Paracoccidioides brasiliensis exoantigens: recognition by IgG from patients with different clinical forms of paracoccidioidomycosis

Serum antibodies against antigens of Paracoccidioides brasiliensis have been one of the major diagnostic indicators of paracoccidioidomycosis (PCM). In the present study, released antigen preparations (exoAg) obtained from P. brasiliensis isolates were characterized in terms of their protein components electrophoretically detectable and recognizable by sera (IgG) of patients. Among five different isolates (DGO, C-9, BAT, Pb-18 and B-339) the electrophoretic profiles of exoAg varied greatly. A total of 28 different components were detected, 11 of them shared by all isolates. The most representative preparation was BAT-exoAg, which presented the largest number of protein bands (23) and the highest frequency of reacting bands (19) with sera from patients with active PCM (n = 40). Six bands reacted with more than 20% of sera. Independently of clinical forms, the sera recognized the 43-kDa (97% of tested sera), 160-kDa (78% of tested sera) and 70-kDa (60% of tested sera) antigens more frequently. Sera from patients with severe forms of acute (n = 14) or chronic (n = 10) PCM recognized a greater number of antigens, with a higher frequency, than those from moderate forms. The most pronounced reduction in reactivity was provided by sera of patients that became asymptomatic at the beginning of treatment. Remnant reactivity with BAT-exoAg was detected after clinical recovery, especially with those of 43, 70 and 160 kDa. The latter presented a stable recognition frequency (60%) during the entire follow-up, allowing us to suppose that the IgG reactivity against the 160-kDa antigen constitutes a possible persistent marker of P. brasiliensis infection.     

Polymerase chain reaction for detection of legionellae DNA in urine samples from patients with community-acquired pneumonia

Polymerase chain reaction (PCR) was used for detectingLegionella DNA in water, sputum, tracheal aspirate and bronchoalveolar lavage fluid. There is paucity of data on the use of PCR for detection ofLegionella in serum and urine samples. In 82 patients admitted with community-acquired pneumonia, urinary PCR was used in addition to urinary antigen assay forLegionella pneumophila serogroup 1 and serological tests (indirect immunofluorescence and ELISA) in paired sera. PCR was positive in urine samples from 21 patients (26 %): in six of seven patients with acute legionellosis by CDC criteria, and 15 patients with negative urine antigen showing no fourfold rise in antibody titers in immunofluorescence test.     

Evaluation of different DNA extraction procedures for the detection of Salmonella from chicken products by polymerase chain reaction

Polymerase chain reaction (PCR) was used after a short pre-enrichment culture to detect Salmonella subspecies in chicken fillets. A direct PCR assay performed with chicken meat inoculated with Salmonella Typhimurium produced no PCR products. Six different DNA extraction protocols were tested to recover efficiently Salmonella DNA after a short incubation period. Three of them gave results that were reliable, rapid and sensitive. Successful protocols used Proteinase K and/or a centrifugation step to concentrate the samples. For reliable detection of Salmonella subspecies, a few thousand bacterial cells per ml must be present. To obtain this number of bacterial cells with an inoculation of about one cell in 25 g of ionized food products, it was necessary to incubate samples for at least 10 h before PCR. A larger inoculum of approximately 10 cells in 25 g of ionized food products, required 8 h in culture broth to give positive results by PCR-based assay.     

Monocyte adherence to Paracoccidioides brasiliensis,zymosan-C3b and erythrocyte-hemolysin in patients with paracoccidioidomycosis

Independent and dependent (C3b/Fc receptors) opsonic adherence ability of monocytes from thirty-three patients with acute or chronic paracoccidioidomycosis and from 13 healthy individuals were studied in the presence of Paracoccidioides brasiliensis (Pb), Paracoccidioides brasiliensis opsonized by patient's serum (PbPS) or normal serum (PbNS), zymosan opsonized by fresh sera from healthy donors (ZyNS) and erythrocytes opsonized by hemolysin (EA). Statistically significant differences concerning the percentage of adhered monocytes to PbPS (number of adhered monocytes/total number of monocytes) were detected between control and chronic (active and inactive) groups. Significant differences in relationship to the mean number of PbPS (number of fungi in monocytes/total number of monocytes) were also observed between control and chronic active mycosis. Present data suggest that patients with chronic disease have more ability in the first step of phagocytic activity, considered as the main effector mechanism to control the dissemination and severity of paracoccidiodomycosis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Homogeneous detection of cyanobacterial DNA via polymerase chain reaction

