients in parallel to parasitemia determined microscopically. This suggests a role of quantitative PCR for the monitoring of patients receiving antimalaria therapy.     

Species-specific PCR detection of malaria parasites by microtiter plate hybridization: clinical study with malaria patients.

A simple and convenient PCR method that amplifies the 18S rRNA genes has been developed for the purpose of detecting and differentiating four species causing malaria in humans. The advantage of the assay is that the biotinylated PCR product is visualized following hybridization with specific probes which are immobilized on plate wells (microtiter plate hybridization). This method has been previously evaluated in a field study and was found to be sensitive and specific for the detection of Plasmodium falciparum and Plasmodium vivax. In the current study, the microtiter plate hybridization PCR method was evaluated by using blood specimens from malaria patients. All of 36 cases of falciparum malaria, 26 of 27 cases of vivax malaria, all of 11 cases of ovale malaria, and 2 cases of malariae malaria were diagnosed species specifically by the PCR method. There were four smear-negative, PCR-positive cases that seemed to correspond to the convalescent stage of malaria. In contrast, 30 cases for which the diagnosis of malaria has been excluded on the basis of microscopy and clinical courses showed negative PCR results. By comparing parasite densities and PCR results following antimalarial treatment of some patients, it was revealed that the PCR results largely paralleled the parasite densities and that PCR could detect as few as 10 parasites per microliter of blood. We conclude that this PCR method is highly sensitive and specific for the detection of all four parasite species and can serve as a useful supplement to microscopy for the clinical management of malaria.     

Real-time PCR for detection and identification of Plasmodium spp

Rapid and accurate detection of malaria parasites in blood is needed to institute proper therapy. We developed and used a real-time PCR assay to detect and distinguish four Plasmodium spp. that cause human disease by using a single amplification reaction and melting curve analysis. Consensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and SYBR Green was used for detection in a LightCycler instrument. Patient specimens infected at 0.01 to 0.02% parasitemia densities were detected, and analytical sensitivity was estimated to be 0.2 genome equivalent per reaction. Melting curve analysis based on nucleotide variations within the amplicons provided a basis for accurate differentiation of Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. For assay validation, 358 patient blood samples from the National University Hospital in Singapore and Evanston Northwestern Healthcare in Illinois were analyzed. Of 76 blinded patient samples with a microscopic diagnosis of P. falciparum, P. vivax, or P. ovale infection, 74 (97.4%) were detected by real-time PCR, including three specimens containing mixed P. falciparum-P. vivax infections. No Plasmodium DNA was amplified in any of the 82 specimens sent for malaria testing but that were microscopically negative for Plasmodium infection. In addition, 200 blood samples from patients whose blood was collected for reasons other than malaria testing were also determined to be negative by real-time PCR. Real-time PCR with melting curve analysis could be a rapid and objective supplement to the examination of Giemsa-stained blood smears and may replace microscopy following further validation.     

Identification of four species of human malaria parasites by fast PCR

Malaria is one of the most prevalent infectious diseases in the world. Accurate identification of four species of human malaria parasite is essential for appropriate treatment. Here, we developed a simple and rapid method of identifying Plasmodium species using a fast polymerase chain reaction (PCR) assay. Based on the previous literature, we amplified small subunit ribosomal RNA genes of four human malaria parasites. To establish a minimum detection limit, a blood sample with a known number of P. falciparum parasites (parasitemia: 3%) was diluted serially(from 0.03% to 0.000003%). We compared the detection limits between single (one-step) PCR and nested (two-step) PCR. Other clinical blood samples, which were infected with P. falciparum (parasitemia: 2.8%), P. vivax (parasitemia: 0.13%), P. ovale (parasitemia: 0.04%), respectively, were also tested by our PCR system. The PCR findings were compared to those of blood film Giemsa staining and rapid diagnostic tests (RDT). The sensitivity of our method is less than one parasite in 1 microl of blood(estimated parasitemia: 0.000003%) for both single PCR and nested PCR, though an increased number of cycles (40 cycles) was required for single PCR. Using clinical samples, it was proven that amplified products by single PCR could clearly distinguish between P. falciparum, P. vivax, and P. ovale. To detect P. vivax and P. ovale, the PCR system was more sensitive than RDT. The total required time for our method was within three to four hours from DNA extraction to PCR detection. Taken together, our method is easier and faster than the previously reported PCR-based malaria parasite identification systems, and is also useful for cases in which diagnosis by Giemsa staining and RDT is difficult.     

