PCR monitoring of parasitemia during drug treatment for canine Chagas disease.

To date, there is no clear standard to monitor drug treatment for canine Chagas disease. We used 2 real-time PCR (rtPCR) assays targeting Trypanosoma cruzi kinetoplast DNA (kDNA) and nuclear satellite DNA (nDNA) to detect T. cruzi in canine whole blood. Samples were collected randomly from 131 untreated dogs with unknown T. cruzi infection status in Texas. The kDNA-based rtPCR was slightly more sensitive (diagnostic sensitivity of kDNA = 49% vs. nDNA = 44%; p = 0.5732) but slightly less specific (diagnostic specificity of kDNA = 96% vs. nDNA = 97%; p > 0.9999) than the nDNA-based rtPCR. However, the differences in sensitivity and specificity between the nDNA- and kDNA-based rtPCR assays were not statistically significant. Using the nDNA- and kDNA-based qualitative rtPCR assays to monitor parasitemia from 137 itraconazole- and amiodarone-treated cases with nDNA- and kDNA-based PCR-positive baselines showed that the PCR positive rate decreased to 0% in 30 d. Using kDNA-based quantitative rtPCR to monitor normalized T. cruzi DNA copies in 4 representative dogs demonstrated that drug treatment could reduce parasite loads within 7-30 d. The kDNA-based qualitative rtPCR may be used for routine parasitemia screening of drug-treated Chagas-positive dogs, whereas nDNA-based qualitative rtPCR may be used for confirmation.

Mitochondrial and satellite real time-PCR for detecting T. cruzi DTU II strain in blood and organs of experimentally infected mice presenting different levels of parasite load.

The choice of cost-effective molecular methods for diagnosing and monitoring of Chagas disease before and after treatment is crucial in endemic countries with high patients' demand and limited financial resources. To this end, a kDNA was compared to a satellite real-time quantitative PCR (sat-qPCR), both amplifications using Sybr Green instead of Taqman hydrolysis probes. Non-isogenic Swiss albino mice were infected with a small inoculum of the highly virulent and partially resistant to benznidazole Y strain, belonging to T. cruzi discrete typing unit II (DTU-II) that predominates in Atlantic and Central Brazil. DNA from EDTA-blood samples and 10 organs of mice containing high, moderate and low parasite load levels were extracted by a highly used commercial kit and tested in triplicate, showing no disagreements between the two qPCRs. The melting temperature of positive samples was 79.8 °C ±â€¯1 °C for satellite-DNA and 78.1 °C ±â€¯1 °C for kDNA. DNA from genetically-related parasites such as Leishmania sp. showed no cross-reactions, but the sympatric T. rangeli was detected by both qPCRs, more effectively by kDNA than the satellite system, which required the equivalent of 50 parasites to give a positive result. Samples from infected mice, regardless of the type of biological matrix (blood or organ samples) or the parasite load gave positive results by both qPCRs. The sensitivity of sat-qPCR was 2 × 10-3 parasite or 240 target copies, and for kDNA, 2 × 10-4 parasite or 24 target copies. Regarding repeatability and reproducibility, the coefficient of variation (CV) was always < 25% in both assays; linearity of sat-qPCR was 0.991 (±0.002) and 0.991 (±0.008) for kDNA qPCR. In most collection times, the median Ct values found in blood and organs provided by sat-DNA and kDNA qPCRs were similar. In conclusion, although kDNA qPCR achieved a better analytical sensitivity, sat-qPCR gave better specificity results. Nevertheless, further research is intended to test other T. cruzi DTUs and chagasic patients' samples before these cost-effective techniques are incorporated into diagnostic routines.

Human circulating ribosomal DNA content significantly increases while circulating satellite III (1q12) content decreases under chronic occupational exposure to low-dose gamma- neutron and tritium beta-radiation.

