Detection of Plasmodium vivax and Plasmodium falciparum DNA in human saliva and urine: Loop-mediated isothermal amplification for malaria diagnosis

This study investigated loop-mediated isothermal amplification (LAMP) detection of Plasmodium falciparum and Plasmodium vivax in urine and saliva of malaria patients. From May to November 2011, 108 febrile patients referred to health centers in Sistan and Baluchestan Province of south-eastern Iran participated in the study. Saliva, urine, and blood samples were analyzed with nested PCR and LAMP targeting the species-specific nucleotide sequence of small subunit ribosomal RNA gene (18S rRNA) of P. falciparum and P. vivax and evaluated for diagnostic accuracy by comparison to blood nested PCR assay. When nested PCR of blood is used as standard, microscopy and nested PCR of saliva and urine samples showed sensitivity of 97.2%, 89.4% and 71% and specificity of 100%, 97.3% and 100%, respectively. LAMP sensitivity of blood, saliva, and urine was 95.8%, 47% and 29%, respectively, whereas LAMP specificity of these samples was 100%. Microscopy and nested PCR of saliva and LAMP of blood were comparable to nested PCR of blood (к = 0.95, 0.83, and 0.94, respectively), but agreement for nested PCR of urine was moderate (к = 0.64) and poor to fair for saliva LAMP and urine LAMP (к = 0.38 and 0.23, respectively). LAMP assay showed low sensitivity for detection of Plasmodium DNA in human saliva and urine compared to results with blood and to nested PCR of blood, saliva, and urine. However, considering the advantages of LAMP technology and of saliva and urine sampling, further research into the method is worthwhile. LAMP protocol and precise preparation protocols need to be defined and optimized for template DNA of saliva and urine.     

环介导等温扩增检测恶性疟原虫的应用研究

目的探讨环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测恶性疟患者血中疟原虫的敏感性和特异性。方法收集恶性疟患者和健康人血液样品,分别用LAMP、镜检法和巢式PCR检测恶性疟原虫,计算LAMP的敏感度、特异度、阳性预测值和阴性预测值,并与巢式PCR进行比较。结果共检测78份恶性疟患者和30份健康人血样,其中72份患者血样LAMP检测为阳性,以镜检法为金标准,LAMP筛检的灵敏度为92.3%,特异度为100%,阳性预测值和阴性预测值分别为100%和83.3%;以巢式PCR为参考,LAMP筛检的灵敏度为97.2%,特异度为94.4%。结论 LAMP方法检测恶性疟原虫的敏感度和特异度与巢式PCR方法相近,可应用于现场恶性疟检测。     

Comparative Diagnosis of Malaria Infections by Microscopy,Nested PCR,and LAMP in Northern Thailand

Three methods, microscopy, nested polymerase chain reaction (nPCR), and loop-mediated isothermal amplification (LAMP) have been applied for malaria diagnosis in 105 human blood samples collected in Northern Thailand. Only Plasmodium falciparum and Plasmodium vivax infections were detected. A total number of 57 positives (54%) could be detected for P. falciparum and 25 (24%) for P. vivax when all samples that were positive in any of the three methods are counted together. The nPCR was used as a reference standard for comparison with the other methods, microscopy and LAMP. The sensitivity of LAMP for P. falciparum was 100%. All nPCR-negative samples for P. falciparum were also negative by both microscopy and LAMP (specificity, 100%). For diagnosis of P. vivax, microscopy detected 15 of 23 nPCR-positive samples (sensitivity, 65%). LAMP detected 22 of 23 nPCR-positives (sensitivity, 96%). Among the 82 nPCR-negative samples microscopy detected two samples (specificity, 98%). All 82 nPCR-negative were also negative by the LAMP method (specificity, 100%). Both Plasmodium genus- and species-specific LAMP primer sets yielded the same results in all samples. There were no significant differences in the prevalence detected by each method. We assume that LAMP was as reliable as nPCR and more reliable than microscopy in the detection of Plasmodium DNA tested in the examined Thai field blood samples. This study further validates LAMP as an alternative molecular diagnostic tool, which can be used in the diagnosis of early infections of malaria cases and together with nPCR can also be used as supplementary methods for clinical and epidemiological use.     