Development of a real-time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for routine clinical diagnosis

A TaqMan-based real-time PCR qualitative assay for the detection of three species of malaria parasites-Plasmodium falciparum, P. ovale, and P. vivax-was devised and evaluated using 122 whole-blood samples from patients who had traveled to areas where malaria is endemic and who presented with malaria-like symptoms and fever. The assay was compared to conventional microscopy and to an established nested-PCR assay. The specificity of the new assay was confirmed by sequencing the PCR products from all the positive samples and by the lack of cross-reactivity with Toxoplasma gondii and Leishmania infantum DNA. Real-time PCR assay showed a detection limit (analytical sensitivity) of 0.7, 4, and 1.5 parasites/ micro l for P. falciparum, P. vivax, and P. ovale, respectively. Real-time PCR, like nested PCR, brought to light errors in the species identification by microscopic examination and revealed the presence of mixed infections (P. falciparum plus P. ovale). Real-time PCR can yield results within 2 h, does not require post-PCR processing, reduces sample handling, and minimizes the risks of contamination. The assay can therefore be easily implemented in routine diagnostic malaria tests. Future studies are warranted to investigate the clinical value of this technique.     

Malaria diagnosis: standardization of a polymerase chain reaction for the detection of Plasmodium falciparum parasites in individuals with low-grade parasitemia

 In Brazil, no study has been done concerning the detection of malaria parasites by polymerase chain reaction (PCR) related to the diagnosis of Plasmodium falciparum malaria. In the present report we describe a highly sensitive methodology for malaria diagnosis using a nested PCR method based on amplification of the p126 P. falciparum gene detected by simple ethidium bromide staining. The P. falciparum Palo Alto strain (culture samples) was serially diluted in blood from an uninfected donor to a final level of parasitemia corresponding to 10^–8% and was processed for PCR amplification. In each of these dilutions a parasitological examination was performed to compare the sensitivity with that of PCR amplification. Blood samples (field samples) were obtained from 51 malarious patients with positive thick blood smears (TBS) who were living in endemic regions of the Brazilian Amazon. They corresponded to 42 P. falciparum and 9 P. vivax cases, with parasitemia levels ranging from only 16 to 20,200 parasites/μl for P. falciparum disease and from 114 to 11,000 parasites/μl for P. vivax malaria. We demonstrate that the use of nested PCR allows the detection of 0.005 parasites/μl without the use of radioactive material. The use of a 1-ml sample volume and the organic DNA extraction method should be suitable in blood banks and for the evaluation of patients during and after drug treatment. Received: 10 January 1996 / Accepted: 10 April 1996     

Natural Plasmodium infections in Anopheles darlingi and Anopheles benarrochi (Diptera: Culicidae) from eastern Peru

Malaria, both Plasmodium falciparum (Welch) and Plasmodium vivax (Grassi & Feletti), has reemerged as a significant public health disease issue in Peru, especially in forested areas in the eastern part of the country. The spread of Anopheles darlingi Root, the principal South American malaria vector, into new areas of Peru is thought to be a factor in this resurgence. However, epidemiological evidence suggests that in malaria endemic areas of eastern Peru where An. darlingi does not occur, other species are involved in malaria transmission. The objective of this study was to analyze Anopheles species collected from 11 provinces within four departments in eastern Peru during 2001 and 2002 for infections with P. falciparum and P. vivax. More than 84,000 Anopheles mosquitoes representing 13 species were tested by enzyme-linked immunosorbent assay for the presence of Plasmodium circumsporozoite (CS) proteins. Of these, only An. darlingi and Anopheles benarrochi Gabaldón, Cova García & López were found positive. In total, 14 (0.98%) of 1,432 pools of An. darlingi were positive for Plasmodium species; specifically 10 (0.70%) pools were positive for P. falciparum, two (0.14%) were positive for P. vivax VK210, and two (0.14%) were positive for P. vivax VK247 proteins. Nine (0.14%) of 6,323 pools of An. benarrochi were positive for Plasmodium; five (0.08%) of 6,323 pools were positive for P. falciparum, two (0.03%) were positive for P. vivax VK247, one (0.02%) was positive for mixed P. vivax VK210/VK247 infections, and one (0.02%) was positive for mixed P. falciparum and P. vivax VK210 CS-proteins. Although infection rates in An. benarrochi were significantly lower (0.14%) than rates found for An. darlingi (0.98%), our data suggest that An. benarrochi may play a role in transmitting and maintaining Plasmodium species in various malaria endemic areas of eastern Peru.     

