A new LAMP-based assay for the molecular diagnosis of toxoplasmosis: comparison with a proficient PCR assay

Toxoplasmosis is generally a benign infection caused by the protozoan parasite Toxoplasma gondii but can have severe consequences in fetuses of mothers infected during pregnancy (congenital toxoplasmosis) and immunocompromised individuals. PCR-based diagnostic tests have become crucial for its diagnosis. However, this molecular diagnosis essentially relies upon laboratory-developed methods and suffers from a lack of standardization, leading to great variation in methods and performance among laboratories. With the need for accreditation of clinical microbiological laboratories, the use of commercial PCR kits has become an attractive alternative; but thorough evaluation of newly commercialized kits by proficient groups is necessary before any recommendation can be made to parasitology laboratories by health authorities or learned societies. Here, we compared the performance of an original commercial method, the Iam TOXO Q-LAMP (DiaSorin®), using Loop-mediated isothermal amplification (LAMP) technology, with our reference laboratory-developed method using real-time PCR. The kit was first tested using amniotic fluid (AF) and plasma samples (either negative or spiked with live T. gondii tachyzoites at different concentrations (from 7 to 10⁵?tachyzoites/mL)). It was then assessed using a cohort of 11 AF, five placental and 32 blood clinical samples preserved at ?20?°C. For the processing of placental/blood samples, a pretreatment step was used, which did not strictly follow the manufacturer’s recommendations. The practical ease of use and compliance with good laboratory practices were also evaluated. Although the LAMP assay was less sensitive than the laboratory-developed method at very low parasite concentrations (0.1?T. gondii genome equivalents/mL), the two methods yielded identical results qualitatively and, in some instances, quantitatively, particularly for AF samples.     

Characterization of ROP18 alleles in human toxoplasmosis

The role of the virulent gene ROP18 polymorphisms is not known in human toxoplasmosis. A total of 320 clinical samples were analyzed. In samples positive for ROP18 gene, we determined by an allele specific PCR, if patients got the upstream insertion positive ROP18 sequence Toxoplasma strain (mouse avirulent strain) or the upstream insertion negative ROP18 sequence Toxoplasma strain (mouse virulent strain). We designed an ELISA assay for antibodies against ROP18 derived peptides from the three major clonal lineages of Toxoplasma. 20 clinical samples were of quality for ROP18 allele analysis. In patients with ocular toxoplasmosis, a higher inflammatory reaction on eye was associated to a PCR negative result for the upstream region of ROP18. 23.3%, 33% and 16.6% of serums from individuals with ocular toxoplasmosis were positive for type I, type II and type III ROP18 derived peptides, respectively but this assay was affected by cross reaction. The absence of Toxoplasma ROP18 promoter insertion sequence in ocular toxoplasmosis was correlated with severe ocular inflammatory response. Determination of antibodies against ROP18 protein was not useful for serotyping in human toxoplasmosis.     

Evaluation of commercial kits for extraction of DNA and RNA from Clostridium difficile

Commercial nucleic acid extraction kits are a cost effective, efficient and convenient way to isolate DNA and RNA from bacteria. Despite the increasing importance of the gastrointestinal pathogen, Clostridium difficile, and the increased use of nucleic acids in its identification, characterization, and investigation of virulence factors, no standardized or recommended methods for nucleic acid isolation exist. Here, we sought to evaluate 4 commercial DNA extraction kits and 3 commercial RNA extraction kits assessing cost, labor intensity, purity, quantity and quality of nucleic acid preparations. The DNA extraction kits produced a range of concentrations (20.9–546 ng/ml) and A_260/280 ratios (1.92–2.11). All kits were suitable for DNA extraction with the exception of the Roche MagNA pure LC DNA isolation kit III which produced DNA of high yield but with substantial shearing, but that did not affect downstream PCR amplifications. For RNA extraction, the Qiagen RNeasy mini kit stood out producing preparations of consistently higher concentrations and higher RNA integrity numbers (RIN). The Roche MagNA pure LC RNA isolation kit produced preparations that could not be properly assigned RINs due to a failure to remove small RNAs which were interpreted as degradation. Good DNA and RNA yield are critical but methods are often overlooked. This study highlights the potential for critical variation between established commercial systems and the need for assessment of any extraction methods that are used.     