Aims: To design a primer set enabling the identification through PCR of high‐quality DNA for routine and high‐throughput genomic screening of a diverse range of cyanobacteria. Methods and Results: A codon‐equivalent multiple alignment of the phycocyanin alpha‐subunit coding sequence (cpcA) of 22 cyanobacteria was generated and analysed to produce a single degeneracy primer set with virtually uniform product size. Also, an 18S ribosomal RNA detection set is proposed for rejecting false positives. The primer sets were tested against five diverse cyanobacteria, Chlorella vulgaris, Saccharomyces cerevisiae, and Escherichia coli. All five cyanobacteria showed positive amplification of cpcA product with homogeneous fragment length, and no products were observed for any other organism. Additionally, the only product formation observed for the 18S rRNA set was in C. vulgaris and S. cerevisiae. Conclusions: The newly proposed primer set served as effective check primers for cyanobacteria. Cyanobacteria gDNA had a positive, homogenous result, while other bacteria, eukaryotes and alga tested were negative. Significance and Impact of the Study: These novel, broad‐spectrum primers will greatly increase the utility of PCR on newly discovered cyanobacterial species.     

Evaluation of the Applied Biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA

In a period where the proportion of culture confirmed cases in the UK has been steadily declining, diagnosis by PCR has been used to increase the number of confirmed cases and provide additional epidemiological data. This report presents a comparative evaluation of the fluorogenic probe-based 5' exonuclease assay (Taqman) using the Perkin-Elmer Applied Biosystems automated sequence detection system 7700 with previously reported polymerase chain reaction enzyme-linked immunosorbent (PCR ELISA) assays for the detection of meningococcal DNA in CSF, plasma and serum samples. Taqman assays developed were based on the detection of a meningococcal capsular transfer gene (ctrA), the insertion sequence IS1106 and the sialytransferase gene (siaD) for serogroup B and C determination and compared with similar assays in a PCR ELISA format. The Taqman ctrA assay was specific for Neisseria meningitidis, however the IS1106 assay gave false positive reactions with a number of non-meningococcal isolates. Sensitivity of the Taqman ctrA, IS1106 and siaD assays testing samples from culture-confirmed cases were 64, 69 and 50%, respectively, compared with 26, 67 and 43% for the corresponding PCR ELISA assays. Improvements to the DNA extraction procedure has increased the sensitivity to 93 and 91% for the TaqMan ctrA and siaD assays, respectively, compared to culture confirmed cases. Since the introduction of Taqman PCR a 56% increase in laboratory confirmed cases of meningococcal disease has been observed compared to culture only confirmed cases. The developed Taqman assays for the diagnosis of meningococcal disease enables a high throughput, rapid turnaround of samples with considerable reduced risk of contamination.     

Cell-free antigens from Paracoccidioides brasiliensis drive IL-4 production and increase the severity of paracoccidioidomycosis

The thermally dimorphic fungus Paracoccidioides brasiliensis (Pb) is the causative agent of paracoccidioidomycosis (PCM), one of the most frequent systemic mycosis that affects the rural population in Latin America. PCM is characterized by a chronic inflammatory granulomatous reaction, which is consequence of a Th1-mediated adaptive immune response. In the present study we investigated the mechanisms involved in the immunoregulation triggered after a prior contact with cell-free antigens (CFA) during a murine model of PCM. The results showed that the inoculation of CFA prior to the infection resulted in disorganized granulomatous lesions and increased fungal replication in the lungs, liver and spleen, that paralleled with the higher levels of IL-4 when compared with the control group. The role of IL-4 in facilitating the fungal growth was demonstrated in IL-4-deficient- and neutralizing anti-IL-4 mAb-treated mice. The injection of CFA did not affect the fungal growth in these mice, which, in fact, exhibited a significant diminished amount of fungus in the tissues and smaller granulomas. Considering that in vivo anti-IL-4-application started one week after the CFA-inoculum, it implicates that IL-4-CFA-induced is responsible by the mediation of the observed unresponsiveness. Further, the characterization of CFA indicated that a proteic fraction is required for triggering the immunosuppressive mechanisms, while glycosylation or glycosphingolipids moieties are not. Taken together, our data suggest that the prior contact with soluble Pb antigens leads to severe PCM in an IL-4 dependent manner.     

