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Genetic studies of the protozoan parasite Toxoplasma gondii have identified three main distinct types according to virulence in some hosts. Several methods have been developed to differentiate genotypes currently dominated by microsatellite markers targeting single-copy loci. We analyzed the possibility of using the 35-fold repetitive B1 gene via high-resolution melting (HRM) curve analysis. Sequencing of the B1 gene of 14 reference strains (four Type I, six Type II, and four Type III strains) identified 18 single nucleotide polymorphisms (SNP). Primers were designed to amplify eight of them for HRM analysis and for relative quantification of each nucleotide variation using SNaPshot mini-sequencing. Genotyping with five microsatellite markers was performed for comparison. Two to four HRM profiles were obtained depending on the SNP tested. The differences observed relied on the different ratios of nucleotides at the SNP locus as evidenced via SNaPshot mini-sequencing. The three main lineages could be distinguished by using several HRM profiles. Some HRM profiles proved more informative than the analysis based on five microsatellite markers, showing additional differences in Type I and Type II strains. Using HRM analysis, we obtained at least an equally good discrimination of the main lineages than that based on five microsatellite markers., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A cytochrome b (cytb) gene quantitative PCR (qPCR) assay was developed to diagnose malaria in travelers. First, manual and automated DNA extractions were compared and automated DNA extraction of 400 μl of blood was found to be more efficient. Sensitivity was estimated using the WHO international standard for Plasmodium falciparum DNA and compared to that of a previously published qPCR targeting the 18S rRNA coding gene (18S qPCR). The limit of detection of the cytb qPCR assay was 20 DNA copies (i.e., 1 parasite equivalent) per 400 μl of extracted whole blood and was comparable for the two qPCR assays. Both qPCR assays were used on blood samples from 265 consecutive patients seen for suspicion of malaria. There were no microscopy-positive and qPCR-negative samples. Positive cytb qPCR results were observed for 51 samples, and all but 1 were also 18S qPCR positive. Eight (16%) of these 51 samples were negative by microscopic examination. The 8 cytb qPCR-positive and microscopy-negative samples were from African patients, 3 of whom had received antimalarial drugs. Three non-P. falciparum infections were correctly identified using an additional qPCR assay. The absence of PCR inhibitors was tested for by the use of an internal control of mouse DNA to allow reliable quantification of circulating DNA. The high analytical sensitivity of both qPCR assays combined with automated DNA extraction supports its use as a laboratory tool for diagnosis and parasitemia determination in emergencies. Whether to treat qPCR-positive and microscopy-negative patients remains to be determined.

Background: Precise genotyping of the intron 8 poly(TG) and poly(T) tracts of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is of clinical relevance in CFTR pathology. The (TG)(m) locus influences the penetrance of the (T)(5) allele, which may be associated with male infertility by congenital bilateral absence of the vas deferens (CBAVD) or other CFTR-related disorders (CFTR-RD), in particular in the context of (TG)(12) and (TG)(13). Simple and accurate genotyping of both loci should thus be routinely offered in laboratories., Methods: We designed a new single test method relying on multiplex allele-specific fluorescent PCR: (T)(5)-, (T)(7)-, and (T)(9)-specific primers, labeled with different fluorophores, in combination with a common primer. Each fluorescent PCR product was identified on a capillary sequencer by its fluorescence color, specific for (T)(n), and size, indicative of the (TG) length. We first validated the assay in 2 different laboratories on 52 DNA samples with already known genotypes. We then evaluated the method prospectively, compared with sequencing, on 62 samples from healthy individuals and 108 samples from patients with CBAVD or other CFTR-RDs., Results: We observed a 100% match in both validation steps. Results found in CBAVD and CFTR-RD patients are in keeping with data in the literature., Conclusions: The assay proved to be simple, rapid, and accurate for single-test (TG)(m)(T)(n) genotyping and suited for analysis in clinical laboratories.

