Specific Polymerase Chain Reaction Assay Research Articles

A real-time polymerase chain reaction (PCR) method for the identification of Nicotiana tabacum in tobacco products

A simple and specific real-time PCR assay based on TaqMan ® technology has been developed for the identification of cultured tobacco ( Nicotiana tabacum) in various commodities such as cigars, cigarettes and reconstituted tobacco. The TaqMan ® assay targets a sequence of the putrescine N-methyltransferase gene family encoding an enzyme that plays a crucial role in the biosynthesis of nicotine. To reduce the possibility of false negatives, universal plant chloroplast primers were also used in a separate real-time PCR reaction to give indication if DNA is amplifiable in the matrix. The TaqMan ® assay successfully identified tobacco in over 40 commercial tobacco products, while negative results were obtained from the assay for DNA extracted from a variety of other botanical products. In our study, two commercial DNA isolation kits were used, namely, the Qiagen DNeasy ® Plant Mini kit and the Qiagen Gentra ® Puregene ® kit. They produced good quality DNAs in sufficient quantities for real-time PCR analysis. In a few cases, an additional purification step with the Promega DNA IQ™ system had to be implemented to obtain amplifiable DNA.

Multicenter Evaluation of an Investigational Prostate Cancer Methylation Assay

Prostate specific antigen tests have low specificity, which frequently results in unnecessary biopsy and typically limits screening to patients with prostate specific antigen greater than 4.0 ng/ml. We evaluated an investigational prostate cancer methylation specific polymerase chain reaction assay that detects aberrant methylation in 3 markers (GSTP1, RARbeta2 and APC) that indicate the presence of prostate cancer. The assay was evaluated in 337 post-digital rectal examination urine samples (178 cancer and 159 noncancer) collected prospectively at a total of 9 clinical sites. Samples were processed wholly or after division into equal portions. Subject prostate specific antigen was 2.0 to 10.0 ng/ml. All subjects underwent transrectal ultrasound guided needle biopsy with 6 or greater cores sampled. Detection of 1 or greater markers indicated positivity. Methylation specific polymerase chain reaction assay performance was better in whole than in divided urine cohorts (p = 0.035). Assay AUC was 0.72 in the whole urine cohort and 0.67 in the combined population. These values were higher than those of prostate specific antigen alone using 4.0 ng/ml as the cutoff (p = 0.00 and 0.01, respectively). Moreover, the assay together with the Prostate Cancer Prevention Trial risk calculator or a standard nomogram significantly improved AUC in the whole urine cohort and the combined population vs predictive algorithms alone (p <0.05). Assay positive predictive value was 54% in whole urine cohort with prostate specific antigen 2.0 to 4.0 ng/ml and negative predictive value was 87% with prostate specific antigen 4.1 to 10.0 ng/ml. Assay positive predictive value was higher in subjects with all 3 methylation markers positive. These data demonstrate that this investigational assay used in conjunction with current screening algorithms may potentially add value to the biopsy decision making process.

Low frequency of MAP kinase pathway alterations in KIT and PDGFRA wild‐type GISTs

Gastrointestinal stromal tumours (GISTs) are commonly driven by oncogenic mutations in KIT and PDGFRA. However, 10-40% of these patients are wild-type for these genes. The prognostic significance of wild-type GISTs is controversial, and they rarely respond to imatinib. The aim of this study was to elucidate the molecular lesions underlying wild-type GISTs tumorigenesis. Twenty-nine KIT and PDGFRA wild-type GISTs were re-assessed for the presence of 'cryptic'KIT exon 11 duplications. Using a specific polymerase chain reaction assay, three previously undetected mutations were identified. In the remaining 26 wild-type GISTs, KIT, stem cell factor (SCF), phospho-KIT and phospho-ERK expression was evaluated by immunohistochemistry. Samples were screened for gain-of-function mutations in the mitogen-activated protein kinase (MAPK) cascade. KIT and SCF co-expression associated with KIT activation was observed in approximately 30% of cases. Furthermore, phospho-ERK expression showed that MAPK is activated in approximately 30% of cases. None of RAS family (H-, K- and N-RAS) oncogenes exhibited activating mutations, whereas BRAF mutations were found in approximately 4% of cases. In the absence of RAS mutations, MAPK could be activated through SCF/KIT autocrine/paracrine mechanisms and/or mutated BRAF in a subset of KIT/PDGFRA wild-type GISTs.

