PCR In Blood Samples Research Articles

Prevalence of Anaplasma phagocytophilum in ruminants, rodents and ticks in Gansu, north-western China

The zoonotic rickettsial pathogen Anaplasma phagocytophilum has a broad geographical distribution and a high degree of biological and clinical diversity. To determine the prevalence of Anaplasma phagocytophilum in the Gannan Tibetan Autonomous Prefecture of Gansu Province, north-western China, four ruminant species, one rodent and one tick species were examined for Anaplasma phagocytophilum infection. DNA from Anaplasma phagocytophilum was detected by nested PCR in blood samples from 21/49 sheep (42.9 %), 35/91 goats (38.5 %), 51/158 yaks (32.3 %) and 7/20 cattle-yaks (35.0 %), and in spleen samples from 2/12 rodents (16.7 %). For samples from tick larvae and nymphs, 105 pools were tested; one of 46 larval tick pools was positive and seven of 59 nymphal tick pools were positive. For adult ticks, 40/598 female ticks (6.7 %) and 26/528 male ticks (4.9 %) were positive. The prevalence of Anaplasma phagocytophilum in female ticks was higher than that in males, although the difference was not statistically significant (P>0.05). Sequences analysis based on the 16S rRNA gene indicated that the strains in the study area were distinct from previously reported Anaplasma phagocytophilum in other continents. These results add new information on the epidemiology of Anaplasma phagocytophilum and indicate the tick-animal cycle of anaplasmosis in the area. To the best of our knowledge, this is the first report of Anaplasma phagocytophilum infection in Gansu Province in north-western China.

Detection ofToxoplasma gondiiDNA by PCR in blood samples collected from pregnant Saudi women from the Aseer region, Saudi Arabia

BACKGROUND AND OBJECTIVESToxoplasmosis, caused by Toxoplasma gondii, is diagnosed mainly by serological methods that are hindered by insufficient sensitivity. When it fails, it becomes necessary to rely on either direct detection of the parasite or DNA detection by polymerase chain reaction (PCR). We aimed to establish molecular tools for toxoplasmosis research in the country by using PCR targeting the B1 gene and compare it with ELISA results.DESIGN AND SETTINGConducted at the College of Science, King Khalid University, Abha, Saudi Arabia between January 2009 and April 2010 on Saudi pregnant women attending three major hospitals in the Aseer region.PATIENTS AND METHODSPeripheral blood samples (n=137) were collected from patients. DNA was extracted and the B1 T gondii gene was amplified by PCR. The amplicons were visualized and sequenced, and the results were analyzed. For comparison, sera were tested for anti-T gondii IgG and IgM by enzyme-linked immunosorbent assay (ELISA).RESULTSOf the 137 samples tested, the B1 gene could be amplified in 56 cases (41%) by PCR. DNA sequencing confirmed these results. IgM-ELISA assay detected 9 (6.5%) of these cases. The results of immunoglobulin G detection were positive in 53 (38.6%) of the patients.CONCLUSIONThe present study showed the need for PCR as a confirmatory assay in addition to serological assays to detect recent infection. We recommend national implementation of these molecular diagnostic tools.

Molecular Detection of Theileria Annulata in Egyptian Buffaloes and Biochemical Changes Associated with Particular Oxidative Changes

This investigation is performed on 100 buffaloes native Breed in Kaliobia governorate Egypt aged from 1-3years, these animals suffered from fever (41℃) enlargement lymph node, inappetence, pale mucous membrane, increase lacrimation, emaciation and corneal opacity in progressive stages when these animals were subjected to microscopic examination the degree of parasitaema was recorded as the percentage of infected red blood cells in 1000red blood cell counted .We report the detection of Theileria annulata by PCR in blood samples obtained from buffaloes supposed to be infected. The assay employs primers specific for the gene encoding Theileria annulata 185RNA biochemical changes associated with Theileria annulata in these buffaloes particular emphasis to the oxidative stress the reduce TAC level may reflect adecrease in antioxidant capacity . Blood collected from all animals on EDTA to microscopic examination and PCR to determine type of Theileria.