Evaluation of a Strategy for Toxoplasma gondii Oocyst Detection in Water

Several recent outbreaks of toxoplasmosis were related to drinking water. We propose a strategy for Toxoplasma oocyst detection as part of an approach to detecting multiple waterborne parasites, including Giardia and Cryptosporidium spp., by the U.S. Environmental Protection Agency method with the same sample. Water samples are filtered to recover Toxoplasma oocysts and purified on a sucrose density gradient. Detection is based on PCR and mouse inoculation (bioassay) to determine the presence and infectivity of recovered oocysts. In an experimental seeding assay with 100 liters of deionized water, a parasite density of 1 oocyst/liter was successfully detected by PCR in 60% of cases and a density of 10 oocysts/liter was detected in 100% of cases. The sensitivity of the PCR assay varied from less than 10 to more than 1000 oocysts/liter, depending on the sample source. PCR was always more sensitive than mouse inoculation. This detection strategy was then applied to 139 environmental water samples collected over a 20-month period. Fifty-three samples contained PCR inhibitors, which were overcome in 39 cases by bovine serum albumin addition. Among 125 interpretable samples, we detected Toxoplasma DNA in 10 cases (8%). None of the samples were positive by mouse inoculation. This strategy efficiently detects Toxoplasma oocysts in water and may be suitable as a public health sentinel method.     

Development of new procedures for the isolation of phytoplankton DNA from fixed samples

Phytoplankton samples collected for routine monitoring programmes have traditionally been preserved with fixatives before subsequent analytical procedures such as microscope-based identification, or simply to permit transport between laboratories. In recent years, to simplify identification and enumeration, the use of DNA or RNA probes coupled with the PCR assay has progressed and now represents a routine procedure for screening cultured and field samples. However, the phytoplankton cells have often still to be treated as fixed samples.The extraction of genomic DNA from fixed cultures of Alexandrium minutum cultures was compared using two new methods based on Magnetisable Solid Phase Support (MSPS) techniques with that using three commercial kits. Genomic DNA recovery and PCR amplification were observed and the results obtained from culture samples were validated using field samples. Among the DNA extraction techniques considered, the MSPS methods provided the best results.     

Physical lysis only (PLO) methods suitable as rapid sample pretreatment for qPCR assay

Quantitative PCR (qPCR) enables rapid and sensitive gene quantification and is widely used in genomics, such as biological, medical, environmental, and food sciences. However, sample pretreatment requires the use of conventional DNA extraction kits which are time-consuming and labor intensive. In this study, we investigated four physical lysis only (PLO) methods which are rapid and could serve as alternatives to conventional DNA extraction kits. These PLO methods are bead mill, heating, sonication, and freeze–thaw. Using ethidium bromide-based assay, their performance was evaluated and compared. The effects of cell debris and its removal were also investigated. Bead mill method without cell debris removal appeared to yield the best qPCR results among the four PLO methods. In addition, bead mill method also performed better than conventional DNA extraction kits. It is probably due to the substantial loss of DNA material during the extensive purification of the conventional DNA extraction kits. The bead mill method has been demonstrated to successfully quantify 10² to 10⁷ copies of the PAH-RHD_α gene of Pseudomonas putida.     

Comparison of five commercial DNA extraction kits for the recovery of Yersinia pestis DNA from bacterial suspensions and spiked environmental samples

Aim:  To evaluate commercial DNA extraction kits for their ability to isolate DNA from Yersinia pestis suspensions and spiked environmental samples.
Methods and Results:  Five commercially available DNA extraction kits were evaluated: the ChargeSwitch gDNA Mini Bacteria Kit, the IT 1-2-3 Sample DNA Purification Kit, the MasterPure Complete DNA and RNA Purification Kit, the QIAamp DNA Blood Mini Kit and the UltraClean Microbial DNA Isolation Kit. The extraction methods were performed upon six Y. pestis strains and spiked environmental specimens, including three swab types and one powder type. Taqman real-time PCR analysis revealed that the use of the MasterPure kit resulted in DNA with the most consistently positive results and the lowest limit of detection from Y. pestis suspensions and spiked environmental samples.
Conclusion:  Comparative evaluations of the five commercial DNA extraction methods indicated that the MasterPure kit was superior for the isolation of PCR-amplifiable DNA from Y. pestis suspensions and spiked environmental samples.
Significance and Impact of the Study:  The results of this study can assist diagnostic laboratories with selecting the best extraction method for processing environmental specimens for subsequent detection of Y. pestis by real-time PCR.     