Polymorphisms in the Enhancer Region of the Thymidylate Synthase Gene Are Associated with Thymidylate Synthase Levels in Normal Tissues but Not in Malignant Tissues of Patients with Colorectal Cancer

The enhancer region of the thymidylate synthase (TS) gene (TSER) contains a polymorphic tandem repeat sequence (2 or 3 repeats, 2R or 3R) and a single-nucleotide polymorphism (G > C) within the second repeat of the 3R alleles which might influence TS expression/activity and response to fluoropyrimidines. However, clinical studies in patients with colorectal cancer (CRC) failed to find a consistent relationship between TSER polymorphisms and protein levels as well as with clinical outcome. The analysis of the relationship between TSER genotype and TS mRNA and activity in normal and malignant tissues might explain the previous controversial data and help in the selection of useful markers to predict drug response and/or toxicity. To address this issue, we studied TSER genotype, TS expression, and activity with specific polymerase chain reaction and activity assays (TS catalytic activity and FdUMP binding) in normal (liver, mucosa) and malignant (primary tumor and liver metastasis) tissues from 83 patients with CRC. No correlation between TSER genotype and TS mRNA and protein levels was observed in malignant tissues. In contrast, normal tissues harboring one or two 3RG alleles were characterized by higher TS protein levels (2.4-fold; P = .008) and catalytic activity (P < .05) compared with the other TSER genotypes. These results suggest that TSER polymorphisms do not predict tumoral TS levels possibly depending on altered TS regulation in cancer tissues, and might explain the lack of clear correlation with clinical outcome after chemotherapy with fluoropyrimidines. However, the relationship between TS phenotype and TSER genotype in normal tissues warrants further investigations in large-scale prospective studies evaluating TS genotype and fluoropyrimidine tolerability.

Occurrence and characterisation of enterohaemorrhagic Escherichia coli isolates from diarrhoeic calves

The presence of major virulence factors of enterohaemorrhagic Escherichia coli (EHEC; stx1, stx2, eae, Ehly) were determined among isolates from 158 diarrhoeic calves by multiplex polymerase chain reaction (PCR). Strains positive for virulence factors were subjected to serotype specific PCR assays for O157:H7 and O111 antigens. Additionally, serogroups were determined by three monovalent antisera for O26, O111 and O157 somatic antigens and enterohaemolysin production were also shown phenotypically. Thirteen (8.2%) calves carried strains positive for one or more of the virulence factors tested, and eleven (6.9%) calves harboured the shiga toxin-producing strains (stx1 or stx2). stx1 was detected in eight (5%) and stx2 in three (1.9%) calves. eae and Ehly were observed in the same frequency (6.3%) and were detected in parallel. Of the 13 virulence-positive strains, the predominant genotype was (stx1/eae/Ehly) at 53.8%. None of the EHEC in this study belonged to O157:H7 or O111 serotypes, but four strains (30.7%) belonged to the O26 serogroup. The results show the possible role of stx1/eae in calf diarrhoea and the particular importance of O26 EHEC. Calves can also act as a reservoir for EHEC and in the transmission of the disease to humans.

