Broad-spectrum Primers Research Articles

Clinical characteristics and HPV type in recurrent respiratory papillomatosis in Colombia

Describe factors associated with aggressive forms of recurrent respiratory papillomatosis (RRP). One hundred eighty-nine RRP cases diagnosed between 1985 and 2009 were identified in pathological records. HPV was detected by the SPF-10 method with broad spectrum primers, (version 1). 113 patients had only one surgery (less aggressive) and 76, two or more interventions (more aggressive). The likelihood of aggressive lesions decreased with increasing age at diagnosis and HPV-11 was associated with no significant increase in the risk of aggressiveness. The age at diagnosis was the main determinant of RRP aggressiveness.

Poor Prognosis Associated With Human Papillomavirus α7 Genotypes in Cervical Carcinoma Cannot Be Explained by Intrinsic Radiosensitivity

To investigate the relationship between human papillomavirus (HPV) genotype and outcome after radiation therapy and intrinsic radiosensitivity. HPV genotyping was performed on cervix biopsies by polymerase chain reaction using SPF-10 broad-spectrum primers, followed by deoxyribonucleic acid enzyme immunoassay and genotyping by reverse hybridization line probe assay (LiPA25) (version 1) (n=202). PapilloCheck and quantitative reverse transcription-polymerase chain reaction were used to genotype cervix cancer cell lines (n=16). Local progression-free survival after radiation therapy alone was assessed using log-rank and Cox proportionate hazard analyses. Intrinsic radiosensitivity was measured as surviving fraction at 2 Gy (SF2) using clonogenic assays. Of the 202 tumors, 107 (53.0%) were positive for HPV16, 29 (14.4%) for HPV18, 9 (4.5%) for HPV45, 23 (11.4%) for other HPV genotypes, and 22 (10.9%) were negative; 11 (5.5%) contained multiple genotypes, and 1 tumor was HPV X (0.5%). In 148 patients with outcome data, those with HPVα9-positive tumors had better local progression-free survival compared with α7 patients in univariate (P<.004) and multivariate (hazard ratio 1.54, 95% confidence interval 1.11-1.76, P=.021) analyses. There was no difference in the median SF2 of α9 and α7 cervical tumors (n=63). In the cell lines, 9 were α7 and 4 α9 positive and 3 negative. There was no difference in SF2 between α9 and α7 cell lines (n=14). The reduced radioresponsiveness of α7 cervical tumors is not related to intrinsic radiosensitivity.

Homogeneous detection of cyanobacterial DNA via polymerase chain reaction

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. 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. 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. These novel, broad-spectrum primers will greatly increase the utility of PCR on newly discovered cyanobacterial species.

Type‐specific human papillomavirus distribution in invasive cervical carcinomas in Paraguay. A study of 432 cases

Cervical carcinoma is the most common malignant tumor among woman in Paraguay. Cytological screening programs have not been successful and a plan for human papillomavirus (HPV) based-screening program and/or vaccination is under evaluation. This study aimed to identify the contribution of HPV genotypes in invasive cervical cancer in Paraguay to provide essential background data to guide and assess the introduction and impact of new preventive strategies based on HPV. Four hundred thirty two histologically confirmed cases (1960-2004) were analyzed. HPV detection in paraffin blocks was performed at the Catalan Institute of Oncology using PCR with SPF-10 broad spectrum primers followed by DNA enzyme immunoassay and genotyping with a reverse hybridization line probe analysis. The majority of cases were squamous cell carcinoma (92.8%). Mean patients age was 48 years old. HPV DNA was detected in 73.1% of the cases and single infections were predominant (97.8%). The most common HPV single types were 16, 18, 45, 33, 31, 52, 35, and 39. 73.1% of HPV positive cases had an HPV 16, 18 as single infection. HPV16 was frequent in SCC whereas HPV 18 and 45 were prevalent in glandular tumors. Significant decrease of HPV 16 with age groups (P-trend = 0.022) and increase in other HPV types (P-trend > 0.001) were observed. The potential impact of HPV 16 and 18 for a vaccination program was 73.1%. The study provide a profile of the HPV situation in the country, with robust clinical, pathological and virological data which would permit a better cervical cancer screening and vaccination programs.