Efficacy of chloroquine plus primaquine treatment and pfcrt mutation in uncomplicated falciparum malaria patients in Rangamati, Bangladesh

A combination of chloroquine (CQ) and primaquine (PQ) had been used as the first-line treatment of uncomplicated Plasmodium falciparum malaria in Rangamati, Bangladesh until the end of 2004. Doctors or medical staffs had felt that CQ plus PQ had become less effective against uncomplicated falciparum malaria patients, but that it was more effective against the minority-indigenous patients than the Bengali patients. The efficacy of CQ plus PQ and the mutation status of the CQ resistance transporter ( pfcrt) gene of infecting P. falciparum were, thus, investigated for 45 uncomplicated falciparum malaria patients in Rangamati in 2004. The total failure rate was 57.8%. One or two pfcrt sequences (C IETH and SMN TH at positions 72, 74–76, and 97, mutation underlined) with K76T mutation known to be related to CQ-resistant phenotype were detected in 38 patients' blood samples. Of the 38 patients, in total 15 patients (14/25 minority-indigenous and 1/13 Bengali patients) resulted in adequate clinical and parasitological response (ACPR). There was a statistically significant difference in ACPR rate between the minority-indigenous patients and the Bengali patients. P. falciparum with mutant or resistant pfcrt ( pfcrt-resistant) was detected by PCR in blood samples on day 28 for 10 ACPR minority-indigenous patients but not for the only one Bengali ACPR patient, who all were infected with pfcrt-resistant P. falciparum on day 0. The minority-indigenous patients, but not Bengalis, are suggested to be often cured by CQ plus PQ, leaving a very few parasites detectable only by PCR, even when they are infected with pfcrt-resistant P. falciparum.

Phase II trial of topical heparin as treatment for patients (pts) with hand-foot syndrome (HFS) induced by capecitabine (CAP).

e19671 Background: Molecular physiopathology of HFS induced by CAP is unclear. Interruption or dose reduction of CAP is the only effective strategy. Aims: to evaluate efficacy and safety of a topical heparin Menaven gel® and to analyse expression levels of four biomarkers. Methods: This is a Simon 2-stage phase 2 trial: if >3 responses were achieved in the first 12 pts, the study proceeded to its second stage. Key entry criteria: Age >18 years, diagnosis of cancer in treatment with CAP, as single-agent or in combination, and presence of HFS grade (G) 1 or 2 (NCI-CTCAE version 3.0). Topical heparin was administered in palms and soles four times/day for three weeks (w). No other concurrent treatment for HFS was allowed. Physical examination and Visual Analogic Pain Scale (VAS) were assessed at baseline, and weekly. Changes were photographically documented by investigator. Skin biopsies of palms and blood samples were obtained at baseline and after 3w. Expression of thymidine phosphorylase (TP), dihydropyrimidine-dehydrogenase (DPD), Fibroblast Growth Factor (FGF) and Keratinocyte Growth Factor (KGFR) were analyzed by immunohistochemistry (IHC) and immunoperoxydase revelation in skin. TP and DPD mRNA expression was determined by quantitative real-time PCR in blood samples. Results: Between Oct/2010 and Jan/2011, 21 pts were included. So far, 16 pts are evaluable: 6 males, median age 61 y. Breast cancer (n=8), colorectal cancer (n=5) and others (n=3). Baseline HFS was G1 18% and G2 82%. CAP was administered as single-agent (n=4) and combination (n=12). Improvement of HFS was observed in 68% and 75% pts in w3 and 6, respectively. Median VAS decreased from 5,6 at baseline to 1,1 in w3, p<0,001. No changes were observed in the skin biopsies. However, high expression of TP was found in conjunction of decreased DPD in blood samples in 12pts with clear response. Definitive results from 21 subjects will be presented at the meeting. Conclusions: Our preliminary results suggest that topical heparin may be effective to relief CAP-induced HFS. A study evaluating topic heparin as a preventive therapy of HFS is planned.