Detection of Mixed-Species Infections of Plasmodium falciparum and Plasmodium vivax by Nested PCR and Rapid Diagnostic Tests in Southeastern Iran

Coexistence of two species of Plasmodium in a single host has disrupted the diagnosis and treatment of malaria. This study was designed to evaluate the ability of rapid diagnostic test (RDT) kits for the diagnosis of mixed-species malaria infections in southeastern Iran. A total of 100 malaria patients were included in the study out of 164 randomly suspected symptomatic malaria patients from May to November 2012. Nested polymerase chain reaction (PCR) was also used to judge the ability of microscopy versus RDT kits for detecting mixed species. The sensitivity of light microscopy for the detection of mixed-species malaria infections was 16.6% (95% confidence interval [CI] = 3–49.1). Nested PCR revealed 12 patients with mixed-species infection. The CareStart Pv/Pf Combo kit detected 58% of the mixed-species infections, which were determined by nested PCR (sensitivity = 58.3%; 95% CI = 28.5–83.5). For identifying P. falciparum, P. vivax, and mixed-species infections, the concordance rates (kappa statistics) of microscopy and CareStart Pv/Pf Combo kit with nested PCR were 0.76 and 0.79, respectively (P = 0.001). This study underlines the effectiveness of RDT kits to improve the differentiation of mixed-species malaria infections in endemic areas where the prevalence of chloroquine resistance is high.     

A new visually improved and sensitive loop mediated isothermal amplification (LAMP) for diagnosis of symptomatic falciparum malaria

Molecular diagnosis of malaria by nucleotide amplification requires sophisticated and expensive instruments, typically found only in well-established laboratories. Loop-mediated isothermal amplification (LAMP) has provided a new platform for an easily adaptable molecular technique for molecular diagnosis of malaria without the use of expensive instruments. A new primer set has been designed targeting the 18S rRNA gene for the detection of Plasmodium falciparum in whole blood samples. The efficacy of LAMP using the new primer set was assessed in this study in comparison to that of a previously described set of LAMP primers as well as with microscopy and real-time PCR as reference methods for detecting P. falciparum. Pre-addition of hydroxy napthol blue (HNB) in the LAMP reaction caused a distinct color change, thereby improving the visual detection system. The new LAMP assay was found to be 99.1% sensitive compared to microscopy and 98.1% when compared to real-time PCR. Meanwhile, its specificity was 99% and 100% in contrast to microscopy and real-time PCR, respectively. Moreover, the LAMP method was in very good agreement with microscopy and real-time PCR (0.94 and 0.98, respectively). This new LAMP method can detect at least 5 parasites/μL of infected blood within 35 min, while the other LAMP method tested in this study, could detect a minimum of 100 parasites/μL of human blood after 60 min of amplification. Thus, the new method is sensitive and specific, can be carried out in a very short time, and can substitute PCR in healthcare clinics and standard laboratories.     

A highly sensitive, nested polymerase chain reaction based method using simple dna extraction to detect malaria sporozoites in mosquitos

Dried Anopheles farauti mosquitos caught in Solomon Islands in 1990 were examined for malaria sporozoites by ELISA and nested polymerase chain reaction (PCR). Only heads and thoraces were used. Plasmodium genus-specific nested PCR amplifications were carried out on all samples. Of the 402 pools of mosquitos that were processed, 30 were positive for malaria. Nest 1 products of positive samples were subjected to further PCR amplifications with species-specific primers for P. falciparum and P. vivax. Twenty pools were positive for P. vivax by PCR while only 7 were positive by ELISA. For P. falciparum 2 pools were positive by both ELISA and PCR, and one of these was a pool which was positive for P. vivax by PCR and ELISA. Thus the sensitivity of PCR for P. vivax was 100% while the specificity was 96.7%. For P. falciparum the sensitivity and specificity were 100%. The PCR technique is highly sensitive and can be used on dried mosquitos which makes it a valuable tool for determining sporozoite rates of mosquitos, even in remote areas.     