Toxoplasma gondii: Sensitive and rapid detection of infection by loop-mediated isothermal amplification (LAMP) method

Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high specificity, sensitivity and rapidity. In this study, we used a conserved sequence in the 200- to 300-fold repetitive 529 bp gene of Toxoplasma gondii to design primers for LAMP test. Detection limit of T. gondii LAMP assay with the primers is 1 pg/μL of T. gondii DNA, which was evaluated using 10-fold serially diluted DNA of cultured parasites. Furthermore, LAMP and conventional PCR methods were applied for amplification of the T. gondii DNA extracted from the lymph nodes taken from pigs which were suspected to be Toxoplasma infection. As a result, 76.9% (70/91) and 85.7% (78/91) of the samples were positive on PCR and LAMP analyzes, respectively. Therefore, the LAMP has a potential to be applied as an alternative molecular diagnostic tool for detection of T. gondii infection from veterinary samples. This is the first study, which applies the LAMP method to diagnose Toxoplasma from veterinary samples.     

Comparison of DNA extraction methods for multiplex polymerase chain reaction

We compared six DNA extraction methods for obtaining DNA from whole blood and saliva for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate saliva sampling as an alternative to blood sampling to obtain DNA for molecular diagnostics, genetic genealogy, and research purposes. The DNA quantity, DNA purity (A_260/280), PCR inhibition ratio, and mitochondrial DNA/genomic DNA ratio were measured to compare the extraction methods. The different extraction methods resulted in variable DNA quantity and purity, but there were no significant differences in the efficiency of multiplex PCR and oligomicroarray signals after single-base extension on the arrayed primer extension 2 (APEX-2).     

Comparing DNA Extraction Methods for Analysis of Botanical Materials Found in Anti-Diabetic Supplements

A comparative performance evaluation of DNA extraction methods from anti-diabetic botanical supplements using various commercial kits was conducted, to determine which produces the best quality DNA suitable for PCR amplification, sequencing and species identification. All plant materials involved were of suboptimal quality showing various levels of degradation and therefore representing real conditions for testing herbal supplements. Eight different DNA extraction methods were used to isolate genomic DNA from 13 medicinal plant products. Two methods for evaluation, DNA concentration measurements that included absorbance ratios as well as PCR amplifiability, were used to determine quantity and quality of extracted DNA. We found that neither DNA concentrations nor commonly used UV absorbance ratio measurements at A ₂₆₀/A ₂₈₀ between 1.7 and 1.9 are suitable for globally predicting PCR success in these plant samples, and that PCR amplifiablity itself was the best indicator of extracted product quality. However, our results suggest that A ₂₆₀/A ₂₈₀ ratios below about 1.3 and above 2.3 indicated a DNA quality too poor to amplify. Therefore, A ₂₆₀/A ₂₈₀ measurements are not useful to identify samples that likely will amplify but can be used to exclude samples that likely will not amplify reducing the cost for unnecessarily subjecting samples to PCR. The two Nucleospin^® plant II kit extraction methods produced the most pure and amplifiable genomic DNA extracts. Our results suggest that there are clear, discernable differences between extraction methods for low quality plant samples in terms of producing contamination-free, high-quality genomic DNA to be used for further analysis.     

Contrasting Effect of Prepulse Signals on Performance of Toxoplasma-Infected and Toxoplasma-Free Subjects in an Acoustic Reaction Times Test

Background

About 30% of people on Earth have latent toxoplasmosis. Infected subjects do not express any clinical symptoms, however, they carry dormant stages of parasite Toxoplasma for the rest of their life. This form of toxoplasmosis is mostly considered harmless, however, recent studies showed its specific effects on physiology, behaviour and its associations with various diseases, including psychiatric disorders such as schizophrenia. Individuals who suffer from schizophrenia have about 2.7 times higher prevalence of Toxoplasma-seropositivity than controls, which suggests that some traits characteristic of schizophrenic patients, including the sex difference in schizophrenia onset, decrease of grey matter density in specific brain areas and modification of prepulse inhibition of startle reaction could in fact be caused by toxoplasmosis for those patients who are Toxoplasma-seropositive.