Human Coinfection with Bartonella henselae and Two Hemotropic Mycoplasma Variants Resembling Mycoplasma ovis

Two variants of an organism resembling the ovine hemoplasma, Mycoplasma ovis, were detected by PCR in blood samples from a veterinarian in Texas. Coinfection with similar variants has been described in sheep. This represents the first report of human infection with this organism. The veterinarian was coinfected with Bartonella henselae.

Detection of a Theileria species in dogs in South Africa

A Theileria species was detected by PCR in blood samples collected from dogs in the Pietermaritzburg area and was also found in dogs presented at the Outpatients Clinic of the Onderstepoort Veterinary Academic Hospital (OVAH), in the Pretoria area, South Africa. In the Pietermaritzburg area, 79 of the 192 samples were positive, while 3 out of 1137 of the Onderstepoort samples were positive. Three positive samples from Pietermaritzburg were co-infected with Ehrlichia canis. PCR positive samples were further analysed by the Reverse Line Blot (RLB) and sequence analysis. Phylogenetic analysis of the 18S rRNA full-length gene sequences of one sample (VT12) from Pietermaritzburg and two samples from OVAH (BC281 and BC295) revealed a close relationship with sequences of Theileria species (sable). Clinical signs of the dogs that were examined at Pietermaritzburg and OVAH included an immune-mediated condition with severe thrombocytopenia. These findings identify a Theileria sp. in dogs for the first time in South Africa and add yet another microorganism to the growing list of haemoprotozoan parasites infecting dogs worldwide. The clinical significance of this infection in dogs is poorly resolved.

Tuberculosis miliar en paciente tratado con instilaciones intravesicales de bacilo de Calmette-Guérin

Tuberculosis miliar is a very rare complication of bacillus Calmette-Guérin (BCG) immunotherapy that is associated to high mortality. Thus, early diagnosis and treatment is essential. At present, there is a polymerase chain reaction technique (PCR) which is a rapid diagnostic method with elevated sensitivity. Treatment with tuberculostatic agents should be initiated as soon as possible, the combination of corticosteroids and cycloserine in serious cases being advisable. A case of a 75 year old patient with intravesical BCG instillations after a transurethral resection of bladder cancer is presented. He was admitted to the Intensive Care Unit (ICU) due to severe acute respiratory failure evolving to acute respiratory distress syndrome (ARDS). Mycobacterium bovis DNA was identified by PCR in blood samples and bronchoaspirate (BAS). He was treated with tuberculostatic agents and corticosteroids, dying due to respiratory failure.

Global Gene Expression Profiling of Circulating Endothelial Cells in Patients with Metastatic Carcinomas

Increased numbers of endothelial cells are observed in peripheral blood of cancer patients. These circulating endothelial cells (CECs) may contribute to the formation of blood vessels in the tumor or reflect vascular damage caused by treatment or tumor growth. Characterization of these cells may aid in the understanding of the angiogenic process and may provide biomarkers for treatment efficacy of angiogenesis inhibitors. To identify markers typical for CECs in cancer patients, we assessed global gene expression profiles of CD146 immunomagnetically enriched CECs from healthy donors and patients with metastatic breast, colorectal, prostate, lung, and renal cancer. From the generated gene profiles, a list of 61 marker genes for CEC detection was generated, and their expression was measured by real-time quantitative PCR in blood samples from 81 metastatic cancer patients and 55 healthy donors that were immunomagnetically enriched for CECs. A set of 34 genes, among which novel CEC-associated genes, such as THBD, BST1, TIE1, POSTN1, SELE, SORT1, and DTR, were identified that were expressed at higher levels in cancer patients compared with healthy donors. Expression of the VWF, DTR, CDH5, TIE, and IGFBP7 genes were found to discriminate between cancer patients and "healthy" donors with a receiver operating characteristic curve accuracy of 0.93. Assessment of the expression of these genes may provide biomarkers to evaluate treatment efficacy.

Validation of a PCR for diagnosis of typhoid fever and salmonellosis by amplification of the hilA gene in clinical samples from Colombian patients.