Visceral and cutaneous leishmaniases are heterogenous entities. The Leishmania species that a given patient harbors usually cannot be determined clinically, and this identification is essential to prescribe the best species-specific therapeutic regimen. Our diagnosis procedure includes a real-time PCR assay targeted at the 18S rRNA gene, which detects all Leishmania species but which is not specific for a given Leishmania species. We developed a species identification based on sequencing of the cytochrome b (cyt b) gene directly from the DNA extracted from the clinical specimen. The sequences were analyzed using the Sequence Analysis/Seqscape v2.1 software (Applied Biosystems). This software is designed to automatically identify the closest sequences from a reference library after analysis of all known or unknown polymorphic positions. The library was built with the Leishmania cyt b gene sequences available in GenBank. Fifty-three consecutive real-time PCR-positive specimens were studied for species identification. The cyt b gene was amplified in the 53 specimens. Sequencing resulted in the identification of six different species with >or=99% identity with the reference sequences over 872 nucleotides. The identification was obtained in two working days and was in accordance with the multilocus enzyme electrophoresis identification when available. Real-time PCR followed by sequencing of the cyt b gene confirmed the diagnosis of leishmaniasis and rapidly determined the infecting species directly from the clinical specimen without the need for the isolation of parasites. This technique has the potential to significantly accelerate species-adapted therapeutic decisions regarding treatment of leishmaniasis.

Background: Multiple in-house polymerase chain reaction (PCR) assays for the diagnosis of parasitic and fungal diseases have been reported. Encouraging results have been published to anticipate or improve the diagnosis. However, the absence of standardized methods has led to discrepant results. As a consequence, these tests are not recognized as consensual diagnostic criteria., Methods: The major breakthrough for improving the results of these methods is the emergence of real-time technologies. This markedly improves the reliability of the PCR results by dramatically decreasing the risk of false positive results due to PCR products carryover. Moreover, the quantitative results provided by these techniques allow to compare rapidly the efficiency of primers, probes, and DNA extraction. Therefore, one can expect a more consensual method to implement comparisons between laboratories. Automated DNA extraction should also be useful to achieve this goal. Whatever sophisticated technology is used, the meaning of detecting nucleic acids in a given clinical sample still needs to be defined. This requires well-designed studies with clinical consensual criteria and PCR techniques that are as similar as possible., Conclusions: The development of real-time technology should improve our knowledge in order to give the clinicians informative clues for decision-making.

Positive galactomannan (GM) anti-genemias are included as a microbiological item in the diagnosis of probable or possible invasive aspergillosis (IA). Because false-positive GM results frequently occur, at least two positive results on two different samples are required. Waiting for clinical specimens can delay the initiation of treatment. As an alternative, we wondered whether detection of circulating Aspergillus DNA on the first positive GM serum sample could aid in diagnosing IA. Therefore, we retrospectively screened the first GM-positive serum samples from 29 patients from our hematology unit for Aspergillus DNA using real-time PCR. We compared the real-time PCR results with the final classification of proven, probable, and possible IA according to consensual criteria. No clear correlation between PCR results and the classification with the medical files could be shown. However, a positive PCR result was associated with a poor prognosis (Fisher's test; P=0.01). Our preliminary data suggest that a positive PCR result could indicate a more advanced stage of the disease. Therefore, concomitant positive PCR and GM results may justify the initiation of antifungal therapy in neutropenic patients. In contrast, a negative PCR on the first positive GM sample may argue for postponing costly antifungal administration until additional arguments for the diagnosis of IA are presented.

A lot of in-house polymerase chain reaction assays have been reported for diagnosis of invasive aspergillosis and disseminated candidosis. Encouraging results have been published to anticipate the diagnosis over the conventional microbiological methods. However, the absence of standardized methods has led to diverging results. As a consequence, these tests are not recognized as consensual diagnostic criteria, in contrast with some antigenemia detection kits. The major breakthrough for improving the results of these methods is the emergence of real-time technologies. This markedly improves the reliability of the PCR results by dramatically decreasing the risk of false positive results due to PCR products carryover. Moreover, using the quantitative results provided by this technique, this allows to rapidly compare the efficiency of primers, probes, and DNA extraction methods. Therefore, the hope is to identify the more specific and sensitive parameters to implement comparative studies. Automated DNA extraction should also be useful to achieve this goal. Whatever sophisticated technology is used, we still have to define the meaning of detecting nucleic acids in a given clinical sample. This seems simple in normally sterile anatomical sites but less obvious for example in respiratory specimens for invasive aspergillosis or in blood for candidosis in heavily colonized patients. Additional studies of the kinetics of fungal DNA are needed. The development of real-time technology should improve our knowledge in order to give the clinicians informative clues for making a decision.