Evaluation of three polymerase chain reaction techniques for detection of Brucella DNA in peripheral human blood

Brucellosis is a widespread zoonosis. Currently the diagnosis of this zoonosis is based on microbiological and serological laboratory tests. Polymerase chain reaction (PCR) has been used to detect DNA from Brucella. Different target genes, primer pairs, PCR techniques, and extraction procedures have previously been published for Brucella detection. But only a few of these primers have been used in human samples, and only one study has been carried out to compare sensitivity between them. In the present study, 3 sets of primers and 3 different PCR protocols amplifying 3 different regions of the Brucella genome were compared for detection of Brucella DNA in a peripheral-blood PCR assay to conclude which is most suitable for the clinical diagnostic laboratory. These 3 pairs of primers amplify 3 different fragments included in (i) a gene encoding a 31 kDa Brucella abortus antigen (B4/B5), (ii) a sequence 16S rRNA of B. abortus (F4/R2), and (iii) a gene encoding an outer membrane protein (omp-2) (JPF/JPR). Some modifications on the reported techniques were applied during the present work to improve the outcome. The results showed that the B4/B5 primer pair had the highest sensitivity for detection of positive samples (98%), the JPF/JPR primer pair detected 88.4% of positive samples, whereas F4/R2 primer pair was the least sensitive, being able to detect only 53.1% of positive samples. The specificity of the 3 techniques was 100%. The B4/B5 primer pair was also able to detect the smallest number of bacteria (700 cfu/mL), whereas JPF/JPR was able to detect 7 x 105 cfu/mL and F4/R2 was able to detect 7 x 107 cfu/mL. It is thus concluded that using the B4/B5 primer PCR with the suggested modifications is a robust assay, which meets the sensitivity requirements to be used for testing of human blood samples for brucellosis in the diagnostic laboratory.     

An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprint analysis

A rapid direct-extraction method was used to obtain DNA from environmental soil samples. Heat, enzymes, and guanidine isothiocyanate were utilized to lyse cells. The DNA was purified by agarose gel electrophoresis, amplified with 16S rRNA-based primers by use of the polymerase chain reaction, and then digested with the restriction endonucleasePalI. The extraction method was used to obtain DNA from a variety of plants, bacteria, and fungi includingGossypium hirsucum (cotton),Pseudomonas, Bacillus, Streptomyces, andColletotrichum. Up to 100 g DNA/g (wet weight) of soil and 400 g DNA/g of plant material were recovered. Restriction endonuclease analysis patterns of amplified rDNA from pure microbial cultures and plant species contained three to five different DNA fragments. Amplified rDNA of mixed population DNA extracts from soil samples, digested with the restriction endonucleasePalI, contained 12–20 DNA fragments, appearing as sample fingerprints. Results from eight environmental soil samples that were analyzed suggest that the amplified rDNA fingerprints can be used to help characterize the genetic and biological diversity of the microbial populations in these samples.     

Rapid detection of Salmonella in field samples by nested polymerase chain reaction

A simple and universal protocol for the rapid detection of Salmonella spp. in various samples using nested polymerase chain reaction (PCR) was developed. The protocol takes advantage of the rapid purification and concentration of Salmonella by centrifugation onto a layer of 60% sucrose solution. DNA was released by treatment with proteinase K and Triton X-100. Even without pre-enrichment, the detection limit for the nested PCR was 10(5) CFU g-1 of faeces. After pre-enrichment, it was possible to detect Salmonella in faeces where their original concentration was approximately 10(2) CFU g-1 of faeces and in meat samples, it was possible to detect Salmonella in samples where their original concentration was less than 10 cells g-1 of minced meat.     