A single exposure to ionizing radiation (IR) results in an elevated cell-free DNA (cfDNA) content in the blood plasma. In this case, the cfDNA concentration can be a marker of the cell death in the organism. However, a chronic exposure to a low-dose IR enhances both the endonuclease activity and titer of antibodies to DNA in blood plasma, resulting in a decrease of the total concentration of circulating cfDNA in exposed people. In this case, the total cfDNA concentration should not be considered as a marker of the cell death in an exposed body. We assumed that a pool of the cfDNA circulating in the exposed people contains DNA fragments, which are resistant to a double-strand break formation in the environment of the elevated plasma endonuclease activity, and can be accumulated in the blood plasma. In order to test this hypothesis, we studied the content of GC-rich sequences (69%GC) of the transcribed region of human ribosomal repeat (rDNA), as well as the content of AT-rich repeat (63%AT) of satellite III (1q12) in the cfDNA samples obtained from 285 individuals. We have found that a chronic exposure to gamma-neutron radiation (N=88) and tritium ß-radiation (N=88) evokes an increase of the rDNA content (RrDNA index) and a decrease of the satellite III content (RsatIII index) in the circulating cfDNA as compared with the cfDNA of non-exposed people (N=109). Such index that simultaneously displays both the increase of rDNA content and decrease of satellite III content in the cfDNA (RrDNA/RsatIII) can be recommended as a marker of chronic processes in the body that involve the elevated cell death rate and/or increased blood plasma endonuclease activity.

New Scheme of Intermittent Benznidazole Administration in Patients Chronically Infected with Trypanosoma cruzi: a Pilot Short-Term Follow-Up Study with Adult Patients.

There is a clinical need to test new schemes of benznidazole administration that are expected to be at least as effective as the current therapeutic scheme but safer. This study assessed a new scheme of benznidazole administration in chronic Chagas disease patients. A pilot study with intermittent doses of benznidazole at 5 mg/kg/day in two daily doses every 5 days for a total of 60 days was designed. The main criterion of response was the comparison of quantitative PCR (qPCR) findings prior to and 1 week after the end of treatment. The safety profile was assessed by the rate of suspensions and severity of adverse effects. Twenty patients were analyzed for safety, while qPCR was tested for 17 of them. The average age was 43 ± 7.9 years; 55% were female. Sixty-five percent of treated subjects showed detectable qPCR results prior to treatment of 1.45 (0.63 to 2.81) and 2.1 (1.18 to 2.78) parasitic equivalents per milliliter of blood (par.eq/ml) for kinetoplastic DNA (kDNA) qPCR and nuclear repetitive sequence satellite DNA (SatDNA) qPCR, respectively. One patient showed detectable PCR at the end of treatment (1/17), corresponding to 6% treatment failure, compared with 11/17 (65%) patients pretreatment (P = 0.01). Adverse effects were present in 10/20 (50%) patients, but in only one case was treatment suspended. Eight patients showed mild adverse effects, whereas moderate reactions with increased liver enzymes were observed in two patients. The main accomplishment of this pilot study is the promising low rate of treatment suspension. Intermittent administration of benznidazole emerges a new potential therapeutic scheme, the efficacy of which should be confirmed by long-term assessment posttreatment.

DYZ1 arrays show sequence variation between the monozygotic males.

BACKGROUND: Monozygotic twins (MZT) are an important resource for genetical studies in the context of normal and diseased genomes. In the present study we used DYZ1, a satellite fraction present in the form of tandem arrays on the long arm of the human Y chromosome, as a tool to uncover sequence variations between the monozygotic males. RESULTS: We detected copy number variation, frequent insertions and deletions within the sequences of DYZ1 arrays amongst all the three sets of twins used in the present study. MZT1b showed loss of 35 bp compared to that in 1a, whereas 2a showed loss of 31 bp compared to that in 2b. Similarly, 3b showed 10 bp insertion compared to that in 3a. MZT1a germline DNA showed loss of 5 bp and 1b blood DNA showed loss of 26 bp compared to that of 1a blood and 1b germline DNA, respectively. Of the 69 restriction sites detected in DYZ1 arrays, MboII, BsrI, TspEI and TaqI enzymes showed frequent loss and or gain amongst all the 3 pairs studied. MZT1 pair showed loss/gain of VspI, BsrDI, AgsI, PleI, TspDTI, TspEI, TfiI and TaqI restriction sites in both blood and germline DNA. All the three sets of MZT showed differences in the number of DYZ1 copies. FISH signals reflected somatic mosaicism of the DYZ1 copies across the cells. CONCLUSIONS: DYZ1 showed both sequence and copy number variation between the MZT males. Sequence variation was also noticed between germline and blood DNA samples of the same individual as we observed at least in one set of sample. The result suggests that DYZ1 faithfully records all the genetical changes occurring after the twining which may be ascribed to the environmental factors.