Comparing the results of light microscopy with the results of PCR method in the diagnosis of Plasmodium vivax

BACKGROUND AND OBJECTIVES: Although polymerase chain reaction (PCR) is a new technique in the diagnosis of malaria with very high accuracy; light microscopy is still conventional diagnostic method in Iran. In this study we checked the accuracy of light microscopy using the results of PCR as gold standard in Iran. METHODS: The blood samples were collected from 124 febrile cases in Kahnooj district. The blood slides were read by microscopists, and double checked by experts in provincial referral laboratory. DNA samples were processed by PCR to amplify species-specific sequences of 18s subunit ribosomal ribonucleic acid (18ssrRNA) genes of Plasmodium vivax and P. falciparum. RESULTS: The sensitivity and specificity of microscopy in the detection of Plasmodium spp infection were 77% (95% CI: 46-94%) and 100% (95% CI: 95-100%), correspondingly. Also, the estimated positive and negative predictive values were 100% (95% CI: 66-100%) and 97% (95% CI: 91-99%), respectively. INTERPRETATION AND CONCLUSION: According to these results, we believe that the accuracy of light microscopy in the diagnosis of malaria in Kahnooj was acceptable. Expert micorscopists in endemic areas of Iran such as Kahnooj and available equipments in one hand and expensive PCR test on the other hand may convince that in current situation we do not have to change the diagnostic method.     

Performance of a histidine-rich protein 2 rapid diagnostic test, Paracheck Pf®, for detection of malaria infections in Ugandan pregnant women

Improved laboratory diagnosis is critical to reduce the burden of malaria in pregnancy. Peripheral blood smears appear less sensitive than Plasmodium falciparum histidine-rich protein 2-based rapid diagnostic tests (RDTs) for placental malaria infections in studies conducted at delivery. In this study, 81 women in Uganda in the second or third trimester of pregnancy were followed-up until delivery. At each visit, peripheral blood was tested by blood smear, RDT, and nested species-specific polymerase chain reaction (PCR). Sensitivity and specificity of the tests was calculated with PCR, which detected 22 infections of P. falciparum, as the gold standard. The sensitivity and specificity of blood smears were 36.4% (95% confidence interval [CI] = 18.0-59.2%) and 99.6% (95% CI = 97.7-100%), respectively. The corresponding values for RDT were 31.8% (95% CI = 14.7-54.9%) and 100% (95% CI = 98.3-100%). The RDTs could replace blood smears for diagnosis of malaria in pregnancy by virtue of their relative ease of use. Field-based sensitive tests for malaria in pregnancy are urgently needed.     

环介导等温扩增法检测人粪样中美洲钩虫的方法建立

目的 建立一种快速、高效的环介导等温扩增方法(loop-mediated isothermal amplification, LAMP)检测粪样中美洲钩虫虫卵。方法 根据美洲钩虫ITS基因序列,设计4条LAMP引物,建立LAMP检测法。利用LAMP法检测日本血吸虫、肝吸虫、鞭虫、猪蛔虫、蛲虫的DNA样本以验证LAMP方法的特异度。以美洲钩虫成虫DNA样本,倍比稀释以评价LAMP法的DNA最低检出量。现场采集55份人粪DNA样本,采用改良加藤法、LAMP法和普通PCR方法同时检测,评价LAMP法的敏感性和特异性。结果 LAMP法可特异性扩增美洲钩虫DNA样本。LAMP法可检测最低DNA浓度为1.2 fg/mL。通过现场样本对LAMP进行评价,LAMP法敏感性相对于改良加藤法和PCR法分别是95.24%和100%。特异性分别是97.06%和100%。结论 建立起以虫卵为检测材料的检测美洲钩虫病的LAMP法,为美洲钩虫病提供特异、敏感、高效的检测方法。     

Rapid diagnostic devices for malaria: field evaluation of a new prototype immunochromatographic assay for the detection of Plasmodium falciparum and non-falciparum Plasmodium