Identification and genotyping of human papillomavirus in a Spanish cohort of penile squamous cell carcinomas: Correlation with pathologic subtypes, p16INK4a expression, and prognosis

Penile squamous cell carcinoma (PSCC) is a tumor with a high metastatic potential. In PSCC the attributable fraction to human papillomavirus (HPV) is not well established. We sought to provide novel data about the prevalence of HPV in a large series of penile intraepithelial neoplasia (PeIN) and invasive PSCC, correlating the results with the histologic subtype, p16(INK4a) immunostaining, and prognosis. A total of 82 PSCC were included in the study, 69 invasive and 13 PeIN. HPV detection was performed by polymerase chain reaction with SPF-10 broad-spectrum primers followed by DNA enzyme immunoassay and genotyping with a reverse hybridization line probe assay. P16(INK4a) immunohistochemical expression on tissue microarrays was also analyzed. HPV DNA was identified in 31 of 77 (40.2%) PSCC (22 of 67 invasive and 9 of 10 PeIN). In 25 of 31 (80.6%) cases HPV-16 was identified. HPV detection was significantly associated with some histologic subtypes: most basaloid and warty tumors were high-risk HPV (hrHPV) positive, whereas only 15% of usual PSCC were hr-HPV positive. All hrHPV-positive PSCC had an adjacent undifferentiated PeIN. Strong p16(INK4a) immunostaining correlated with hrHPV infection. Most undifferentiated PeIN showed p16(INK4a) immunohistochemical overexpression. Both hrHPV-positive and p16(INK4a)-positive tumors showed a better overall survival without reaching statistical significance. This was a retrospective study. Our results suggest that most hrHPV-positive PSCC develop from undifferentiated hrHPV-positive PeIN. P16(INK4a) immunostaining may be useful in identifying both etiologically related hrHPV-positive tumors and those with better outcome. The routine use of p16(INK4a) staining should be incorporated in histologic evaluation of PSCC.

Comparison of 2 Different PCR-Based Technologies for the Detection of Human Papilloma Virus from Paraffin-Embedded Tissue

The great interest in molecular epidemiology of human papilloma virus (HPV) in cervical cancer led us to perform a thorough evaluation of 2 polymerase chain reaction (PCR)-based methods for the detection of HPV in archival formalin-fixed paraffin-embedded (FFPE) samples. Thus, the aim of this study was to compare HPV detection in FFPE samples that have histopathologic diagnosis of invasive cervical cancer using SPF10 broad-spectrum primers PCR followed by DNA enzyme immunoassay and LiPA25 (version 1: Labo Biomedical products, Rijswijk, The Netherlands version 1) and the Papillomavirus Clinical Arrays technique (Genómica, Tres Cantos, Madrid, Spain). In this study, 235 biopsies with histopathologic diagnosis of invasive cervical cancer were analyzed for the detection and genotyping of HPV by LiPA25 SPF10-PCR System (version 1) and Papillomavirus Clinical Arrays technique. The detection of HPV DNA with Genómica technique was 75.1%, and 91.9% with LiPA25 SPF10-PCR. The Genómica technique detected a higher percentage of multiple infections (35%) than LiPA25 (8.9%), with a very low agreement for the detection of multiple infections between them (P>0.05). Our study highlights an important difference between 2 PCR-based methods for detection and genotyping of HPV. LiPA25 SPF10-PCR technology may be more adequate than Genómica for the detection of HPV DNA when using FFPE tissue.

P5-05-05: Human Papiloma Virus Identification in Intraductal Papilloma and Breast Cancer Using Broad-Spectrum Primers.

Abstract Background There is limited data about Human Papiloma Virus (HPV) presence in intraductal papilloma, and there are some conflicted studies about the effects of HPVs on breast cancer. Most of these studies have methodological pitfalls as well. We conducted a retrospective paraffin block study to understand the presence of HPV in papilloma and cancer tissues with broad-spectrum primers. Methods: Twenty seven intraductal papilloma and 18 breast cancer patients’ paraffin blocks were obtained from the patients who underwent ductoscopy aided papillomectomy or mastectomy. In totally 87 histopathological samples, HPV-DNA were identified by real time PCR and broad-spectrum genotyping was performed by in situ hybridization. The cancer patients were evaluated according to the status of Estrojen Reseptor (ER), Progesteron Reseptor (PR) and ErbB-2 positivity. Results: The mean age±SD was 49±16 in papilloma and 52±14 in breast cancer patients. We found that 29.6% (8 of 27) of intraductal papillomas and 44.4% (8 of 18) of breast cancer paraffin blocks had HPV-DNA positivity by PCR in situ hybridization with broad spectrum primers. Fifty percent (4 of 8) of HPV-DNA positive papillomas had only type 11; in one, only type 6; in one, mixture of type 6,11; in one, mixture of type 11, 39; and in one, mixture of type 11, 39, 82 was detected. Breast cancer lesions had only type 11 of HPV-DNA positivity in 62.5% (5 of 8) of cases; in one, mixture of type11, 39; in one, mixture of type 11, 82; in one, mixture of type 6, 11, 39 was detected. In seven HPV-DNA positive cancer patients, ER/PR and/or ErbB-2 was positive, but it was only positive in one triple negative patient. Conclusion: HPVs may have a role in developing papilloma and breast cancer. Validation of HPVs role in the etiology of breast cancer/papilloma, require further studies to understand whether the HPVs are episomal or integrated during the tumorigenesis. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-05-05.