[Transcripts of satellite DNA in blood plasma: probable markers of tumor growth].

A recent study of human normal and tumor tissues revealed a high transcriptional activity of pericentromeric satellite DNA repeats (they produce half of all transcripts in tumor cells that is many times higher than in normal ones). It was found also that the two subtypes of satellite DNA (HSATII and GSATII) are transcribed reciprocally, i.e. there is a sharp prevalence of HSATII transcription in tumors, while GSATII transcription prevails in the corresponding normal tissues. As different RNAs are present in blood plasma, and some of them serve as effectivetumor markers, we attempted for the first time to evaluate satellite HSATII and GSATII RNAs in the blood plasma of healthy donors and cancer patients. The RT-PCR protocol designed for this purpose allowed us to detect transcripts of both HSATII and GSATII repeats. As it has been shown, HSATII transcripts are more abundant than GSATII ones in plasma of healthy donors and vice versa in plasma of cancer patients; these ratios being diametrically opposed to those that exist within the cells. Some suggestions concerning origins of circulating satellite RNAs and their probable role as tumor markers are discussed.

[The changing of cell-free DNA properties of peripheral blood and TCR-mutant cell frequency in individuals exposed to ionizing radiation].

Some properties of the cell-free DNA (cfDNA) of peripheral blood plasma were assessed in 153 employees of atomic industry enterprises. The contents of ribosomal repeat (rDNA) and its concentration in plasma increased in cfDNA of the group of persons in comparison with non-irradiated individuals. The contents of satellite III in cfDNA of donors and of irradiated persons do not differ and less than in DNA nucleus. The correlation between cumulative dose of radiation, contents of rDNA in cfDNA and the frequency of lymphocytes bearing mutations at T-cell receptor (TCR) locus was obtained. The definition of three indications in irradiated persons: the contents of ribosomal genes in cfDNA, TCR-mutant cell frequency and concentration of ribosomal genes in blood plasma--may be useful for revealing individuals in organism of which an intensive cell apoptosis takes place and there is an increased probability of carcinogenesis and of progress of disease of immune system.

In utero transplantation of human hematopoetic stem cells into fetal goats under B-type ultrasonographic scan: an experimental model for the study of potential prenatal therapy.

OBJECTIVE: Using fetal goats as animal models, to establish the methodology of in utero transplantation of human hematopoeitic stem cell (HSC) under B-scan ultrasonographic guidance for prenatal therapy. STUDY DESIGN: Human HSC were directly injected into the peritoneal cavities of the recipient fetal goats at 45-55 days of gestation (term: 145 days) under the guidance of B-type ultrasound scan. After birth, the peripheral blood was collected for fluorescence assisted cell sorting (FACS), quantitative real-time PCR and fluorescence in situ hybridization (FISH) to detect and analyze the presence of human cells in the recipients. RESULTS: The 32 recipients were born alive except one miscarriage. To test for the presence of human-goat chimeras, cells from 13 randomly selected transplanted goats were collected. FACS analyses showed the presence of human cells in all the transplanted goats tested. The average proportion of CD34+ cells and GPA+(glycophorin A) cells in the peripheral blood were 1.34 +/- 1.10% and 2.80 +/- 2.10%, respectively. No CD34+ or GPA+ cells were found in the non-transplanted goats tested. The results of the quantitative real-time PCR in three engraftment goats were 1.2 x 10(4), 2.9 x 10(4), and 3.2 x 10(4) copies of human GPA DNA per mug of genomic DNA. FISH experiments showed that cells containing human specific alpha-satellite DNA sequence were present in the peripheral blood of the transplanted goats. CONCLUSIONS: The method described herein is safe and reliable, with low miscarriage risk and high chimerism rate. This approach may provide a promising animal model for potential prenatal treatment.