Objective: Evaluation of the use of short probes as a detection system in real-time PCR., Design and Method: Comparison of results obtained with hybridization probes with and without locked nucleic acid (LNA) residues in the detection of fetal DNA in maternal serum., Results: The use of chimeric LNA/DNA probes led to a slight but significantly higher level of sensitivity as well as a higher level of fluorescence signal., Conclusion: Chimeric LNA/DNA probes offer an interesting alternative detection method in real-time PCR.

Rapid identification of patients infected with clarithromycin-resistant Helicobacter pylori without the need for culture can help to avoid useless prescriptions of clarithromycin. We developed and tested a routine real-time quantitative PCR assay dedicated to that purpose. One hundred ninety-six consecutive gastric biopsy specimens were examined by culture, histology performed by a trained physician, and rapid PCR with the LightCycler apparatus. Infection was defined as (i) positivity of culture, (ii) positivity of histology, or (iii) positivity of PCR if confirmed by positivity of a concomitant indirect test (serology or urea breath test). Susceptibility to clarithromycin was tested by E-test and PCR. The prevalence of infection was 33.7% (66 of 196 samples). The sensitivities of culture, histology, and PCR were 90.9% (60 of 66 samples), 87.9% (58 of 66 samples), and 97.0% (64 of 66 samples), respectively. The specificity of PCR was 94.6% (123 of 130 samples). The linearity of the PCR results was achieved over a 6-log range of input DNA, and we were able to accurately quantify as few as 300 bacteria and to qualitatively detect as few as 30 bacteria per DNA sample. For clarithromycin susceptibility testing, there was 98.2% (55 of 56 samples) concordance between E-test and PCR. Forty-eight strains were clarithromycin susceptible, and 9 strains were clarithromycin resistant. The single discrepancy concerned a culture which was a mixture of mutant and wild type, with a susceptible-to-resistant ratio of 11.5: the resistant population was detected by E-test but not by PCR. Our PCR assay is accurate for fast detection of H. pylori as well as of clarithromycin resistance and is also able to objectively determine bacterial density.

Fetal DNA and cells present in maternal blood have previously been used for non-invasive prenatal diagnosis. However, some fetal cells can persist in maternal blood after a previous pregnancy. Fetal rhesus status and sex determination have been performed by using amplification by real-time polymerase chain reaction (PCR) of fetal DNA sequences present in maternal circulation; no false-positive results related to persistent fetal DNA from a previous pregnancy have been reported. This idea has recently been challenged. An SRY real-time PCR assay was performed on the serum of 67 pregnant women carrying a female fetus but having previously given birth to at least one boy and on the serum of 30 healthy non-pregnant women with a past male pregnancy. In all cases, serum was negative for the SRY gene. These data suggest that fetal DNA from a previous pregnancy cannot be detected in maternal serum, even by using a highly sensitive technique. Therefore, non-invasive prenatal diagnosis by fetal sex determination for women at risk of producing children with X-linked disorders, and fetal RHD genotyping is reliable and secure as previously demonstrated.

To improve the diagnosis of invasive aspergillosis (IA), we developed a LightCycler PCR assay targeted to Aspergillus fumigatus and A. flavus mitochondrial DNA. To avoid contamination, fully automated nucleic acid extraction with the MagNA Pure LC apparatus was used. The linearity of the results was achieved over a 6-log range of input A. fumigatus DNA, from 0.3 ng to 3 fg/10 microl of water. We retrospectively compared the LightCycler PCR and an enzyme-linked immunosorbent assay for the detection of galactomannan (GM) in serum from 14 patients with IA. The GM assay was more frequently positive (57 of 109; 52%) than the PCR assay (49 of 109; 45%). The LightCycler PCR assay, combined with automated DNA extraction, could be used in association with the GM assay to improve the reliability of IA diagnosis.