Detection of Coxiella burnetii specific DNA in blood samples from Japanese patients with chronic nonspecific symptoms by nested polymerase chain reaction

Sprague Dawley rats, previously infected with Phase-I Bordetella pertussis , developed more severe abnormal respiratory sounds than normal animals, but not coughing, when exposed to aerosolized capsaicin, one of several cough-inducing agents tested. Stethoscope examination suggested that greater production of pulmonary mucus might be occurring after capsaicin challenge of the infected animals, compared to the uninfected controls. Rats of three other strains gave characteristically different responses from the Sprague Dawleys. The administration of capsaicin to B. pertussis -infected rats may provide useful insights into the pathophysiology of excess mucus secretion in human pertussis.     

Use of the polymerase chain reaction for Salmonella detection

J. KWANG, E.T. LITTLEDIKE AND J.E. KEEN. 1996. A primer set of oligonucleotides (S18 and S19) from the _omp C gene of Salmonella has been evaluated for specific detection of Salmonella by polymerase chain reaction (PCR). This primer set successfully amplified 40 Salmonella serovars (60 isolates), but not 24 non-Salmonella bacteria (42 isolates) that have been tested so far. The uniqueness of these primer sequences was also confirmed. The sensitivity of PCR detection in extracted chromosomal DNA for Salm. typhimurium was 1 pg. The sensitivity for boiled whole bacteria was 400 cells. The detection of Salm. typhimurium in ground beef samples required 4–6 h enrichment with an initial inocula of 100 bacteria.     

A rapid method for detection of Y-chromosomal DNA from dried blood specimens by the polymerase chain reaction

Summary The alphoid satellite family is the only repetitive DNA family showing chromosome specificity. We have developed a simple, rapid, and reliable test for sex diagnosis based on detection of these sequences in undigested genomic DNA using the polymerase chain reaction. In our test, dried blood specimens were the source of DNA. When female DNA was used as a template for the reaction, only the expected 130-bp X-chromosome-specific fragment was detected, while with male DNA both the expected 170-bp Y-chromosome-specific and X-chromosome-specific fragments were detected. The Y-chromosome-specific fragment was further characterized by restriction enzyme analysis. The Y fragment was detectable when DNA obtained from an equivalent of 10 l of spotted blood was used in the reaction, whereas detection of the X fragment was possible with DNA from an equivalent of 5 l of blood. Our test may find various applications in newborn screening and in forensic science.     

Improved detection of Dirofilaria repens DNA by direct polymerase chain reaction

Diagnosis of human infection by Dirofilaria repens, depends mainly on microscopic evaluation of tissue cross-sections and the macroscopic characteristics of the worm. Tissue degeneration and/or poor specimen preparation practices however, often render many cases of subcutaneous dirofilariasis elusive to such morphological diagnostic approaches. The early PCR protocols, developed to satisfy these complex diagnostic needs, failed to amplify dirofilariae DNA from formalin preserved material. To overcome these difficulties, we developed an improved PCR protocol using a set of primers designed to amplify a rather stable, highly repetitive D. repens-specific genomic DNA target. We report the performance of this protocol with a large variety of dirofilariae infected DNA specimens, including those extracted from formalin preserved biological material for up to 20 days. Our findings support its potential application to routine clinical diagnosis.     

Recombinant DNA probes and polymerase chain reaction for detection of Mycoplasma gallisepticum strains

Abstract Two recombinant DNA clones, pMG286.2 and pMG301.1, were isolated from the partial genomic library of Mycoplasma gallisepticum strain S6. Recombinant M. gallisepticum specific fragments were used as probes in Southern hybridisation with 10 M. gallisepticum strains whose DNA was digested by Eco RI, Hin dIII, Bgl II, Rsa I and Bam HI. The 1.5 kb fragment pMG301.1 did not show polymorphism in hybridisation patterns with M. gallisepticum strains, while the 3.5 kb fragment pMG286.2 enabled differentiation of M. gallisepticum strains into clusters. The DNA sequence of pMG301.1 was used to design a pair of 27-mer oligonucleotides flanking a 1.3 kb genomic region. These two primers directed specific in vitro amplification of all M. gallisepticum strains assayed giving an expected 1.3 kb product. Digestion of polymerase chain reaction products by Dde I enabled simple differentiation between clusters of M. gallisepticum strains and may be useful for improved epizootiological studies of M. gallisepticum infections in poultry.     