Methodology/Principal Findings

We measured the effect of prepulse inhibition/facilitation of the startle reaction on reaction times. The students, 170 women and 66 men, were asked to react as quickly as possible to a startling acoustic signal by pressing a computer mouse button. Some of the startling signals were without the prepulse, some were 20 msec. preceded by a short (20 msec.) prepulse signal of lower intensity. Toxoplasma-seropositive subjects had longer reaction times than the controls. Acoustic prepulse shorted the reaction times in all subjects. This effect of prepulse on reaction times was stronger in male subjects and increased with the duration of infection, suggesting that it represented a cumulative effect of latent toxoplasmosis, rather than a fading out after effect of past acute toxoplasmosis.

Conclusions

Different sensitivity of Toxoplasma-seropositive and Toxoplasma-seronegative subjects on effect of prepulses on reaction times (the toxoplasmosis-prepulse interaction) suggested, but of course did not prove, that the alternations of prepulse inhibition of startle reaction observed in schizophrenia patients probably joined the list of schizophrenia symptoms that are in fact caused by latent toxoplasmosis.     

Toxoplasma gondii: Congenital transmission in a hamster model

The objective of the research was to test the hamster for a model of transmission of congenital toxoplasmosis. A non-invasive method for the diagnosis of pregnancy in hamsters was designed, with a specificity and a sensitivity of 70.2 and 94.7%, respectively (n = 168). Of 32 females with a chronic toxoplasma infection, 3 transmitted Toxoplasma congenitally during their first pregnancy, but not during the subsequent pregnancy. Congenital transmission rates of infections initiated during pregnancy with 2 stages of 2 strains of Toxoplasma were in the range of 33 to 100% of the 76 females inoculated. Only 1 of 17 females transmitted the parasite exclusively via milk. It was concluded that the hamster is a promising species for a model of transmission of congenital toxoplasmosis.     

Validation of the isothermal Schistosoma haematobium Recombinase Polymerase Amplification (RPA) assay,coupled with simplified sample preparation,for diagnosing female genital schistosomiasis using cervicovaginal lavage and vaginal self-swab samples

BackgroundFemale genital schistosomiasis (FGS) is a neglected and disabling gynecological disease that can result from infection with the parasitic trematode Schistosoma haematobium. Accurate diagnosis of FGS is crucial for effective case management, surveillance and control. However, current methods for diagnosis and morbidity assessment can be inaccessible to those at need, labour intensive, costly and unreliable. Molecular techniques such as PCR can be used to reliably diagnose FGS via the detection of Schistosoma DNA using cervicovaginal lavage (CVL) samples as well as lesser-invasive vaginal self-swab (VSS) and cervical self-swab samples. PCR is, however, currently unsuited for use in most endemic settings. As such, in this study, we assessed the use of a rapid and portable S. haematobium recombinase polymerase amplification (Sh-RPA) isothermal molecular diagnostic assay, coupled with simplified sample preparation methodologies, to detect S. haematobium DNA using CVL and VSS samples provided by patients in Zambia.Methodology/Principal findingsVSS and CVL samples were screened for FGS using a previously developed Sh-RPA assay. DNA was isolated from VSS and CVL samples using the QIAamp Mini kit (n = 603 and 527, respectively). DNA was also isolated from CVL samples using two rapid and portable DNA extraction methods: 1) the SpeedXtract Nucleic Acid Kit (n = 223) and 2) the Extracta DNA Tissue Prep Kit (n = 136). Diagnostic performance of the Sh-RPA using VSS DNA extacts (QIAamp Mini kit) as well as CVL DNA extracts (QIAamp Mini kit, SpeedXtract Nucleic Acid Kit and Extracta DNA Tissue Prep Kit) was then compared to a real-time PCR reference test.Results suggest that optimal performance may be achieved when the Sh-RPA is used with PuVSS samples (sensitivity 93.3%; specificity 96.6%), however no comparisons between different DNA extraction methods using VSS samples could be carried out within this study. When using CVL samples, sensitivity of the Sh-RPA ranged between 71.4 and 85.7 across all three DNA extraction methods when compared to real-time PCR using CVL samples prepared using the QIAamp Mini kit. Interestingly, of these three DNA extraction methods, the rapid and portable SpeedXtract method had the greatest sensitivity and specificity (85.7% and 98.1%, respectively). Specificity of the Sh-RPA was >91% across all comparisons.Conclusions/SignificanceThese results supplement previous findings, highlighting that the use of genital self-swab sampling for diagnosing FGS should be explored further whilst also demonstrating that rapid and portable DNA isolation methods can be used to detect S. haematobium DNA within clinical samples using RPA. Although further development and assessment is needed, it was concluded that the Sh-RPA, coupled with simplified sample preparation, shows excellent promise as a rapid and sensitive diagnostic tool capable of diagnosing FGS at the point-of-care in resource-poor schistosomiasis-endemic settings.     