Detection of multiple sequence variants of Grapevine rupestris stem pitting-associated virus using primers targeting the polymerase domain and partial genome sequencing of a novel variant

Grapevine rupestris stem pitting-associated virus (GRSPaV) is a member of the genus Foveavirus within the new family Betaflexiviridae. GRSPaV is distributed among grapevines worldwide and is implicated in the disease rupestris stem pitting (RSP) of the rugose wood complex and two other disorders. GRSPaV is composed of a wide range of sequence variants, and so far, the complete genomes of five sequence variants have been sequenced. Quick and reliable detection of different GRSPaV variants is a critical step in the elimination and control of GRSPaV. Previously, primers designed from various genomic regions have been used in RT-PCR for the detection of GRSPaV variants. The efficiency of RT-PCR varied widely depending on the spectrum of the primers that were used. In this study, we designed a pair of degenerate primers based on the consensus sequence of the genomic region encoding the highly conserved RNA-dependent RNA polymerase domain from five reference isolates of GRSPaV for which the genome sequence are available. We demonstrate that this set of primers is comparable, if not superior, to the broad-spectrum primers RSP13&14 in detecting multiple GRSPaV variants. Using these degenerate primers, we identified two new and distinct sequence variants. The 3′ terminal genomic region of one of the new variants, GRSPaV-ML, spanning the 3′ part of ORF1, through the entire open reading frames 2–4, and the 5′ region of ORF5 were sequenced. Sequence comparison demonstrates that GRSPaV-ML is distinct from each of the five reference isolates.

Evaluation of a Novel Multiplex Human Papillomavirus (HPV) Genotyping Assay for HPV Types in Skin Warts

Human papillomaviruses (HPV) of the genera alpha, mu, and nu induce benign tumors of the cutaneous epithelia that constitute a significant burden for immunocompromised adults. Currently, no gold standard for genotyping of these HPV types exists. In this study, we describe the prevalence of genus alpha, mu, and nu HPV types in cutaneous warts. We developed a novel multiplex HPV genotyping assay, BSwart-PCR/MPG (BSwart), to type sensitively and specifically 19 cutaneous HPV types frequently found in warts. BSwart-PCR/MPG is based on a multiplex PCR using broad-spectrum primers and subsequent multiplex hybridization to type-specific probes coupled to Luminex beads. In a first application comprising 100 cutaneous warts, the assay was compared to another, recently described genotyping assay, the HSL-PCR/MPG. When a 10-fold dilution series was used, the detection limit was between 10 and 100 HPV genomes per PCR. When comparing the two assays, there was an excellent agreement in detecting dominant HPV types; however, we also obtained evidence for a higher sensitivity of the BSwart assay for multiple infections in these cutaneous warts. Using BSwart, HPV was found in 95% of wart preparations, with HPV1 being most prevalent, followed by types 27, 57, and 2. Both novel BSwart and HSL-PCR/MPG HPV genotyping assays are powerful high-throughput tools that could be used to learn more about the natural history of cutaneous HPV. They would be advantageous to monitor the efficacy of future skin HPV vaccines and to identify novel HPV vaccine candidates.