Specific detection of Neospora caninum oocysts in fecal samples from experimentally-infected dogs using the polymerase chain reaction

Neospora caninum oocysts, passed in the feces of a definitive host (dog), were isolated, and genomic DNA was extracted. A polymerase cahin reaction (PCR) targeting the N. caninum-specific Nc 5 genomic sequence was performed using the isolated DNA. A synthesized competitor molecule containing part of the Nc 5 sequence was included in the assay as a check against false-negative PCR results and to quantify N. caninum oocyst DNA in fecal samples. A standard curve of the ratio of fluorescence intensity of PCR-amplified competitor to that of oocyst DNA was constructed to compare oocyst equivalents from fecal samples containing unknown numbers of N. caninum oocysts and to assess the sensitivity of the assay. The specificity of the assay was determined using the Nc 5-specific primers in PCR assays against other parasites likely to be found in canine feces. Genomic DNA sequences from the canine coccidians Hammondia heydorni, Cryptosporidium parvum, Sarcocystis cruzi, S. tenella, and Isospora ohioensis and the canine helminth parasites Strongyloides stercoralis, Toxocara canis, Dipylidium caninum, and Ancylostoma caninum were not amplified. In addition, genomic DNA sequences from oocysts of coccidian parasites that might contaminate dog feces, such as Hammondia hammondi, Toxoplasma gondii, or Eimeria tenella, were not amplified in the PCR assay. The assay should be useful in epidemiological surveys of both domestic and wild canine hosts and in investigations of oocyst biology in experimental infections.     

Quantification of serum HBXAP DNA in lung cancer patients by quantitative fluorescent polymerase chain reaction

Hepatitis B virus x associated protein (HBXAP), as a subunit of chromatin remodeling and spacing factor, plays a critical role in cancer development through gene amplification. In this study, we aimed to quantify the levels of serum HBXAP DNA, to analyze and compare its diagnostic value with existing clinical parameters in lung cancer, and to potentially provide a novel tumor marker for lung cancer. Serum HBXAP DNA from 65 lung cancer patients and 20 healthy controls was quantified using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) analysis. The data were analyzed by statistical software SPSS 13.0. We found that serum HBXAP DNA levels in lung cancer patients were higher compared to healthy controls (u = 219.0, p = 0.001) and were closely associated with TNM stage and lymph node metastasis (p = 0.015 and p = 0.016, respectively). However, serum HBXAP DNA levels were not associated with patient age, gender, smoking status, histological type, or tumor size (p > 0.05). We identified a sensitivity of 61.9 % and a specificity of 93.7 % for the ability of HBXAP DNA levels to detect lung cancer at a cutoff value of 1,557.6 copies/μl. The sensitivity for existing lung-tumor markers, such as squamous cell carcinoma antigen, cytokeratin fragment 21-1, and neuron specific enolase, was increased from 35.7 %, 53.5 %, and 56.0 % to 75.0 %, 86.0 %, and 80.0 %, respectively, by inclusion of serum HBXAP DNA. Taken together, quantification of serum HBXAP DNA by FQ-PCR could potentially serve as a novel complementary tool for the clinical screening and detection of lung cancer.     

DNA extraction from coral reef sediment bacteria for the polymerase chain reaction

A rapid and effective method for the direct extraction of high molecular weight amplifiable DNA from two coral reef sediments was developed. DNA was amplified by the polymerase chain reaction (PCR) using 16S rDNA specific primers. The amplicons were digested with HaeIII, HinP1I and MspI and separated using polyacrylamide gel electrophoresis and silver staining. The resulting amplified ribosomal DNA restriction analysis (ARDRA) patterns were used as a fingerprint to discern differences between the coral reef sediment samples. Results indicated that ARDRA is an effective method for determining differences within the bacterial community amongst different environmental samples.