Development and Validation of a HPV-32 Specific PCR Assay

BackgroundHuman Papillomavirus-32 (HPV-32) has traditionally been associated with focal-epithelial-hyperplasia (FEH). It is also present in 58% of oral warts of HIV-positive individuals whose prevalence is increasing. Current methods for the detection of HPV-32 are labor-intensive and insensitive so the goal of this work was to develop a highly sensitive and easy to use specific polymerase chain reaction (PCR) assay.Materials and methodsAn HPV-32 L1 specific PCR assay was developed and optimized. The sensitivity and specificity was compared to previous assays utilized for detection (PGMY and MY09/11 PCR with dot blot hybridization) using cloned HPV-32 L1, the closely related HPV-42 L1 as well as clinical samples (oral swabs and fluids from 89 HIV-positive subjects).ResultsThe HPV-32 specific PCR assay showed improved sensitivity to 5 copies of HPV-32 as compared to the PGMY PCR, MY09/11 PCR and dot blot which had a limit of detection of approximately 3,000 copies. Using the HPV-32 dot blot hybridization assay as the gold standard, the HPV-32 specific PCR assay has a sensitivity of 95.8% and 88.9% by sample and subject, respectively, and specificity was 87.8% and 58.8% by sample and subject, respectively. The low sensitivity is due to the HPV-32 specific PCR assays ability to detect more HPV-32 positive samples and may be the new gold standard.ConclusionDue to the ease, sensitivity, and specificity the HPV-32 specific PCR assay is superior to previous assays and is ideal for detection of HPV-32 in large cohorts. This assay provides an excellent tool to study the natural history of HPV-32 infection and the development of oral warts.

Identification of Nutrient-Responsive Arabidopsis and Rapeseed MicroRNAs by Comprehensive Real-Time Polymerase Chain Reaction Profiling and Small RNA Sequencing

Comprehensive expression profiles of Arabidopsis (Arabidopsis thaliana) MIRNA genes and mature microRNAs (miRs) are currently not available. We established a quantitative real-time polymerase chain reaction platform that allows rapid and sensitive quantification of 177 Arabidopsis primary miR transcripts (pri-miRs). The platform was used to detect phosphorus (P) or nitrogen (N) status-responsive pri-miR species. Several pri-miR169 species as well as pri-miR398a were found to be repressed during N limitation, whereas during P limitation, pri-miR778, pri-miR827, and pri-miR399 species were induced and pri-miR398a was repressed. The corresponding responses of the biologically active, mature miRs were confirmed using specific stem-loop reverse transcription primer quantitative polymerase chain reaction assays and small RNA sequencing. Interestingly, the latter approach also revealed high abundance of some miR star strands. Bioinformatic analysis of small RNA sequences with a modified miRDeep algorithm led to the identification of the novel P limitation-induced miR2111, which is encoded by two loci in the Arabidopsis genome. Furthermore, miR2111, miR169, a miR827-like sequence, and the abundances of several miR star strands were found to be strongly dependent on P or N status in rapeseed (Brassica napus) phloem sap, flagging them as candidate systemic signals. Taken together, these results reveal the existence of complex small RNA-based regulatory networks mediating plant adaptation to mineral nutrient availability.

Specific PCR assays for the detection of Trichoderma harzianum causing green mold disease during mushroom cultivation

We have developed a polymerase chain reaction (PCR)-based detection method for Trichoderma harzianum, which causes green mold disease in mushroom cultivation fields and facilities. Based on the sequence data of the internal transcribed spacer (ITS) region of T. harzianum strains and several other species, six primers consisting of three forward and three reverse primers were designed. Among the nine possible combinations of these primers, PCR with the pair THITS-F2 and THITS-R3 distinguished most T. harzianum strains from other Trichoderma species. The optimal annealing temperature for detection of T. harzianum strains was from 62° to 63°C with this primer combination. We designed new primers derived from THITS-F2 and THITS-R3. Annealing temperatures to detect T. harzianum ranged from 64° to 67°C using the new primers. The detection limit of T. harzianum DNA was 50fg by nested PCR with THITS-F1 and LR1-1 for the first PCR and the new primers for the second PCR. T. harzianum was readily detectable in contaminated cultures of Lentinula edodes by this method.