Validation of a PCR test to detect hilA gene sequences of Salmonella spp. was performed in blood and faeces samples from typhoid fever and salmonellosis patients. Sensitivity (S), specificity (SP), positive predictive value (PPV) and negative predictive value (NPV) of the PCR in blood samples were performed by testing: 37 patients with clinical diagnosis of typhoid fever, 34 of them confirmed by isolation of S. Typhi from blood cultures; 35 patients infected with other pathogens corroborated by blood culture (Klebsiella pneumoniae, 9; Serratia marcescens, 5; Escherichia coli, 4; Pseudomonas aeruginosa, 9; Providencia alcalifaciens, 4 and Enterobacter cloacae, 4) and blood samples from 150 healthy volunteers. To evaluate S, SP, PPV and NPV of the PCR in faeces samples we studied: 34 patients with enteritis due Salmonella spp. (S. Typhimurium, 21; S. Enteritidis, 9; S. Choleraesuis, 3 and S. Agona, 1); faeces samples from 35 patients with enteric infection due to Shigella sonnei (8), Shigella flexneri (10), enteropathogenic E. coli (12), Aeromonas hydrophila (5) and faeces samples from 150 healthy volunteers. The S, SP, PPV and NPV of the PCR in blood samples were all 100 %. PCR detected three patients with clinical diagnosis of typhoid fever and negative blood cultures. In faeces samples, S was 97 %, SP 100 %, PPV 100 % and NPV 99 %. The lowest number of c.f.u. ml(-1) detected by PCR in blood samples was 1 x 10(1) and in faeces samples 4 x 10(2).

Desarrollo y evaluación de una prueba de reacción en cadena de la polimerasa (PCR), utilizando la secuencia del gen hilA para diagnóstico de fiebre entérica por Salmonella spp.

Typically, diagnosis of enteric fever due to Salmonella spp. is by bacterial isolation from blood culture; however, the blood culture method is slow, not always available, and not informative in patients with antibiotic treatment. Salmonella spp. uses the hilA gene (component of the pathogenicity island I) to invade epithelial cells and produce infection. Using the hilA gene sequence a PCR test was designed to detect Salmonella in blood samples. The sensitivity (S), specificity (SP), positive predictive value (PPV) and negative predictive value (NPV) of the PCR method were obtained by testing the blood samples from 34 patients with suspected of enteric fever. Presence of S. typhi was confirmed by blood culture. Blood samples were also tested from 35 patients with infections due to other non-Salmonella pathogens, again corroborated by blood culture (Klebsiella pneumoniae, 9; Serratia marcescens, 5; Escherichia coli, 4; Pseudomonas aeruginosa, 9; Providencia alcalifaciens, 4; Enterobacter cloacae, 4). Control samples were obtained from 150 healthy volunteers. The S, SP, PPV and NPV for the PCR method were all 100%. The lowest number of colony forming units/ml detected by PCR in blood samples was 10.

Enzootic Transmission of Anaplasma bovis in Nantucket Cottontail Rabbits

Serological studies of cottontail rabbits sampled from Nantucket Island, Mass., have suggested exposure to at least two ehrlichiae. The agent of human granulocytic ehrlichiosis (Anaplasma phagocytophilum) is intensely enzootic in rabbits there, but the identity of the other ehrlichial infection remains undescribed. We sampled rabbits over five transmission seasons and tested their blood and tissues for evidence of infection using PCR targeting an Ehrlichia genus-wide 16S rDNA target. Sequence analysis of positive amplicons revealed the presence of Anaplasma bovis, an agent not known to be present in North America. The average annual prevalence of A. bovis within rabbits, as determined by PCR of blood samples, was 18%. Haemaphysalis leporispalustris appears to serve as vector. The public health (human or veterinary) significance of this finding remains speculative.

Natural Ehrlichia phagocytophila transmission coefficients from sheep 'carriers' to Ixodes ricinus ticks vary with the numbers of feeding ticks.