Molecular diagnosis of primary liver cancer by microsatellite DNA analysis in the serum.

Frequent loss of heterozygosity of microsatellites markers on specific chromosomal region have been reported in various types of primary human cancer. The same loss of heterozygosity has also been identified in the matched plasma/serum DNA. Using 109 microsatellite markers representing 24 chromosomal arms, we have examined the loss of heterozygosity in 21 cases of hepatocellular carcinoma, six of cholangiocarcinoma, and 27 cases of chronic hepatitis or cirrhosis. All cases of the hepatocellular carcinoma showed deletion from two to 10 chromosomal arms, while deletion of chromosomes from two to eight regions was detected in five of six cholangiocarcinoma patients. One or more loss of heterozygosity in the paired serum DNA could be detected in 16 of 25 (76.2%) hepatocellular carcinoma patients. In contrast, no alterations in serum DNA test could be found in cholangiocarcinoma patients. Five of seven (71.4%) hepatocellular carcinoma patients with alpha-fetoprotein levels less than 20 ng ml(-1) produced positive serum DNA test. The profiles of 19 microsatellite markers gave a 100% positive predictive value and an 80.8% negative predictive value for hepatocellular carcinoma. In conclusion, we have determined a profile of microsatellite markers appropriate for differential diagnosis of primary liver cancer. The discovery may permit a high-throughput screening of hepatocellular carcinoma at an early stage of disease.

Genetic diversity and assignment tests among seven French cattle breeds based on microsatellite DNA analysis.

Genetic variability and relationships among six native French cattle breeds (Abondance, Tarentaise, Villard de Lans, Montbéliarde, Limousin, and Charolais) and one foreign breed (Holstein) were investigated using 23 microsatellite markers. These breeds were also compared with four Swiss breeds genotyped in a previously published study. Interestingly, the French alpine breeds have smaller population sizes but showed higher genetic variability than the larger Holstein breed. Neighbor-joining trees and PCA (principal components analysis) showed that alpine breeds tend to cluster together. Abondance and Tarentaise breeds were closely related, whereas the Holstein was highly differentiated from all breeds analyzed. Two different assignment tests for determining the breed of origin of individuals were compared: "direct" and "exclusion-simulation" approaches. The exclusion-simulation significance test correctly assigns fewer individuals than the direct approach but provides a confidence level (e.g., P < 0.01) for each individual being assigned. Accurate assignment with high statistical confidence is required for animal traceability. Unfortunately, the accuracy of assignment greatly decreases as the threshold level of confidence of assignment increases (e.g., from P < 0.05 to P < 0.001). Assignment accuracy also greatly declines as the level of population differentiation decreases below the level often found between related breeds (e.g., F(ST) < 0.1).

Evidence of abundant constitutive alkali-labile sites in human 5 bp classical satellite DNA loci by DBD-FISH.

Human blood leukocytes within an agarose matrix were deproteinized and exposed to an alkaline denaturation that generates single-stranded DNA (ssDNA) starting from the ends of spontaneous basal DNA breaks and alkali-labile sites. Since the amount of ssDNA produced within a specific sequence area may be detected by hybridization with a specific probe, we quantified this in situ in different satellite DNA loci (DBD-FISH: DNA Breakage Detection FISH). The DBD-FISH signal, corrected for the respective FISH signals in metaphase, was remarkably strong in the 5bp classical satellite DNA domains analyzed (D1Z1, D9Z3, DYZ1), intermediate in the classical satellite 1 DNA sequences, and low in the alphoid satellite regions (D1Z5, DXZ1, all centromeres). This result is evidence of a high density of constitutive alkali-labile sites, probably abasic sites, within the 5bp satellite DNA sequences in human blood leukocytes. The presence and relative abundance of alkali-labile sites could explain the high frequency of spontaneous breakage and rearrangements in pericentromeric heterochromatin of chromosomes 1, 9, and 16, but not in Yqh, when this chromatin is undercondensed through spontaneous or induced demethylation, i.e. ICF syndrome or 5-azacytidine treatment.