Detection of Known and Novel Adenoviruses in Cattle Wastes via Broad-Spectrum Primers

The critical assessment of bovine adenoviruses (BAdV) as indicators of environmental fecal contamination requires improved knowledge of their prevalence, shedding dynamics, and genetic diversity. We examined DNA extracted from bovine and other animal waste samples collected in Wisconsin for atadenoviruses and mastadenoviruses using novel, broad-spectrum PCR primer sets. BAdV were detected in 13% of cattle fecal samples, 90% of cattle urine samples, and 100% of cattle manure samples; 44 percent of BAdV-positive samples contained both Atadenovirus and Mastadenovirus DNA. Additionally, BAdV were detected in soil, runoff water from a cattle feedlot, and residential well water. Overall, we detected 8 of 11 prototype BAdV, plus bovine, rabbit, and porcine mastadenoviruses that diverged significantly from previously reported genotypes. The prevalence of BAdV shedding by cattle supports targeting AdV broadly as indicators of the presence of fecal contamination in aqueous environments. Conversely, several factors complicate the use of AdV for fecal source attribution. Animal AdV infecting a given livestock host were not monophyletic, recombination among livestock mastadenoviruses was detected, and the genetic diversity of animal AdV is still underreported. These caveats highlight the need for continuing genetic surveillance for animal AdV and for supporting data when BAdV detection is invoked for fecal source attribution in environmental samples. To our knowledge, this is the first study to report natural BAdV excretion in urine, BAdV detection in groundwater, and recombination in AdV of livestock origin.

Identification and characterization of one Roseomonas strain

Objective To identify one runny mucoid-like Gram-negative bacteria with pink pigment isolated from clinical pus sample. Methods The pus sample was aseptically extracted from a deep lesions of one patient, then stored in Amies medium at room temperature for transportation. One sheep blood plate and one chocolate plate were used to detect the possible pathogens from the specimens. After inoculation, the plates were placed in a humidified incubator with 5% CO2 at 35 ℃. To identify the obtained isolates, we used the commercial Vitek2 and API systems, combining some traditional morphological examination and classical biochemical and physiological characteristics. For pure cultures, the cellular fatty acids were extracted, methylated, and determined by gas chromatography method. The 16S rRNA gene was amplified and sequenced by a commercial broad-spectrum PCR primers. The phylogenetic tree based on 16S rRNA gene was constructed by Mega 4.1 software using the neighbour-joining methods with 1 000 bootstrap replications. Results One runny mucoid-like Gram-negative bacterium, named K8756, was isolated both on sheep blood and chocolate plates after 72 h incubation. The API 20NE profile was 1245045 after a 3-day culture, which would be identified as Ochrobactrum anthropi with a good confidence of 98% probability. It was identified as Ralstonia pickettii and Bordetella bronchiseptica by VITEK 2 GN kits. However, further comparative 16S rRNA gene sequences showed that strain K8756 was closely related to the valid published Roseomonas mucosa MDA 5527 with 100% identity. Colonial morphologic features, phenotypic characteristics and major cellular fatty acid composition were also with high similarity to Roseomonas mucosa. Conclusions Strain K8756( = GIMCC 1.0030 ) is identified as Roseomonas mucosa by the polyphasic phenotypic and genotypic characteristics. The comparative analysis based on 16S rRNA gene sequences is a useful method for identifying the problematic and newly named bacteria. Key words: RNA,ribosomal,16S; Sequence analysis,RNA

Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study

Knowledge about the distribution of human papillomavirus (HPV) genotypes in invasive cervical cancer is crucial to guide the introduction of prophylactic vaccines. We aimed to provide novel and comprehensive data about the worldwide genotype distribution in patients with invasive cervical cancer. Paraffin-embedded samples of histologically confirmed cases of invasive cervical cancer were collected from 38 countries in Europe, North America, central South America, Africa, Asia, and Oceania. Inclusion criteria were a pathological confirmation of a primary invasive cervical cancer of epithelial origin in the tissue sample selected for analysis of HPV DNA, and information about the year of diagnosis. HPV detection was done by use of PCR with SPF-10 broad-spectrum primers followed by DNA enzyme immunoassay and genotyping with a reverse hybridisation line probe assay. Sequence analysis was done to characterise HPV-positive samples with unknown HPV types. Data analyses included algorithms of multiple infections to estimate type-specific relative contributions. 22,661 paraffin-embedded samples were obtained from 14,249 women. 10,575 cases of invasive cervical cancer were included in the study, and 8977 (85%) of these were positive for HPV DNA. The most common HPV types were 16, 18, 31, 33, 35, 45, 52, and 58 with a combined worldwide relative contribution of 8196 of 8977 (91%, 95% CI 90-92). HPV types 16 and 18 were detected in 6357 of 8977 of cases (71%, 70-72) of invasive cervical cancer. HPV types 16, 18, and 45 were detected in 443 of 470 cases (94%, 92-96) of cervical adenocarcinomas. Unknown HPV types that were identified with sequence analysis were 26, 30, 61, 67, 69, 82, and 91 in 103 (1%) of 8977 cases of invasive cervical cancer. Women with invasive cervical cancers related to HPV types 16, 18, or 45 presented at a younger mean age than did those with other HPV types (50·0 years [49·6-50·4], 48·2 years [47·3-49·2], 46·8 years [46·6-48·1], and 55·5 years [54·9-56·1], respectively). To our knowledge, this study is the largest assessment of HPV genotypes to date. HPV types 16, 18, 31, 33, 35, 45, 52, and 58 should be given priority when the cross-protective effects of current vaccines are assessed, and for formulation of recommendations for the use of second-generation polyvalent HPV vaccines. Our results also suggest that type-specific high-risk HPV-DNA-based screening tests and protocols should focus on HPV types 16, 18, and 45.