The NOW ICT Malaria P.f./P.v. for Whole Blood (Binax, Inc., Portland, ME) is a new malaria rapid diagnostic device that represents a technical advance over previous assays, such as ICT Malaria P.f./P.v. and ICT Malaria P.f.. We evaluated this device in March 2001 in symptomatic patients at malaria clinics in Maesod, Thailand. Microscopic examination of Giemsa-stained blood smears was the reference standard. In 246 patients, microscopy showed 32 (13.0%) infected with Plasmodium falciparum, 63 (25.6%) with P. vivax, 6 (2.4%) with mixed infections of P. falciparum and P. vivax, 5 (2.0%) with P. malariae, and 140 (56.9%) negative. Sensitivity for P. falciparum was 100% and specificity was 96.2% (200 of 208; 95% confidence interval [CI] = 92-98). For P. vivax, sensitivity was 87.3% (55 of 63; 95% CI = 77-93) and specificity was 97.7% (173 of 177; 95% CI = 95-99), but all the four false-positive results were microscopically positive for P. malariae; thus, specificity for non-falciparum Plasmodium was 100%. These results suggest improved performance over NOW ICT predecessors.     

Development of a Polymerase Chain Reaction (PCR) method based on amplification of mitochondrial DNA to detect Plasmodium falciparum and Plasmodium vivax

In this study we standardized a new technical approach in which the target mitochondrial DNA sequence (mtDNA) is amplified using a simple but sensitive PCR method as a tool to detect Plasmodium falciparum and Plasmodium vivax. Specific primers were designed to hybridize with cytochrome c oxidase genes of P. falciparum (cox III) and P. vivax (cox I). Amplification products were obtained for all positive samples, presenting homology only for species-specific mtDNA. Sensitivity and specificity were 100%. The applicability of the method was tested in a cross-sectional study, in which 88 blood samples from individuals naturally exposed to malaria in the Brazilian Amazon region were analyzed. Based on the results, the sensitivity and specificity were 100% and 88.3%, respectively. This simple and sensitive PCR method can be useful in specific situations and in different settings of malaria management, in endemic as well as non-endemic areas (travelers), and in clinical or epidemiological studies, with applications in malaria control programs.     

Evaluation of loop-mediated isothermal amplification (LAMP) for malaria diagnosis in a field setting

We used the loop-mediated isothermal amplification (LAMP) method developed by our group for malaria diagnosis with genus-specific and species-specific primers for the four human malaria parasites at a field clinic in comparison with standard microscopy. Among 110 blood samples collected from the malaria clinic in Thailand, LAMP detected 59 of 60 samples positive by microscopy (sensitivity = 98.3%) and none of the 50 microscopy-negative samples (specificity = 100%). Negative predictive value (NPV) and positive predictive value (PPV) of LAMP were 98% and 100%, respectively. These results indicate that LAMP is an effective tool for malaria diagnosis at a field clinic in a field setting.     

Sensitivity and specificity of an antigen detection ELISA for malaria diagnosis

Enzyme-linked immunosorbent assays (ELISAs) allow for the testing of large numbers of samples within a short time frame. We tested the sensitivity and specificity of a histidine-rich protein 2 (HRP2)-based, commercially available ELISA antigen detection assay for Plasmodium falciparum (Malaria Antigen CELISA; Cellabs, Sydney, Australia). A total of 700 whole blood samples obtained from symptomatic outpatients of malaria clinics along the Thai-Myanmar border were tested relative to blinded duplicate expert microscopy adjusted with species-specific polymerase chain reaction (PCR). PCR-adjusted microscopy showed that 79 (11.3%) were infected with P. falciparum, 118 (16.9%) with P. vivax, 1 (0.1%) with P. malariae, 7 (1.0%) with mixed infections (P. falciparum and P. vivax), and 495 (70.7%) were negative. The geometric mean parasite density for P. falciparum was 7547/muL (range: 12-363,810/muL). The overall sensitivity of the HRP2 ELISA for P. falciparum malaria was 98.8% (95% CI, 93.6-100%) and the specificity was 100% (95% CI, 99.5-100%). The positive and negative predictive values for the ELISA were 100% (95% CI, 96.5-100%) and 99.8% (95% CI, 99.1-100%), respectively. The results for P. falciparum were clearly superior to expert microscopy alone, particularly in mixed infections. Microscopy combined with ELISA reaches a sensitivity and specificity similar to PCR-adjusted microscopy for the diagnosis of P. falciparum while being considerably less expensive and faster. We conclude that ELISA serves as an excellent tool to augment microscopy as the gold standard for P. falciparum diagnosis in research settings and should be further evaluated for screening in blood banks.     