Microsatellite alterations and TP53 mutations in plasma DNA of small-cell lung cancer patients: follow-up study and prognostic significance.

BACKGROUND: Small-cell lung cancer (SCLC), one of the major types of lung cancer, is associated with many different somatic molecular genetic changes. These alterations, observed in tumor DNA, have also been identified in the plasma DNA of patients. We undertook the present study to make a prospective investigation into the correlation between abnormal plasma DNA and patient survival. PATIENTS AND METHODS: Thirty-five patients with SCLC were selected after histological diagnosis. Polymorphic markers (ACTBP2, UT762 and AR) were chosen for their reported high rate of alterations in SCLC and analyzed in tumor tissue, normal blood cells and plasma DNA. Furthermore, we looked for mutations of the TP53 gene in tumor and plasma DNA. RESULTS: In 25 patients (71%) at least one molecular change precisely matching that of the primary tumor was detected in the plasma DNA. No difference in survival was observed between patients with aberrant plasma DNA and patients without plasma DNA alterations. However, patients with microsatellite modifications and TP53 mutations concomitantly, showed a significant difference (P = 0.02) in survival compared with patients bearing only one of these molecular changes. In 15 cases it was possible to find a correlation either between tumor response and disappearance of abnormal plasma DNA, or tumor progression and persistence of plasma DNA alterations. CONCLUSIONS: Free plasma DNA with molecular alterations is present to a high degree in plasma DNA of SCLC patients and may have a role as a prognostic factor.

Report of the European DNA Profiling Group (EDNAP)--an investigation of the hypervariable STR loci ACTBP2, APOAI1 and D11S554 and the compound loci D12S391 and D1S1656.

This paper describes the results of three collaborative exercises which continues the EDNAP theme to explore whether uniformity of DNA profiling results could be achieved between European laboratories using STRs. In an earlier exercise, complex hypervariable AAAG-repeat STR loci were investigated, but reproducibility was found to be poor because of the variation of techniques used by participating laboratories. In the exercise reported here, an internal allelic ladder composed of ACTBP2 and D11S554 fragments was distributed. This ladder was used to size ACTBP2 analysed by a "singleplex" PCR amplification and D11S554 combined with APOAI1 in a separate "duplex" reaction. Laboratories were asked to test 7 blood stains, one of which was a known control, and to report the results to the co-ordinating laboratory. The exercise demonstrated that ACTBP2 showed good reproducibility between laboratories, whereas further testing would be needed to validate APOAI1 and D11S554 for interlaboratory comparisons. In separate exercises, the simple loci D12S391 and D1S1656 were tested; both of these showed excellent reproducibility between laboratories.

Somatic mutation processes at a human minisatellite.

Germline instability at human minisatellites frequently involves complex inter-allelic transfers of repeat units usually restricted to one end of the repeat array and apparently regulated by flanking DNA. In contrast, nothing is known about the structural basis of somatic instability at minisatellites. An electrophoretic size-enrichment strategy was therefore developed at minisatellite MS32 (D1S8) to enable rare abnormal-length mutants to be detected, validated and quantitated in blood DNA by single molecule PCR. Structural analysis of rare mutant alleles in blood revealed simple deletions/duplications of repeat unit blocks located at random along the tandem repeat array, a mode of mutation completely different from that seen in sperm. Furthermore, allele-specific suppression of sperm instability at MS32 did not affect somatic instability. These data suggest that conversion-based minisatellite mutation in sperm is completely germline-specific and most likely meiotic in origin. Somatic instability appears to occur by a separate pathway involving replication slippage or, more likely, intra-allelic unequal crossing over.

Microsatellite alterations in plasma DNA of small cell lung cancer patients.