Comparison of human papillomavirus detection between freshly frozen tissue and paraffin embedded tissue of invasive cervical cancer

BackgroundHuman Papillomavirus (HPV) detection results comparing paraffin embedded cervical tissue and other cervical specimens have been done with varying degrees of agreement. However, studies comparing freshly frozen specimens and paraffin embedded specimens of invasive cervical carcinomas are lacking. The aim of the study was to compare HPV detection using SPF10 broad-spectrum primers PCR followed by DEIA and genotyping by LiPA25 (version 1) between freshly frozen cervical tissue samples and paraffin embedded blocks of cervical tissue from the same patient. There were 171 pairs of paraffin embedded and freshly frozen samples analyzed from cervical carcinoma cases from Kampala, Uganda.Results88.9% (95% CI: 83.2%-93.2%) of paraffin embedded samples were HPV positive compared with 90.1% (95% CI: 84.6%-94.1%) of freshly frozen samples, giving an overall agreement in HPV detection between fresh tissue and paraffin embedded tissue at 86.0% (95% CI: 79.8%-90.8%). Although the proportion of HPV positive cases in freshly frozen tissue was higher than those in paraffin blocks, the difference was not statistically significant (p > 0.05). In both types of tissues, single HPV infections were predominant, with HPV16 accounting for 47% of positive cases. Comparison in the overall agreement, taking into accounts not only positivity in general, but also HPV types, showed a 65% agreement (complete agreement of 59.7%, partial agreement of 5.3%) and complete disagreement of 35.0%. HPV detection in squamous cell carcinomas (SCC) and adenocarcinomas (ADC) was similar in fresh tissue or paraffin blocks (p ≥ 0.05).p16 immunostaining in samples that had at least one HPV negative results showed that 24 out of 25 cases had an over-expressed pattern.ConclusionsHPV DNA detection was lower among ADC as compared to SCC. However, such differences were minimized when additional p16 testing was added, suggesting that the technical issues may largely explain the HPV negative cases.

Human Papilloma Virus prevalence and type-specific relative contribution in invasive cervical cancer specimens from Italy

BackgroundCervical cancer represents an important global public health problem. It is the 2nd most common cancer among women worldwide. Human Papillomavirus (HPV) infection is now well-established as a necessary cause of invasive cervical cancer (ICC) development. Only a few studies on HPV prevalence and type-specific distribution in ICC have been conducted in Italy.AimTo describe the prevalence of HPV and the HPV type-specific distribution in ICC cases identified in Rome, Italy.Methods140 paraffin embedded tissue blocks of primary ICC diagnosed between 2001 and 2006 were identified at the Regina Elena Cancer Institute in Rome (Italy). HPV was detected through amplification of HPV DNA using SPF-10 HPV broad-spectrum primers followed by DEIA and then genotyping by LiPA25 (version 1).Results134 cases were considered suitable for HPV DNA detection after histological evaluation; and overall, 90.3% (121/134) HPV prevalence was detected. 111 cases had a single HPV type, 4 cases had an uncharacterized type (HPVX) and 6 cases had multiple HPV infections. The five most common single HPV types among positive cases were: HPV16 (71/121; 58.7%), HPV18 (12/121; 9.9%), HPV31, HPV45 and HPV58 (5/121; 4.1% each). 2 (1.5%) of the single infections and 2 (1.5%) of the multiple infections contained low risk types. Statistically significant differences in the relative contribution of HPV18 were found when comparing squamous cell carcinomas with adenocarcinomas.ConclusionsHPV16 and HPV18 accounted for almost 70% of all the HPV positive ICC cases. The study provides baseline information for further evaluation on the impact of recently introduced HPV vaccines in Italy.