Patterns of Gene Transcript Abundance in the Blood of Children with Severe or Uncomplicated Dengue Highlight Differences in Disease Evolution and Host Response to Dengue Virus Infection

DNA microarrays and specific reverse-transcription polymerase chain reaction assays were used to reveal transcriptional patterns in the blood of children presenting with dengue shock syndrome (DSS) and well-matched patients with uncomplicated dengue. The transcriptome of patients with acute uncomplicated dengue was characterized by a metabolically demanding "host-defense" profile; transcripts related to oxidative metabolism, interferon signaling, protein ubiquination, apoptosis, and cytokines were prominent. In contrast, the transcriptome of patients with DSS was surprisingly benign, particularly with regard to transcripts derived from apoptotic and type I interferon pathways. These data highlight significant heterogeneity in the type or timing of host transcriptional immune responses precipitated by dengue virus infection independent of the duration of illness. In particular, they suggest that, if transcriptional events in the blood compartment contribute to capillary leakage leading to hypovolemic shock, they occur before cardiovascular decompensation, a finding that has implications for rational adjuvant therapy in this syndrome.

Specific PCR assays for the detection of &lt;i&gt;Trichoderma harzianum&lt;/i&gt; causing green mold disease during mushroom cultivation

We have developed a polymerase chain reaction (PCR)-based detection method for Trichoderma harzianum, which causes green mold disease in mushroom cultivation fields and facilities. Based on the sequence data of the internal transcribed spacer (ITS) region of T. harzianum strains and several other species, six primers consisting of three forward and three reverse primers were designed. Among the nine possible combinations of these primers, PCR with the pair THITS-F2 and THITS-R3 distinguished most T. harzianum strains from other Trichoderma species. The optimal annealing temperature for detection of T. harzianum strains was from 62° to 63°C with this primer combination. We designed new primers derived from THITS-F2 and THITS-R3. Annealing temperatures to detect T. harzianum ranged from 64° to 67°C using the new primers. The detection limit of T. harzianum DNA was 50 fg by nested PCR with THITS-F1 and LR1-1 for the first PCR and the new primers for the second PCR. T. harzianum was readily detectable in contaminated cultures of Lentinula edodes by this method.

DETECTION AND IDENTIFICATION OF VIRULENT YERSINIA RUCKERI: THE CAUSATIVE AGENT OF ENTERIC REDMOUTH DISEASE IN RAINBOW TROUT (ONCORHYNCHUS MYKISS) CULTURED IN FARS PROVINCE, IRAN

Summary From the winter 2002 to spring 2006, 126 moribund rainbow trout with clinical signs of external body haemorrhages around and within the oral cavity were sampled from 10 rainbow trout farms situated in the northwest and west of Fars province, Iran and examined for the detection and identification of Yersinia ruckeri, the causative agent of enteric redmouth disease. Fish kidneys were cultured aseptically on brain heart infusion (BHI) agar plates and incubated at 25oC for 48 h. Using conventional biochemical tests, Y. ruckeri was detected in 7 fish (5.5% of total fish sampled). This was also confirmed using specific polymerase chain reaction (PCR) assay. The 16S rDNA PCR assays produced amplicons of 409 bp when applied to Y. ruckeri isolates as well as a reference strain. Results of antibiogram tests on Y. ruckeri isolates showed a high susceptibility to enrofloxacin, norfloxacin, ofloxacin, trimethoprim and oxytetracycline. In pathogenicity tests, dilution of 4 × 10 8 colony forming unit/ml of Y. ruckeri by immersion route in challenge experiments showed 70 ± 8.2% mortality during 14 days post-infection. Experimentally infected fish showed typical haemorrhages in mouth, blackening of skin, exophthalmia and a wide haemorrhages on the internal organs.