First Colombian multicentric newborn screening for congenital toxoplasmosis

Aims

To determine the incidence of congenital toxoplasmosis in Colombian newborns from 19 hospital or maternal child health services from seven different cities of five natural geographic regions (Caribbean, Central, Andean, Amazonia and Eastern).

Materials and Methods

We collected 15,333 samples from umbilical cord blood between the period of March 2009 to May 2010 in 19 different hospitals and maternal-child health services from seven different cities. We applied an IgM ELISA assay (Vircell, Spain) to determine the frequency of IgM anti Toxoplasma. The results in blood cord samples were confirmed either by western blot and repeated ELISA IgM assay. In a sub-sample of 1,613 children that were negative by the anti-Toxoplasma IgM assay, the frequency of specific anti-Toxoplasma IgA by the ISAGA assay was determined. All children with positive samples by IgM, IgA, clinical diagnosis or treatment during pregnancy were recalled for confirmatory tests after day 10 of life.

Results

61 positive samples for specific IgM (0.39%) and 9 positives for IgA (0.5%) were found. 143 questionnaires were positive for a clinical diagnosis or treatment for toxoplasmosis during pregnancy. 109 out of the 218 children that had some of the criteria for postnatal confirmatory tests were followed. Congenital toxoplasmosis infection was confirmed in 15 children: 7 were symptomatic, and three of them died before the first month of life (20% of lethality). A significant correlation was found between a high incidence of markers for congenital toxoplasmosis and higher mean annual rainfall for the city.

Conclusions

Incidence for congenital toxoplasmosis is significantly different between hospitals or maternal child health services from different cities in Colombia. Mean annual rainfall was correlated with incidence of congenital toxoplasmosis.     

Long-Term Frozen Storage of Urine Samples: A Trouble to Get PCR Results in Schistosoma spp. DNA Detection?

Background

Human schistosomiasis remains a serious worldwide public health problem. At present, a sensitive and specific assay for routine diagnosis of schistosome infection is not yet available. The potential for detecting schistosome-derived DNA by PCR-based methods in human clinical samples is currently being investigated as a diagnostic tool with potential application in routine schistosomiasis diagnosis. Collection of diagnostic samples such as stool or blood is usually difficult in some populations. However, urine is a biological sample that can be collected in a non-invasive method, easy to get from people of all ages and easy in management, but as a sample for PCR diagnosis is still not widely used. This could be due to the high variability in the reported efficiency of detection as a result of the high variation in urine samples’ storage or conditions for handling and DNA preservation and extraction methods.

Methodology/Principal Findings

We evaluate different commercial DNA extraction methods from a series of long-term frozen storage human urine samples from patients with parasitological confirmed schistosomiasis in order to assess the PCR effectiveness for Schistosoma spp. detection. Patientś urine samples were frozen for 18 months up to 7 years until use. Results were compared with those obtained in PCR assays using fresh healthy human urine artificially contaminated with Schistosoma mansoni DNA and urine samples from mice experimentally infected with S. mansoni cercariae stored frozen for at least 12 months before use. PCR results in fresh human artificial urine samples using different DNA based extraction methods were much more effective than those obtained when long-term frozen human urine samples were used as the source of DNA template.