Utility of PCR Amplification and DNA Microarray Hybridization in 16S rDNA for Rapid Diagnosis of Bacteremia Associated with Hematological Malignancies.

Abstract 1635Poster Board I-661 IntroductionThe rapid diagnosis of bacteremia is important for patient management and the choice of antimicrobial and antifungal therapy, especially in hematological malignancies. We describe a rapid detection and identification system that uses universal PCR primers and DNA microarray hybridization compared to the conventional culture method. This new DNA-microarray method could be performed within only 24 hours. Eighty-four microorganism probes are spotted onto a microarray plate containing most causal clinical pathogens (Patent No.WO2003/106676). We can detect 93% of pathogen of blood culture by the frequency of upper 28 microorganisms. Patients and MethodsWe used universal PCR primers to amplify a variable region of bacterial 16S ribosomal DNA or fungal ITS (internal transcribed spacer region) of ribosomal DNA, followed by the reverse hybridization of products to a panel of oligonucleotides. Genomic DNA was extracted from blood with a DNA isolation kit (DNeasy; QIAGEN, CA, USA) according to the manufacturer's instructions. The 16S ribosomal ribonucleic acid gene (16S rDNA) was amplified from genomic DNA using broad-spectrum PCR primers; 5'-AGAGTTTGATCCTGGCT-CAG-3', corresponding to Escherichia coli 16S rDNA positions 8 through 27, and 5'-GTATTACCGCGGCTGCTGG-3', corresponding to Escherichia coli 16S rRNA gene positions 517 through 535. On the other hand, the PCR primers; 5'-TCCGTAGGTGAACCTGCGG-3' and 5'-GCTGCGTTCTTCATCGATGC-3' are used to amplify the ITS 1 region. Thirty-five cycles of amplification were performed in a thermal cycler (Gene Amp PCR system 9600, Perkin-Elmer Cetus, CT, USA). The PCR products from samples were labeled by Cy5- primers and applied to the microarray. The microarray was scanned using a ScanArray 4000 (Axon Instruments, CA, USA) to determine the amount of Cy5-labeled PCR products. Fluorescence intensities were calculated with DNASIS Array software (HITACHI Soft Engineering, Japan). This new identification system using DNA microarray hybridization and the conventional blood culture method (BacT/ALERT, bioMerieux and Bactec, Becton-Dickinson, USA) were applied for 336 infectious episodes from 100 patients with hematological malignancy except 2 patients with aplastic anemia retrospectively from May 2004 to February 2007. The hematological malignancies included 152 episodes of acute myeloid leukemia (45.2%), 106 of non-Hodgkin lymphoma (31.5%), 38 of acute lymphoid leukemia (11.3%), 17 of multiple myeloma (5%), 12 of myelodysplastic syndrome (3.6%), 4 of aplastic anemia (1.2%) etc. ResultsThirty-nine episodes (11.6%) were positive after a few days of culturing, and 41 episodes (12.2%) were identified by the new microarray method within 24 hours. Both methods yielded positive results 10.7% (36 episodes). Pathogens in most cases were identical at the species level (21/36 episodes 58.3%), some cases were identical at the genus level (6/36 16.7%), in the other cases, several bacteria were detected by each method. A positive DNA microarray and negative blood culture occurred at a rate of 1.5% (5 episodes), with pathogens in these cases being Bacillus cereus, Staphylococcus epidermidis, Chryseobacterium indologenes, and Enterococcus faecium. A negative DNA microarray and positive blood culture occurred at a rate of 0.9% (3 episodes), and with pathogens comprising Staphylococcus aureus, á-Streptococcus, and Pseudomonas aeruginosa. Both methods yielded negative results at a rate of 86.9% (292 episodes). The sensitivity and specificity of this new microarray method were 92.3 and 98.3%, respectively, compared with the conventional blood culture method. Regarding fungal infection, only one episode out of one patient was found to be positive microarray and negative blood culture of Yeast like organism. ConclusionThis new DNA microarray method has the potential to diagnose bacteremia quickly and detect pathogens at least at the same rate as the conventional blood culture method. Regarding the possibility of improving the sensitivity and specificity, or application for fungal infection further study should be needed. In conclusion, DNA microarray is a useful method for the management of febrile patients with hematological malignancy. DisclosuresIwasaki:The ministry of Health, Labour and Welfare of Japan: Research Funding.