Evaluation of a Colorimetric PCR-Based Assay To Diagnose Plasmodium falciparum Malaria in Travelers

New diagnostic tests are needed to facilitate the diagnosis of Plasmodium falciparum malaria in the returned traveler. We performed a blinded evaluation of a nonisotopic colorimetric PCR-based assay (Digene SHARP Signal System) and compared the results with those obtained by microscopy and nested PCR for the detection of P. falciparum malaria in 150 febrile travelers. By using nested PCR as the reference standard, the colorimetric assay had a sensitivity of 100% and a specificity of 95.4% for the detection of P. falciparum. This PCR-based nonisotopic assay is a rapid, sensitive, and specific method for the detection of falciparum malaria in returned travelers.     

Malaria rapid diagnostic devices: performance characteristics of the ParaSight F device determined in a multisite field study

Microscopic detection of parasites has been the reference standard for malaria diagnosis for decades. However, difficulty in maintaining required technical skills and infrastructure has spurred the development of several nonmicroscopic malaria rapid diagnostic devices based on the detection of malaria parasite antigen in whole blood. The ParaSight F test is one such device. It detects the presence of Plasmodium falciparum-specific histidine-rich protein 2 by using an antigen-capture immunochromatographic strip format. The present study was conducted at outpatient malaria clinics in Iquitos, Peru, and Maesod, Thailand. Duplicate, blinded, expert microscopy was employed as the reference standard for evaluating device performance. Of 2,988 eligible patients, microscopy showed that 547 (18%) had P. falciparum, 658 (22%) had P. vivax, 2 (0.07%) had P. malariae, and 1,750 (59%) were negative for Plasmodium. Mixed infections (P. falciparum and P. vivax) were identified in 31 patients (1%). The overall sensitivity of ParaSight F for P. falciparum was 95%. When stratified by magnitude of parasitemia (no. of asexual parasites per microliter of whole blood), sensitivities were 83% (>0 to 500 parasites/microl), 87% (501 to 1,000/microl), 98% (1,001 to 5,000/microl), and 98% (>5,000/microl). Device specificity was 86%.     

Comparison of two commercial assays with expert microscopy for confirmation of symptomatically diagnosed malaria

Conventional light microscopy has been the established method for malaria diagnosis. However, recently several nonmicroscopic rapid diagnostic tests have been developed for situations in which reliable microscopy may not be available. This study was conducted to evaluate the diagnostic performance of a recently introduced ICT Malaria Pf/Pv test. This assay detects Plasmodium falciparum histidine-rich protein 2 antigen (PfHRP-2) for P. falciparum diagnosis and pan-malarial antigen for P. vivax diagnosis. In this study we compared the performance of ICT Malaria Pf/Pv with microscopy of Giemsa-stained blood films and with an OptiMAL test that detects Plasmodium lactate dehydrogenase (pLDH) antigen. A total of 750 clinically suspected malaria patients were examined at local health centers in Kuwait. Both the antigen tests had a high degree of specificity (>98%) for detection of malaria infection. However, they were less sensitive than microscopy. Compared with microscopy the ICT Malaria PF/pf test failed to detect malaria infection in 93 (34%) of 271 malaria patients (11% of patients with P. falciparum and 37% of patients with P. vivax) and the OptiMAL test failed to detect malaria infection in 41 (15%) of 271 malaria patients (7% of patients with P. falciparum and 13% of patients with P. vivax). The sensitivities of the ICT Malaria Pf/Pv and OptiMAL tests for detection of P. falciparum infection were 81 and 87%, and those for detecting P. vivax were 58 to 79%, respectively. The sensitivity of the ICT Malaria Pf/Pv and OptiMAL tests decreased significantly to 23 and 44%, respectively, at parasite densities of <500/ micro l. Both of the tests also produced a number of false-positive results. Overall, the performance of the OptiMAL test was better than that of the ICT Malaria Pf/Pv test. However, our results raise particular concern over the sensitivity of the ICT Malaria Pf/Pv test for detection of P. vivax infection. Further developments appear necessary to improve the performance of the ICT Malaria Pf/Pv test.     