Polymerase chain reaction (PCR) testing may be a rapid and reliable means of diagnosing toxoplasmosis in fetuses. Toxoplasmosis is a parasitic infection that can lead to serious neurologic disorders in children who acquire the infection before birth from their mothers. PCR is a genetic analysis technique. A total of 339 women with toxoplasmosis had testing to determine if their fetuses were infected. Researchers compared PCR testing with conventional prenatal diagnostic tests such as ultrasound, amniocentesis and fetal-blood sampling performed between 18 and 38 weeks' gestation. Thirty-four fetuses tested positive for infection by conventional methods, and PCR results were also positive in all 34 cases. Three other cases were diagnosed by PCR that were initially missed by conventional testing. PCR testing yielded one false negative result but no false positives.

We designed a single nucleotide primer extension ( SNaPshot) assay for Pneumocystis jirovecii genotyping, targeting mt85 SNP of the mitochondrial large subunit ribosomal RNA locus, to improve minority allele detection. We then analyzed 133 consecutive bronchoalveolar lavage ( BAL) fluids tested positive for P. jirovecii DNA by quantitative real-time PCR, obtained from two hospitals in different locations (Hospital 1 [ n = 95] and Hospital 2 [ n = 38]). We detected three different alleles, either singly ( mt85C: 39.1%; mt85T: 24.1%; mt85A: 9.8%) or together (27%), and an association between P. jirovecii mt85 genotype and the patient's place of hospitalization ( p = 0.011). The lowest fungal loads (median = 0.82 × 103 copies/μl; range: 15-11 × 103) were associated with mt85A and the highest (median = 1.4 × 106 copies/μl; range: 17 × 103-1.3 × 107) with mt85 CTA ( p = 0.010). The ratios of the various alleles differed between the 36 mixed-genotype samples. In tests of serial BALs (median: 20 d; range 4-525) from six patients with mixed genotypes, allele ratio changes were observed five times and genotype replacement once. Therefore, allele ratio changes seem more frequent than genotype replacement when using a SNaPshot assay more sensitive for detecting minority alleles than Sanger sequencing. Moreover, because microscopy detects only high fungal loads, the selection of microscopy-positive samples may miss genotypes associated with low loads. [ABSTRACT FROM AUTHOR]

The purpose of this study was to determine the accuracy of the none-invasive prenatal determination of polymerase chain reaction (PCR)-based fetal RhD genotyping. Study design: A prospective case series was undertaken on all RhD-negative pregnant women presenting for genetic counseling in our prenatal diagnosis center from January 2001 until December 2002. Results were compared with serologic RhD typing of the newborns. Results: Among the 285 pregnant women who participated in the study, fetal RhD status could be determined for 283 patients. In 2 cases, the RhD-negative phenotype of the mother was not the result of a complete RHD gene deletion, and therefore, the status of the fetus could not be determined. Neither false-negative nor false-positive results were observed. Conclusion: The present report demonstrates that a reliable fetal RHD genotype determination can be achieved with 100% accuracy. It is therefore possible to consider that such an assay could be systematically proposed to all RhD-negative pregnant women in order to more effectively utilize RhD prophylaxis. [ABSTRACT FROM AUTHOR]