Human Papillomavirus Types in Invasive Cervical Cancer Specimens From Turkey

The main aim of the study is to describe the human papillomavirus (HPV) type-specific distribution in invasive cervical cancer (ICC) specimens from Turkey. Paraffin-embedded ICC specimens were identified from the histopathologic archives of the Hacettepe University Medical School in Turkey. HPV detection was carried out through amplification of HPV DNA by a SPF-10 broad-spectrum primer polymerase chain reaction and subsequently followed by DNA enzyme immunoassay and genotyping by LiPA25 (version 1). Two hundred seventy-seven ICC cases diagnosed between 1993 and 2004 were retrieved. After histologic evaluation and human beta-globin gene analysis for sample quality, 248 cases were considered suitable for HPV/DNA testing. HPV prevalence was 93.5% (232/248; 95% confidence interval: 90.5%-96.6%). The five most common HPV types identified as single types among HPV-positive cases were HPV16 (64.7%), HPV18 (9.9%), HPV45 (9.9%), HPV31 (3.0%), and HPV33 (2.2%). The study shows that in Turkey, HPV16/HPV18 accounted for 75.4% (95% confidence interval: 69.9%-81.0%) of HPV-positive ICC cases. This information is essential to evaluate the potential impact of the HPV vaccines in the country.

Abundance of Multiple High-Risk Human Papillomavirus (HPV) Infections Found in Cervical Cells Analyzed by Use of an Ultrasensitive HPV Genotyping Assay

PCR methods enable the detection of a large variety of human papillomavirus (HPV) genotypes that infect the anogenital tract. However, PCR with consensus primers, general primers, and, to a lesser extent, broad-spectrum primers may underrepresent the true prevalence of HPV, especially the true prevalence of multiple infections. We compared the rate of HPV positivity determined by a broad-spectrum PCR with primers BSGP5+ and BSGP6+ (BS-PCR) coupled to an established bead-based multiplex HPV genotyping (MPG) assay with the rate of HPV positivity determined by a multiplex PCR with type-specific primers (TS-PCR) coupled to a newly developed MPG assay for 735 selected cervical scraping samples. While the primers used for the BS-PCR are located within the L1 region of the HPV genome, the primers used for the TS-PCR target the E7 gene. The overall rates of positivity for the 19 HPV types included in both assays were 60.9% and 72.2% by the BS-PCR and the TS-PCR, respectively, and the two assays found multiple infections in 34.8% and 58.0% of the specimens, respectively. Both HPV detection assays allowed the semiquantitative detection of HPV types and identified the same dominant HPV type in 66.6% of the multiple infections. In conclusion, the TS-PCR-MPG assay significantly increased the rate of detection of HPV DNA and the number of infections with multiple HPV types detected and demonstrated that the prevalence of low-copy-number HPV infections in the anogenital tract may be strongly underestimated by conventional HPV amplification methods, especially in cases of multiple infections. As a consequence, PCR-TS-MPG appears to be highly suited for analysis of the significance of multiple infections in the development of cervical cancer and for the study the natural history and the latency of HPV.

Detection and Genotyping of Human Rotavirus VP4 and VP7 Genes by Reverse Transcriptase PCR and Reverse Hybridization