Conclusions/Significance

Long-term frozen human urine samples are probably not a good source for DNA extraction for use as a template in PCR detection of Schistosoma spp., regardless of the DNA method of extraction used.     

Evaluation of Five Methods for Total DNA Extraction from Western Corn Rootworm Beetles

Background

DNA extraction is a routine step in many insect molecular studies. A variety of methods have been used to isolate DNA molecules from insects, and many commercial kits are available. Extraction methods need to be evaluated for their efficiency, cost, and side effects such as DNA degradation during extraction.

Methodology/Principal Findings

From individual western corn rootworm beetles, Diabrotica virgifera virgifera, DNA extractions by the SDS method, CTAB method, DNAzol® reagent, Puregene® solutions and DNeasy® column were compared in terms of DNA quantity and quality, cost of materials, and time consumed. Although all five methods resulted in acceptable DNA concentrations and absorbance ratios, the SDS and CTAB methods resulted in higher DNA yield (ng DNA vs. mg tissue) at much lower cost and less degradation as revealed on agarose gels. The DNeasy® kit was most time-efficient but was the costliest among the methods tested. The effects of ethanol volume, temperature and incubation time on precipitation of DNA were also investigated. The DNA samples obtained by the five methods were tested in PCR for six microsatellites located in various positions of the beetle''s genome, and all samples showed successful amplifications.

Conclusion/Significance

These evaluations provide a guide for choosing methods of DNA extraction from western corn rootworm beetles based on expected DNA yield and quality, extraction time, cost, and waste control. The extraction conditions for this mid-size insect were optimized. The DNA extracted by the five methods was suitable for further molecular applications such as PCR and sequencing by synthesis.     

An optimized DNA extraction and purification method from dairy manure compost for genetic diversity analysis

An unbiased DNA extraction protocol is necessary for analysis of genetic diversity, particularly, of genes in complex environmental samples by nucleic acid techniques. In the present study, three manual extraction methods and two commonly used commercial kits, which were accompanied by two DNA purification strategies, were compared based on cell lysis efficiency, DNA and humic acid yields, PCR amplification and denaturing gradient gel electrophoresis (DGGE) analysis. The results show that in spite of higher cell lysis efficiencies of the two commercial kits, the purified DNA yields were only one-third of that obtained by the two manual methods of FTSP (Freeze–thaw–SDS–Protein K) and FTSPP (Freeze–thaw–SDS–Protein K-Polyvinylpolypyrrolidone). The purified DNA from all five methods was pure enough for successful PCR and real-time PCR amplifications in the presence of 1 μg μL^?1 BSA. However, the FTSPP extraction method with DNA purification by a Wizard^® kit yielded the largest number of 16S rRNA gene copies and ribotypes or bands in DGGE profiles, which indicated a superiority over the other four methods. The development of this optimized DNA extraction and purification method may provide a valuable tool for further molecular analysis of compost.     

Application of gelatin-coated magnetic particles for isolation of genomic DNA from bacterial cells

Gelatin-coated magnetic particles were implemented for bacterial genomic DNA isolation in this study. Based on structural differences in the cell wall, the standard strains Staphylococcus aureus and Escherichia coli were selected. The quantity, quality, and timing process for DNA extraction using gelatin-coated magnetite were compared to reference phenol-chloroform extraction and a commercially available kit. Approximately twice as much DNA was recovered with the use of coated magnetite, providing greater yields than other DNA extraction methods. In addition, the DNA quality was determined using 16S ribosomal DNA (rDNA) gene amplification by polymerase chain reaction (PCR). The described technique is rapid, simple, and a well-suited method to use with PCR for diagnosis of bacterial infections.     

International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients

Background

A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation.

Methodology/Findings

An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05–0.5 parasites/mL whereas specific kDNA tests detected 5.10⁻³ par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3–94.4%, specificity of 85–95%, accuracy of 86.8–89.5% and kappa index of 0.7–0.8 compared to consensus PCR reports of the 16 good performing tests and 63–69%, 100%, 71.4–76.2% and 0.4–0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the performance obtained by the participating laboratories.

Conclusion/Significance

This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.