POLYMERASE CHAIN REACTION CONFIRMATORY METHOD FOR MICROBIOLOGICAL DETECTION OF BRETTANOMYCES BRUXELLENSIS IN WINES

ABSTRACTBrettanomyces bruxellensis is one of the major causes of contamination in wine and is an important source of economic losses in this industry. In this work we developed a specific polymerase chain reaction (PCR) assay for the detection of B. bruxellensis to be used in the confirmation stage of the microbiological analysis. From a random amplification analysis using 40 primers in various B. bruxellensis strains and other yeasts that are generally present in must and wine, we designed the primers E09F and E09R that amplified a 450 bp product only in B. bruxellensis strains. We determined that the concentration of the PCR components and the annealing temperature are relevant factors in the PCR reaction, which was optimized using the response surface methodology. The protocol developed confirmed the contamination by B. bruxellensis in wines obtained from cellars, showing the capacity and speed of the technique to specifically confirm the results of the microbiological analysis.PRACTICAL APPLICATIONS Brettanomyces bruxellensis is responsible for the “Brett” character in wine that results in phenolic or medicinal aromas, leading to a decrease in its quality. The most utilized microbiological analysis for the detection of this yeast is based on the sequential use of selective media and confirmatory physiological tests that are unspecific and take up to 3 weeks to complete. However, its low cost means that this type of analysis is still widely used in the industry. On the other hand, molecular techniques are fast and specific in the amplification of DNA sequences. The polymerase chain reaction assay development in this work is specific for B. bruxellensis and allowed the confirmation of the microbiological analysis in only 2 days, compared with how long it currently takes (5–10 days), and at a lower cost than other molecular techniques.

Phenotypic and genotypic identification of Candida dubliniensis from subgingival sites in immunocompetent subjects in Argentina

It is generally recognized that Candida dubliniensis is commonly found in immunocompromised patients, such as those with advanced human immunodeficiency virus infection, at sites of periodontal disease. Since there are no data available for Argentina, the aim of this study was to determine the prevalence of and to identify C. dubliniensis in periodontal pockets from immunocompetent subjects living in Buenos Aires, Argentina, through a comparison of phenotypic and molecular assays. Yeasts recovered from subgingival plaque samples were studied for 180 immunocompetent non-smoking patients with periodontal disease. Yeasts were identified by conventional mycological methods and by specific polymerase chain reaction (PCR) assay. Fluconazole and voriconazole susceptibility studies were performed in keeping with the Clinical and Laboratory Standards Institute. Among 76 yeasts isolated, C. dubliniensis comprised 10.5% (n = 8; 95% confidence interval 4.7-19.7), which corresponded to 4.4% of patients studied (8/180). C. albicans was the most frequently isolated species of yeast. A great majority of C. dubliniensis isolates was susceptible with only one isolate resistant to both antifungals. Micromorphology on Staib agar was the phenotypic method that was most concordant with PCR and it was useful for selecting presumptive C. dubliniensis. This is the first report to use PCR to identify C. dubliniensis in subgingival fluid from immunocompetent individuals with periodontal disease in Argentina. On the basis of the findings presented here, we confirm that C. dubliniensis can colonize periodontal pockets of immunocompetent patients with periodontal disease.

CagA, vacA, and iceA genotypes of Helicobacter pylori in Korean children

Several putative virulence factors for Helicobacter pylori have been identified including cagA, vacA, and iceA. The aims of the present study were to study the distribution of cagA, vacA, and iceA genotypes in children with H. pylori gastritis and to examine the association of genotypes with severity of gastritis. H. pylori DNA was extracted from antral biopsy specimens from 33 children with H. pylori gastritis. Specific polymerase chain reaction assays were used for three genes: cagA, vacA, and iceA. The features of gastritis were graded in accordance with the updated Sydney System. Of the 33 children, 31 (94%) were cagA positive. Twenty-four (72%) had s1c genotype and nine (27%) had s1a. The m1 genotype was seen in 27 (82%) and m2 was found in five (15%). The iceA1 genotype was detected in 25 (76%). Scores of neutrophil activity, chronic inflammation, and H. pylori density were independent of cagA, vacA and iceA status. The cagA-positive vacA s1c/m1 iceA1 genotype was predominant in Korean children with recurrent abdominal pain and H. pylori gastritis. The cagA, vacA and iceA genotype were not associated with the severity of gastritis.