田鼠巴贝虫感染FTA-环介导等温扩增检测技术的建立

目的 建立敏感、特异、高效的检测田鼠巴贝虫 FTA-环介导等温扩增技术(LAMP)。方法 根据GenBank公布的田鼠巴贝虫基因序列,就细胞色素B基因保守序列设计了多套LAMP引物,利用LAMP RealTime Turbidimeter LA-320 仪筛选最佳引物、反应条件,建立LAMP检测方法,从保存有血液样本的FTA卡片中提取田鼠巴贝虫 DNA进行LAMP,观察检测效果。结果 设计的4组引物做LAMP试验,其中以引物1,最佳反应温度为64 ℃,恒温下作用,敏感性高,可在1 h内完成,其检出限量为0.687 fg/μL,而PCR的最低检测量为0.687 pg/μL,与间日疟原虫、恶性疟原虫、卵形疟原虫、三日疟原虫、诺氏疟原虫、冈地弓形虫、利什曼原虫、冈比亚锥虫均无交叉反应。以建立的FTA-LAMP法检测20份PCR检测阳性的田鼠巴贝虫感染者血样,其阳性符合率100%。结论 成功建立了检测田鼠巴贝虫特异性细胞色素B基因的FTA-LAMP方法,该法特异性强、灵敏度高、简便、快速、适于现场应用。     

Usefulness of polymerase chain reaction to supplement field microscopy in a pre-selected population with a high probability of malaria infections

This study determines the use of nested PCR as a diagnostic tool to supplement field microscopy in symptomatic individuals suspected of being positive for malaria, and it explores its role in active case detection to identify asymptomatic parasite carriers. In symptomatic individuals, compared with PCR, microscopy had a sensitivity of 86.6% (95% confidence interval [CI] = 77.8-92.4) and specificity of 100% (95% CI = 96.9-100). During active case detection, two asymptomatic persons were diagnosed as having vivax malaria by polymerase chain reaction (PCR) but not microscopy. Currently, PCR is being carried out in Sri Lanka only for population surveys to estimate the hidden reservoir of malaria. Based on the results of this study and because of cost considerations, pooled PCR will be used in the future to screen samples from clinically suspected foci to increase the proportion of malaria cases detected. This strategy will assist the success of the malaria elimination program in Sri Lanka.     

Development and evaluation of novel loop-mediated isothermal amplification for rapid detection of bla(IMP-1) and bla(VIM-2) genes

Loop-mediated isothermal amplification (LAMP) amplifies a target gene with high specificity and rapidity under isothermal conditions. LAMP assays were developed for the rapid detection of metallo-beta-lactamase (MBL) genes such as bla(IMP-1)) and bla(VIM-2). We initially designed specific primers to detect MBL genes for LAMP assays and evaluated the specificity and sensitivity of these assays. LAMP assays amplified MBL genes under a constant temperature of 63 degrees C within 1 hour, and were compared to PCR in MBL-producing strains. The results of MBL genes typing by LAMP assays agree completely with PCR results. The lower detection limits of bla(IMP-1)- and bla(VIM-2)-LAMP assays using real-time turbidimeters were 30cfu/test and 3cfu/test. After amplification, products were directly observed by the naked eye with a fluorescent detection reagent. In conclusion, LAMP assays are convenient, rapid, and fully feasible for detecting MBL genes in ordinary clinical microbiology laboratories without special apparatus.     

An Update on Malaria Rapid Diagnostic Tests

Purpose of Review

Modern advances in malaria rapid diagnostic test (RDT) technology have increased demand for low-cost, easy-to-use assays in areas endemic for malaria. Substantial developments in diagnostic sensitivity and specificity, improvements in non-falciparum RDTs, and novel biotechnological innovations are gradually aligning the performance of RDTs with reference-level diagnostics including PCR and expert microscopy gold standards.