Microsatellite instability is an important characteristic of many tumor types especially those associated with hereditary non-polyposis colorectal carcinoma (HNPCC) syndrome. Microsatellite alterations in 50% of primary small cell lung carcinoma (SCLC) have been found. These alterations were also found in the sputum. Because neoplastic characteristics such as decreased strand stability9 and ras mutations have been found in the plasma DNA of cancer patients, we looked for microsatellite alterations in the plasma of SCLC patients. A microsatellite alteration was present in 16 out of 21 (76%) SCLC tumors and in 15 out of 21 (71%) plasma samples. In one case, the alteration was present only in the plasma DNA. If confirmed in larger studies, microsatellite analysis of plasma DNA might constitute a new tool for tumor staging, management and, possibly, detection.

Microsatellite alterations in serum DNA of head and neck cancer patients.

Microsatellite DNA alterations are an integral part of neoplastic progression and are valuable as clonal markers for the detection of human cancers. Moreover, recent evidence suggests that senescent tumor cells may release DNA into the circulation, which is subsequently carried by and therefore enriched in the serum and plasma. We tested 21 patients with primary head and neck squamous cell carcinoma (HNSCC) by polymerase chain reaction (PCR)-based microsatellite analysis of DNA from lymphocytes and paired serum samples. Patients were scored for alterations as defined by the presence of new alleles (shifts) or loss of heterozygosity (LOH) in serum at each of 12 markers and then compared with primary tumor DNA. Six out of 21 patients (29%) were found to have one or more microsatellite alterations in serum precisely matching those in the primary tumors. All six patients had advanced disease (stage III or IV); five of these patients had nodal metastases, three later developed distant metastases, and four died of disease. Microsatellite analysis of serum represents a novel method for the detection of circulating tumor cell DNA. If these results are confirmed in larger studies, microsatellite markers may be useful in assessing tumor burden in cancer patients.

Microsatellite polymorphism in commercial broiler and layer lines estimated using pooled blood samples.

For 17 microsatellite markers, allele frequencies were determined in nine highly selected commercial broiler and six highly selected commercial layer lines using pooled blood samples from 60 animals. The average number of marker alleles was 5.8 over all lines, 5.2 over broiler lines, and 3.0 over layer lines. The average number of marker alleles within a line was 2.9, 3.6, and 2.0 for all, broiler, and layer lines, respectively. Over all 15 lines, the average percentage of heterozygosity was 42, whereas the heterozygosity in the broiler lines was 53% and in the layer lines only 27%. In broiler lines, 50% of the marker-line combinations showed a heterozygosity above 60%, whereas this was only 5% in layer lines. Estimation of allele frequencies with microsatellite markers was first assessed in pooled and individual samples before usage in the commercial lines. Allele frequencies for 19 microsatellite markers were estimated in chicken pooled blood samples and compared with allele frequencies from individual typed animals. Similar results were obtained when pooled blood samples (heterozygosity of 35.3%) or individual typed animals (heterozygosity of 34.2%) were used. The method to determine allele frequencies using pooled blood samples is faster, cheaper, and as reliable and repeatable as determining allele frequencies using individual typings.

Characterization of four microsatellites in an Italian population and their application to paternity testing.

Microsatellites have recently been used for linkage analysis of genetic diseases and for DNA fingerprinting in forensic medicine. In the present study the heterozygosity, PIC values and allele distributions of four microsatellites, D8S85, D8S88, D5S346 and D7S460, in an Italian population have been investigated. After amplification with primers specific for each locus, alleles were separated and detected by denaturing gel electrophoresis and ethidium bromide staining. High heterozygosity and PIC values were observed for all microsatellites in accordance with data in other Caucasian populations. However, different allele distributions for D8S85, D8S88 and D5S346, due to the presence of additional bands or to different frequencies, were found. D7S460, which has never been fully characterized before, appeared to have five alleles in the range 172 to 188 bp. When used for paternity testing, all microsatellites gave results which were consistent with those obtained with established markers, including apo B 3'HVR, D1S80 and COL2A1. This indicates that D8S85, D8S88, D5S346 and D7S460 may be useful as additional informative markers or for solving discrepancies in selected cases.