Specific PCR assays for the identification of common anisakid nematodes with zoonotic potential

Based on the sequences of the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) for six taxa of anisakids, namely, Anisakis simplex (s.s.), Anisakis typica, Anisakis pegreffii, Hysterothylacium aduncum, Hysterothylacium sp, and Contracaccum osculatum C, specific primers were designed in the ITS-1 and/or ITS-2 for each of the six anisakid taxa. These specific primers were used to develop polymerase chain reaction (PCR) tools for the identification of these anisakid taxa of sea fish by amplifying partial ITS-1 and/or ITS-2 of rDNA from anisakid nematodes. This approach allowed their specific identification, with no amplicons being amplified from heterogeneous DNA samples, and sequencing confirmed the identity of the DNA fragments amplified. The minimum amounts of DNA detectable using the PCR assays were 0.5-1 ng. These PCR tools were then applied to ascertain the specific identity of 143 anisakid larval samples collected from fish in China, Canada, Thailand, and Indonesia, and these anisakid samples were identified to represent one of the six anisakid taxa. These PCR assays based on ITS sequences should provide useful molecular tools for the accurate identification and molecular epidemiological investigations of anisakid infections in humans and fish.

A Novel Noninvasive Genotyping Method of Helicobacter pylori Using Stool Specimens

The source(s) of the infection and the route(s) of transmission of Helicobacter pylori have not yet been clarified. This is to introduce a noninvasive protocol allowing molecular typing of H pylori using stool specimens. The genotyping method is based on 2 H pylori-specific biprobe real-time polymerase chain reaction assays using fragments of the glmM and the recA genes as target sequences. Discrimination between strains results from differences in the melting temperature during melting curve analysis. In case of identical melting temperatures in both assays, sequence analysis of the glmM amplicon was performed to confirm strain identity. The method was validated using gastric biopsy specimens and stool specimens of 97 unrelated individuals suffering from abdominal pain and stool specimens of members of 10 families in Austria (infected index child and family members) and 8 African households. Of the 97 patients, 27 were infected as shown by culture, histology, and rapid urease test. The sensitivity of each of the assays was 100% in gastric biopsy specimens and 92.2% in stool specimens; the specificity was 100%. The discriminatory capacity of the method was 100%. Clonal identities were found in 9 of 10 (90%) European and 7 of 8 (87.5%) African households. In 2 African households, 2 different clonal lineages each were found. The genotyping protocol introduced allows for both accurate detection and discrimination of H pylori strains in stool samples. Large-scale studies using this protocol may contribute to the clarification of the transmission pathways of infection with H pylori.

Observation of a higher JAK2 V617F homozygous mutated clone in polycythemia vera compared to essential thrombocythemia

Single-tube bi-directional allele specific amplification (SB-ASA) and real-time quantitative polymerase chain reaction (RQ-PCR) assays were developed and performed for JAK2V617F detection on 40 polycythemia vera (PV) samples, 31 essential thrombocythemia (ET) samples, 40 acute leukemia samples, and 40 healthy control samples. Differences between detect limitations of the two assays and their influence on the mutation detection rate were analyzed. The results showed that in some samples, the JAK2V617F burden was as low as nearly 1%, and thus more JAK2V617F-positive samples were detected by RQ-PCR than by SB-ASA assay due to the former higher detect limitation. Mutation allele ratios in PV and ET samples and their relevance to biological characteristics were also analyzed. The results showed that the mutation allele ratio was 0.436 ± 0.261 in PV, higher than the 0.216 ± 0.207 in ET; percentage of certainly homozygous mutation carriers in PV was 40.54%, higher than the 10% in ET. However, statistical analysis showed no relevance between mutation allele burden and sex or age. Our result shows that the pathogenesis of PV and ET may be related to the mutation allele burden of JAK2V617F.