Recent Findings

Trends have emerged in recent malaria RDT literature: (1) improvements in the sensitivity and specificity of RDTs for Plasmodium falciparum diagnosis, making them comparable to expert microscopic examination; (2) reduced false-positive and false-negative reactions with novel antibody development; (3) improved sensitivity and specificity capabilities of Plasmodium vivax-specific RDTs; (4) developing RDTs for co-endemic mixed infection differentiation; (5) significant improvements of RDTs for Plasmodium knowlesi; (6) a global push towards assessing and confronting the growing concerns of widespread pfhrp2 gene deletions; and (7) original innovation in loop-mediated isothermal amplification (LAMP) biotechnological RDT-like platforms that demonstrate promising performance characteristics for P. falciparum, P. vivax, and P. knowlesi infections.

Summary

The past 5 years have been characterized by increasing demand for malaria RDTs, translating into meaningful improvements in performance and novel biotechnological innovation. Future work should facilitate the development of improved RDT platforms for Plasmodium ovale, P. knowlesi, and Plasmodium malariae, and surmount the issue of pfhrp2 gene deletions, while maintaining comparable performance to both PCR and expert microscopy reference standards.

     

Genetic variability in Plasmodium vivax aldolase gene in Korean isolates and the sensitivity of the Binax Now malaria test

Introduction of rapid malaria diagnostic tests (RDT) initiated numerous field evaluations in various epidemiologic settings. But the efficiency of some RTD kits based on aldolase raised reservations for direct implementation of RDT into clinical settings. We performed Binax Now malaria test in 84 Korean Plasmodium vivax isolates and compared it with the traditional Giemsa stain microscopy test as the reference standard. The sensitivity of Binax Now was 62.0% for P. vivax cases (52/84, 95% CI 51.2-71.6%) with 100.0% specificity (50/50, 95% confidence interval 92.9-100%). After the aldolase gene sequence analysis of 84 isolates, two synonymous mutations in aldolase gene were identified in both Binax Now positive and negative samples. No significant association between the mutations and Binax Now malaria tests was found. Thus, the genetic variability would not explain the poor performance of P. vivax RDTs by detecting aldolase in ROK isolates.     

Accuracy of loop-mediated isothermal amplification for diagnosis of human leptospirosis in Thailand

There is a lack of diagnostic tests for leptospirosis in technology-restricted settings. We developed loop-mediated isothermal amplification (LAMP) specific for the 16S ribosomal RNA gene (rrs) of pathogenic and intermediate group Leptospira species. The lower limit of detection was 10 genomic equivalents/reaction, and analytical specificity was high; we observed positive reactions for pathogenic/intermediate groups and negative reactions for non-pathogenic Leptospira species and other bacterial species. We evaluated this assay in Thailand by using a case-control study of 133 patients with laboratory-proven leptospirosis and 133 patients with other febrile illnesses. Using admission blood, we found that the rrs LAMP showed positive results in 58 of 133 cases (diagnostic sensitivity = 43.6, 95% confidence interval [CI] = 35.0-52.5) and in 22 of 133 controls (diagnostic specificity = 83.5, 95% CI = 76.0-89.3). Sensitivity was high for 39 patients who were culture positive for Leptospira spp. (84.6, 95% CI = 69.5-94.1). The rrs LAMP can provide an admission diagnosis in approximately half of patients with leptospirosis, but its clinical utility is reduced by a lower specificity.     

Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection

Entomological monitoring of Leishmania infection in leishmaniasis endemic areas offers epidemiologic advantages for predicting the risk and expansion of the disease, as well as evaluation of the effectiveness of control programs. In this study, we developed a highly sensitive loop-mediated isothermal amplification (LAMP) method for the mass screening of sand flies for Leishmania infection based on the 18S rRNA gene. The LAMP technique could detect 0.01 parasites, which was more sensitive than classical PCR. The method was robust and could amplify the target DNA within 1 h from a crude sand fly template without DNA purification. Amplicon detection could be accomplished by the newly developed colorimetric malachite green (MG)—mediated naked eye visualization. Pre-addition of MG to the LAMP reaction solution did not inhibit amplification efficiency. The field applicability of the colorimetric MG-based LAMP assay was demonstrated with 397 field-caught samples from the endemic areas of Ecuador and eight positive sand flies were detected. The robustness, superior sensitivity, and ability to produce better visual discriminatory reaction products than existing LAMP fluorescence and turbidity assays indicated the field potential usefulness of this new method for surveillance and epidemiological studies of leishmaniasis in developing countries.