Comparison of the OptiMAL Test with PCR for Diagnosis of Malaria in Immigrants

The OptiMAL test (Flow Inc., Portland, Oreg.), which detects a malaria parasite lactate dehydrogenase (pLDH) antigen, has not been evaluated for its sensitivity in the diagnosis of malaria infection in various epidemiological settings. Using microscopy and a PCR as reference standards, we performed a comparison of these assays with the OptiMAL test for the detection of Plasmodium falciparum and Plasmodium vivax infection in 550 immigrants who had come from areas where malaria is endemic to reside in Kuwait, where malaria is not endemic. As determined by microscopy, 125 (23%) patients had malaria, and of these, 84 (67%) were infected with P. vivax and 36 were infected with P. falciparum; in 5 cases the parasite species could not be determined due to a paucity of the parasites. The PCR detected malaria infection in 145 (26%) patients; 102 (70%) of the patients had P. vivax infection and 43 had P. falciparum infection. Of the five cases undetermined by microscopy, the PCR detected P. falciparum infection in two cases, P. vivax infection in two cases, and mixed (P. falciparum plus P. vivax) infection in one case. Correspondingly, the OptiMAL test detected malaria infection in 95 patients (17%); of these, 70 (74%) had P. vivax infection and 25 were infected with P. falciparum. In this study, 61 (49%) of the 125 malaria cases, as confirmed by microscopy, had a degree of parasitemia of <100 parasites per microl, and 23 (18%) of the cases had a degree of <50 parasites per microl. Our results show that the sensitivity of the OptiMAL test is high (97%) at a high level of parasitemia (>100 parasites/microl) but drops to 59% when the level is <100 parasites/microl and to 39% when it is <50 parasites/microl. In addition, the OptiMAL test failed to identify four patients whose blood smears contained P. falciparum gametocytes only. We conclude that the sensitivity and specificity of the OptiMAL test are comparable to those of microscopy in detecting malaria infection at a parasitemia level of >100 parasites/microl; however, the test failed to identify more than half of the patients with a parasitemia level of <50 parasites/microl. Thus, the OptiMAL test should be used with great caution, and it should not replace conventional microscopy in the diagnosis of malaria infection.     

Characterization of peripheral blood lymphocyte subsets in patients with acute Plasmodium falciparum and P. vivax malaria infections at Wonji Sugar Estate, Ethiopia

We investigated the absolute counts of CD4+, CD8+, B, NK, and CD3+ cells and total lymphocytes in patients with acute Plasmodium falciparum and Plasmodium vivax malaria. Three-color flow cytometry was used for enumerating the immune cells. After slide smears were stained with 3% Giemsa stain, parasite species were detected using light microscopy. Data were analyzed using STATA and SPSS software. A total of 204 adults of both sexes (age, >15 years) were included in the study. One hundred fifty-eight were acute malaria patients, of whom 79 (50%) were infected with P. falciparum, 76 (48.1%) were infected with P. vivax, and 3 (1.9%) were infected with both malaria parasites. The remaining 46 subjects were healthy controls. The leukocyte count in P. falciparum patients was lower than that in controls (P=0.015). Absolute counts of CD4+, CD8+, B, and CD3+ cells and total lymphocytes were decreased very significantly during both P. falciparum (P<0.0001) and P. vivax (P<0.0001) infections. However, the NK cell count was an exception in that it was not affected by either P. falciparum or P. vivax malaria. No difference was found in the percentages of CD4, CD8, and CD3 cells in P. falciparum or P. vivax patients compared to controls. In summary, acute malaria infection causes a depletion of lymphocyte populations in the peripheral blood. Thus, special steps should be taken in dealing with malaria patients, including enumeration of peripheral lymphocyte cells for diagnostic purposes and research on peripheral blood to evaluate the immune status of patients.     