Background: Toxoplasmosis is an infectious disease caused by the parasitic protozoan Toxoplasma gondii. It is endemic worldwide and, depending on the geographic location, 15 to 85% of the human population are asymptomatically infected. Routine diagnosis is based on serology. The parasite has emerged as a major opportunistic pathogen for immunocompromised patients, in whom it can cause life-threatening disease. Moreover, when a pregnant woman develops a primary Toxoplasma gondii infection, the parasite may be transmitted to the fetus and cause serious damnage. For these two subpopulations, a rapid and accurate diagnosis is required to initiate treatment. Serological diagnosis of active infection is unreliable because reactivation is not always accompanied by changes in antibody levels, and the presence of IgM does not necessarily indicate recent infection. Application of quantitative PCR has evolved as a sensitive, specific, and rapid method for the detection of Toxoplasma gondii DNA in amniotic fluid, blood, tissue samples, and cerebrospinal fluid. Methods: Two separate, real-time fluorescence PCR assays were designed and evaluated with clinical samples. The first, targeting the 35-fold repeated B1 gene, and a second, targeting a newly described multicopy genomic fragment of Toxoplasma gondii. Amplicons of different intragenic copies were analyzed for sequence heterogeneity. Results: Comparative LightCycler experiments were conducted with a dilution series of Toxoplasma gondii genomic DNA, 5 reference strains, and 51 Toxoplasma gondii-positive amniotic fluid samples revealing a 10 to 100-fold higher sensitivity for the PCR assay targeting the newly described 529-bp repeat element of Toxoplasma gondii. Conclusion: We have developed a quantitative LightCycler PCR protocol which offer rapid cycling with real-time, sequence-specific detection of amplicons. Results of quantitative PCR demonstrate that the 529-bp repeat element is repeated more than 300-fold in the genome of Toxoplasma gondii. Since individual intragenic copies of the target are conserved on sequence level, the high copy number leads to an ultimate level of analytical sensitivity in routine practice. This newly described 529-bp repeat element should be preferred to less repeated or more divergent target sequences in order to improve the sensitivity of PCR tests for the diagnosis of toxoplasmosis. [ABSTRACT FROM AUTHOR]

Background: Couples with a risk of transmitting X-linked diseases who are included in a preimplantation genetic diagnosis (PGD) programme need early and rapid fetal sex determination in two situations. The first situation is for the control of embryo sexing after PGD and the second situation is for those couples having a spontaneous pregnancy before the start of their PGD cycle. Among invasive techniques, chorionic villus sampling is the earliest procedure for fetal sex determination and molecular analysis of X-linked genetic disorders during the first trimester but it is associated with a risk of fetal loss. Non-invasive procedures such as ultrasound examination allow reliable fetal sex determination only during the second trimester. Reliable fetal sex determination can now be realised by using SRY gene amplification in maternal blood.Methods and Results: We report the use of fetal sex determination from maternal serum as a diagnostic tool for the control of embryo sexing (two cases) and to manage spontaneous pregnancies in couples included in a PGD programme for X-linked diseases (five cases). Fetal sex determination using SRY gene amplification in maternal serum were in complete concordance with fetal sex observed by cytogenetic analysis or ultrasound examination and at birth. This novel strategy allowed the PGD results to be controlled precociously and avoided the performance of invasive procedures in four cases of female fetus.Conclusions: This rapid fetal sex determination during the first trimester provides advantages to both clinicians and patients in a PGD centre. [ABSTRACT FROM AUTHOR]

MiR-31-3p expression has been shown to be a predictive biomarker for response to anti-epithelial growth factor receptor therapy in patients with RAS wild-type metastatic colorectal cancer (mCRC). To aid in the quantification of miR-31-3p expression in formalin-fixed paraffin-embedded (FFPE) primary tumor samples from patients with mCRC, a reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay was developed and validated. Assay development included the identification of a microRNA reference standard and the determination of an appropriate relative quantification cutoff for differentiating low versus high miR-31-3p expression. Sample specimens for the validation studies included both FFPE slides and shavings. Polymerase chain reaction (PCR) efficiency and linearity, analytical sensitivity and specificity, assay robustness, reproducibility, and accuracy were demonstrated across a number of test conditions and differing quantitative PCR platforms. The data from this study provide evidence as to the feasibility of quantifying the expression of miR-31-3p from FFPE tumor tissue using a standardized RT-qPCR assay. [ABSTRACT FROM AUTHOR]