In a longitudinal study in a UK upland site, 38% of adult sheep were detected as infected with the tick-borne bacterium Ehrlichia phagocytophila by PCR of blood samples. Infection prevalence declined significantly with sheep age but varied significantly and non-linearly with the number of adult Ixodes ricinus ticks feeding per sheep. These findings suggested that under conditions of natural repeated tick-borne challenge sheep remain partially susceptible to re-infections, but the likelihood of re-infection depended on the numbers of feeding ticks. Transmission efficiency from sheep to immature ticks also varied significantly and non-linearly with the number of adult ticks feeding per sheep: transmission efficiency was almost zero in sheep with low adult tick infestations rising to 30% at certain levels of adult tick infestation. Infection intensity in infected engorged immature ticks also varied with the number of adult ticks feeding per sheep, but neither prevalence nor intensity of infection in engorged ticks were related to sheep blood PCR result. These findings suggest that variation in the numbers of ticks feeding per sheep may influence E. phagocytophila transmission by direct effects on transmission at the tick-host interface.

Detection of granulocytic Ehrlichia species DNA by PCR in persistently infected dogs

Three female beagle dogs inoculated with granulocytic Ehrlichia species were monitored for four to six months to determine whether there was evidence that the organisms persisted. The dogs were inoculated...

Allele-related variation in minisatellite repeats involved in the transcription of the blood group ABO gene.

Since the cloning in 1990 of cDNA corresponding to mRNA transcribed at the blood group ABO locus, polymorphisms at the ABO locus and phenotype-genotype correlation have been analysed by several investigators. An enhancer-active minisatellite motif reported to contain four 43-bp repeats has been analysed by PCR in blood samples from 160 random Swedish blood donors. Different sizes of the DNA fragments obtained led to further analysis by direct sequencing. Fragments with either one or four 43-bp repeats were identified. A nucleotide substitution (G-->A) at nt. 41 of 43 was found in all alleles with only one repeat. Correlation with the ABO genotypes of the samples, as determined by a panel of ABO genotyping techniques, revealed an allele-related variable number of tandem repeats (VNTR). The A1 and the infrequent O2 allele had only one repeat whilst A2, B, O1 and O1v had four repeats and thus generated longer (by 129 bp) fragments. A further 74 samples obtained from various geographical areas/ethnic groups indicated a widespread correlation with few exceptions. In conclusion, a novel ABO polymorphism located in the 5'-nontranslated region involved in transcriptional regulation of the ABO gene is reported and its relationship to common alleles at this locus defined.

Detection of Pneumocystis carinii DNA in blood specimens from human immunodeficiency virus-infected patients by nested PCR.

The detection of Pneumocystis carinii DNA in blood by PCR could be useful for studying the natural history of pneumocystosis and could also be a noninvasive diagnostic method. The results of previous studies are nevertheless conflicting. In our study, we compared three commercially available DNA extraction kits (GeneReleaser, QIAamp Tissue Kit, and ReadyAmp Genomic DNA Purification System) and proteinase K and proteinase K-phenol-chloroform treatments for the extraction of P. carinii DNA from dilutions of a P. carinii f. sp. hominis cyst suspension mixed with human whole blood. A rapid and simple nested PCR protocol which amplifies a portion of the mitochondrial large-subunit rRNA gene was applied to all the extraction products. The QIAmp Tissue Kit was the most effective kit for the isolation of amplification-ready P. carinii DNA and was used with nested PCR for the testing of whole-blood specimens from 35 immunocompetent control patients and 84 human immunodeficiency virus (HIV)-infected patients investigated for pulmonary disease and/or fever. In HIV-infected patients, P. carinii DNA was detected by nested PCR in blood samples from 3 of 14 patients with microscopically proven P. carinii pneumonia, 7 of 22 patients who were considered to be colonized with P. carinii, and 9 of 48 patients who were neither infected nor colonized with P. carinii. P. carinii DNA was not detected in blood specimens from the 35 immunocompetent patients. P. carinii DNA in blood might represent viable P. carinii organisms or DNA complexes released from pulmonary phagocytes. In conclusion, P. carinii DNA may be detected in whole blood from HIV-infected patients, but the nature and the meaning of the circulating form of P. carinii remain to be established.