套式/多重PCR检测疟疾感染的应用研究

目的了解标签引物-套式/多重PCR技术在检测疟疾感染中的现场应用价值。方法随机采取我省疟疾流行区有发热症状的居民及家庭成员的耳垂血79份,同时制作厚血片和滤纸血样本各1份,用标签引物-套式/多重PCR检测所采集滤纸血样本中的疟原虫,并与镜检法进行比较。结果79份样本中,用标签引物-套式/多重PCR技术检出间日疟原虫阳性6例,镜检初检出10例间日疟原虫阳性,经复查血片后有4例排除了疟疾感染。镜检复核阳性的6例样本PCR均为阳性。以镜检复核为标准,两法阳性和阴性符合率为100%。结论标签引物-套式/多重PCR检测疟疾感染具高度敏感性和特异性,对疟疾鉴别诊断和明确诊断具有重要价值。     

环介导等温扩增技术检测间日疟原虫方法的建立及应用分析

目的建立一种快速、简便的检测间日疟原虫的环介导等温扩增方法（LAMP）并与常规PCR方法作比较。方法根据间日疟原虫环子孢子蛋白（CSP）基因序列合成2对特异性LAMP引物,优化Mg2＋浓度、dNTPs浓度、BstDNA聚合酶添加量、反应温度、时间以及设计引物缺省试验。评估优化后的LAMP反应的特异性和灵敏性。检测133份患者血样,以显微镜检方法为金标准,比较LAMP和多重PCR法检测间日疟原虫的敏感性和特异性。结果 LAMP法检测重组质粒DNA（Pv-rDNA）的灵敏度达到10-10,为传统PCR方法的100倍。镜检确诊的68例间日疟、43例恶性疟和22例非疟疾患者中,LAMP法和多重PCR检测间日疟原虫的敏感性为98.53%和97.06%,两法基本相当（χ2=0.34,P〉0.05）;特异性为86.15%和100%,LAMP法低于多重PCR法,差异有统计学意义（χ2=9.67,P〈0.05）。LAMP法的阳性预测值和阴性预测值分别为88.16%和98.25%,多重PCR的阳性预测值和阴性预测值分别为100%和97.01%。结论 LAMP法检测间日疟原虫具有快速简便、敏感性高、设备要求低的特点,具有较好的应用前景。     

Devices for rapid diagnosis of Malaria: evaluation of prototype assays that detect Plasmodium falciparum histidine-rich protein 2 and a Plasmodium vivax-specific antigen

The ParaSight F test was developed as a pioneer industry effort in the large-scale, process-controlled production of a device for the rapid diagnosis of malaria. This device performed well in field settings but was limited to the detection of a single malaria species, Plasmodium falciparum. The ParaSight F+V assay advanced upon the ParaSight F test format by incorporating a monoclonal antibody directed against a proprietary Plasmodium vivax-specific antigen, in addition to the antibody directed against P. falciparum histidine-rich protein 2, which was used in the ParaSight F assay. The modified assay was developed to add the capability to detect P. falciparum and P. vivax in a single-test-strip format. The present study evaluated three distinct ParaSight F+V prototypes with samples from symptomatic patients in regions of Thailand and Peru where malaria is endemic. Over a 2-year enrollment period (1998 and 1999), a total of 4,894 patients consented to participation in the study. Compared with the results for duplicate microscopic examinations of Giemsa-stained blood smears as the reference diagnostic standard, each successive prototype showed substantial improvement in performance. The final ParaSight F+V prototype, evaluated in 1999, had an overall sensitivity for detection of asexual P. falciparum parasites of 98%. The sensitivity of the device was 100% for P. falciparum densities of >500 parasites/ micro l, with a sensitivity of 83% for parasite densities of 5,000/ micro l, 92% for parasite densities of 1,001 to 5,000/ micro l, 94% for parasite densities of 501 to 1,000/ micro l, and 55% for parasite densities of 1 to 500/ micro l. The specificity for the exclusion of P. vivax was 87%. The areas under the receiver operating characteristic curves for the diagnostic performance of the assay for the detection of P. falciparum and P. vivax were 0.8907 and 0.8522, respectively. These findings indicate that assays for rapid diagnosis have the potential to enhance diagnostic capabilities in those instances in which skilled microscopy is not readily available.