Background: Rhesus D genotyping with cell-free fetal DNA currently is used throughout the world. Although this technique has spread rapidly, its optimal use is still a matter of debate. This screening test has been introduced mainly for the treatment of RhD-negative pregnant women during the third trimester of pregnancy, thereby avoiding systematic anti-D prophylaxis, yet such a strategy has proved cost-ineffective. Publications reporting on fetal RHD genotyping with cell-free DNA in maternal plasma, specifically during the first trimester of pregnancy, are scarce in the scientific literature.Objective: This study sought to assess the performance of noninvasive fetal Rhesus D genotyping in the first trimester of pregnancy with a single-exon real-time polymerase chain reaction assay.Study Design: This was a retrospective observational multicenter study. Cell-free fetal DNA was extracted from maternal blood of both nonimmunized and immunized women at 10-14 weeks of gestation. RHD sequence was determined by quantitative polymerase chain reaction, with amplification of exon 10. Results were compared with RhD phenotype data that were obtained by cord blood sampling of neonates.Results: In total, 416 serum samples from RhD-negative pregnant women were collected during the first trimester of pregnancy. The test's overall sensitivity and specificity were 100% (95% confidence interval, 96.9-100.0) and 95.2% (95% confidence interval, 90.5-97.6), respectively. The negative and positive predictive values were 99.8% (95% confidence interval, 94.9-100.0) and 97.1% (95% confidence interval, 94.2-98.6), respectively. Fetal RHD status was inconclusive in 9 cases (2.2%).Conclusion: Noninvasive fetal RHD determination by single-exon quantitative polymerase chain reaction during the first trimester of pregnancy exhibits high accuracy. [ABSTRACT FROM AUTHOR]

Both circulating Pneumocystis jirovecii DNA burden and (1→3)-β-d-glucan level are associated with the pulmonary fungal load in bronchoalveolar lavage fluids assessed using quantitative polymerase chain reaction in immunocompromised patients.Circulating Pneumocystis jirovecii DNA and (1→3)-β-d-glucan determined in 70 serum samples from immunocompromised patients were compared to fungal load in bronchoalveolar lavage fluids assessed using quantitative polymerase chain reaction. Both serum biomarkers are influenced by pulmonary fungal load, which should be taken into account when diagnosing Pneumocystis infection. [ABSTRACT FROM AUTHOR]

Coelocentesis may represent the ideal technique for early prenatal diagnosis. This study aimed to quantify the number of cells in coelomic fluid and to investigate the feasibility of interphase fluorescence in-situ hybridization (FISH) in uncultured coelomic cells for chromosomes X and Y in 12 samples of 0.4-0.8 ml of coelomic fluid obtained by transvaginal puncture at 8-9 weeks of gestation. It was found that the density of cells in the coelomic fluid was low and variable ranging from 0 to 10,600 cells/ml. The FISH analysis failed in three cases because of the absence or remarkably low number of cells. Among the remaining nine cases, FISH analysis led to an unambiguous result in all the samples except two in whom the FISH analysis clearly demonstrated a high count of maternal cells whereas the fluid was apparently not blood stained. The presence of such maternal cells, while their source and nature remaining unexplained, stressed the question of the absolute necessity to prove the fetal origin of the cells analysed. Whatever the cytogenetic analysis performed on coelomic fluid, combining a systematic exclusion of significant maternal contamination is recommended, using multiplex polymerase chain reaction for short tandem repeat analysis to cytogenetic analyses. [ABSTRACT FROM AUTHOR]

Abstract: Invading rodent species can harbor parasites with potential transmission to native rodents and/or humans. To investigate trypanosomes prevalence in rodents, the spleen of 76 rodents from Niger identified by their karyotype was used as a DNA source for Trypanosoma detection using a newly developed qPCR assay. Of the invasive black rat, Rattus rattus, 71% (10/14) were PCR positive as well as 6% (4/62) of native African rodents. Sequences of ∼400bp of the SSU rDNA gene identified phylogenetically close Trypanosoma lineages. Trypanosoma lewisi was present in all positive black rats and the sequences displayed 100% similarity with T. lewisi-infected humans in Senegal. T. lewisi was also detected in one Acomys johannis, suggesting a possible transmission to native species. In addition to improved knowledge of Trypanosoma diversity in rodents, our data underscore the introduction of the potentially pathogenic T. lewisi kinetoplastid through the human-mediated invasion of black rats all over West Africa. [Copyright &y& Elsevier]