Atypical presentation of Old‐World cutaneous leishmaniasis, diagnosis and species identification by PCR

The diagnosis of cutaneous leishmaniasis (CL) is traditionally based on microscopic demonstration of amastigote forms in tissue biopsies or smears. However, this method usually presents low sensitivity, and in atypical forms, CL may be overlooked because of similarity to other dermal diseases. Thus, it is necessary to apply specific diagnostic methods as polymerase chain reaction (PCR). To evaluate the possible advantage of PCR in the diagnosis and species identification of CL in patients with atypical clinical presentation. Fifty-one patients clinically suspected of CL with positive and negative controls were tested. After microscopic examination, extraction of DNA was performed on their smears and analysed by two specific PCR assays for diagnosis and species identification. For these methods, conserved and variable regions of kinetoplastic DNA (KDNA) of Leishmania species have been amplified, respectively. Atypical forms of CL were evaluated among PCR-positive patients. PCR results were positive in 37 out of 51 cases (72.5%), among whom microscopic examination revealed Leishmania amastigotes in only 3 (5.9%). Among these patients, 10 (27%) had atypical presentation of CL; using species-specific primers, 6 patients had Leishmania major, 3 had Leishmania tropica and 1 patient had no species diagnosis. None of the samples of other dermal diseases revealed positive results (specificity, 100%). All patients were successfully treated by CL-specific drug regimens. The results showed that KDNA PCR methods have a higher sensitivity compared with microscopic method. Moreover, PCR could identify the parasite species for specific therapy. Microscopic method had low sensitivity and less value in chronic and atypical CL cases.

Molecular Quantification ofGardnerella vaginalisandAtopobium vaginaeLoads to Predict Bacterial Vaginosis

Bacterial vaginosis (BV) is a poorly detected public health problem that is associated with preterm delivery and for which no reliable diagnostic tool exists. Molecular analysis of 231 vaginal samples, classified by Gram stain-based Nugent score, was used to propose molecular criteria for BV; these criteria were prospectively applied to 56 new samples. A quantitative molecular tool targeting 8 BV-related microorganisms and a human gene was developed using a specific real-time polymerase chain reaction assay and serial dilutions of a plasmid suspension. The targeted microorganisms were Gardnerella vaginalis, Lactobacillus species, Mobiluncus curtisii, Mobiluncus mulieris, and Candida albicans (which can be identified by Gram staining), as well as Atopobium vaginae, Mycoplasma hominis, and Ureaplasma urealyticum (which cannot be detected by Gram staining). With use of the Nugent score, 167 samples were classified as normal, 20 were classified as BV, and 44 were classified as intermediate. Except for U. urealyticum, M. mulieris, and Lactobacillus species, DNA of the tested bacteria was detected more frequently in samples demonstrating BV, but the predictive value of such detection was low. The molecular quantification of A. vaginae (DNA level, > or = 10(8) copies/mL) and G. vaginalis (DNA level, > or = 10(9) copies/mL) had the highest predictive value for the diagnosis of BV, with excellent sensitivity (95%), specificity (99%), and positive (95%) and negative (99%) predictive values; 25 (57%) of the samples demonstrating intermediate flora had a BV profile. When applied prospectively, our molecular criteria had total positive and negative predictive values of 96% and 99%, respectively. We report a highly reproducible, quantitative tool to objectively analyze vaginal flora that uses cutoff values for the concentrations of A. vaginae and G. vaginalis to establish the molecular diagnosis of BV.

Failure to detect hepatitis C virus (HCV) genome by polymerase chain reaction in human anti-HCV-positive intravenous immunoglobulins.

The prevalence of HCV antibodies was determined by a second-generation ELISA and a four-antigen recombinant immunoblot assay in nine intravenous immunoglobulin (IVIG) preparations commercially available in Italy. In addition, the clinical safety of six of them was ascertained by polymerase chain reaction (PCR) of HCV RNA and a prospective study in 14 patients with immunodeficiency disorders. Results indicated that all IVIG preparations were anti-HCV-positive. However, there were substantial variations in their anti-HCV antibody titres. The preparations retained IgG subclass reactivities to HCV-associated structural (C22-3) and non-structural (C33c, C100-3) proteins. Our sensitive and specific PCR assay was unable to detect HCV RNA in the six preparations tested. Clinical surveillance of IVIG-treated patients prospectively evaluated over a mean period of 8.3 months failed to detect clinical and/or biochemical evidence of hepatitis.