Rotavirus infections can be diagnosed in stool samples by serological and molecular methods. We developed a novel reverse transcriptase PCR (RT-PCR) method for the amplification of rotavirus RNA and a reverse hybridization assay on a strip to detect amplimers and identify the specific G and P genotypes present in human stool specimens. An additional aim was to permit specific identification of the rotavirus G1P[8] strain, used in the Rotarix vaccine. Novel broad-spectrum PCR primers were developed for both VP4 and VP7, permitting the amplification of a wide range of rotavirus genotypes. Primer sets comprise mixtures of defined primer sequences. For the identification of G and P genotypes, two reverse hybridization strip assays were developed. Both the VP4 and the VP7 strip contain universal probes for the detection of VP4 and VP7 sequences, irrespective of the G or P genotype. The VP4 strip contains type-specific probes for P[4], P[6], P[8], P[9], and P[10]. The VP7 strip contains type-specific probes for G1, G2, G3, G4, G5, G6, G8, and G9. In addition, probes to distinguish between wild-type G1 and G1 vaccine strain sequences were present. Testing by analysis of multiple reference strains confirmed that both RT-PCR methods allowed the detection of a broad spectrum of genotypes. RT-PCR for VP7 was more sensitive than RT-PCR for VP4, but all samples identified as positive for rotavirus antigen by an enzyme-linked immunosorbent assay (ELISA) were also positive for both VP4 and VP7. The high specificity of the reverse hybridization method was confirmed by sequence analysis as well as by type-specific PCR, and the vaccine strain could also be specifically identified. The reverse hybridization method permits accurate identification of mixed infections with different genotypes. Rotavirus genotypes for which no type-specific probes were present on the strip were adequately identified by the universal detection probes. The assay was formally validated by analyses of specificity, sensitivity, precision, accuracy, and robustness. In a panel of 149 ELISA-positive stool samples, comparison with conventional type-specific RT-PCR methods revealed the superiority of the novel method, mainly in cases of mixed rotavirus infections. This novel method permits highly accurate detection and identification of human rotavirus infections in stool samples. This validated assay could be useful for large-scale epidemiological and clinical trials.

Multiplex real-time PCR for detection and quantification of mycotoxigenic Aspergillus, Penicillium and Fusarium

The most agriculturally and economically important classes of mycotoxins are produced by species of Aspergillus, Penicillium, and Fusarium. Rapid methods to detect mycotoxigenic fungi could help prevent mycotoxins from entering the food chain. The purpose of this research was to develop a multiplex real-time PCR assay to detect and quantify multiple species of mycotoxigenic fungi. A pair of broad-spectrum PCR primers was designed for amplification of the internal transcribed spacer (ITS) regions of rDNA from the mycotoxigenic species. An in silico analysis of the primers revealed the presence of amplification in more than 40 Aspergillus species, 23 Fusarium species, and 32 Penicillium species as well as 64 other fungal genera. Genus-specific Taqman probes were designed from the ITS sequences of the most important mycotoxigenic species of Fusarium, Penicillium, and Aspergillus. The specificity of the probes was established against a wide range of fungal species. As a multiplex assay, the linear range of detection was 1 pg to 10 ng of DNA. The assay was validated by analyzing fungal growth in distiller's grain (DG), an animal feedstock that is a by-product when ethanol is produced from corn. This assay could be used as an initial step to evaluate the mycotoxigenic potential of DG and various other agricultural commodities.

5′ Noncoding Region Alone Does Not Unequivocally Determine Genetic Type of Human Rhinovirus Strains

During the last couple of years there has been a delightful increase in interest in genetic typing of human rhinoviruses. This is to a large extent due to the discovery of a proposed novel clade, human rhinovirus C (HRV-C). As a consequence, new methods have been reported aiming at unequivocal distinction of traditional HRV prototype strains as well as the newly found uncultivable HRV-C strains. We read with interest the article by Kiang and coworkers (2) describing reverse transcription-PCR-sequencing applications for genetic typing of human rhinoviruses targeting the 5′noncoding region (5′ NCR). A similar approach with largely similar results was published earlier (6). Relatively conserved areas within this region enable broad-spectrum primer design for sensitive methods. However, there are several issues to consider when using the 5′ NCR for genetic typing of HRV. Current taxonomy and classification of picornaviruses are based on capsid region coding sequences. On the basis of this region, a group of previously characterized novel HRV strains form one distinct clade (5, 7) (Fig. ​(Fig.1A),1A), a fact that has also been the basis of the proposal of the Picornavirus Study Group to form a new species, HRV-C, within the Enterovirus genus (4). However, in the article by Kiang et al., on the basis of the partial 5′ NCR sequences, the designated HRV-C strains clustered within the HRV-A clade (2). In contrast, the strains labeled HRV-C in this article formed a clade of their own. As a consequence, because 5′ NCR sequences do not segregate the designated HRV-C from HRV-A (Fig. ​(Fig.1B),1B), they should not be used for typing of new strains. Nevertheless, this region is quite suitable for selected topics of molecular epidemiology, such as analysis of short-term transmission routes (1, 8) or tentative prediction of genetic type as in human enteroviruses (HEV) (9). The sequences nominated as HRV-C by Kiang et al. (2) and by Lee et al. (6) form a new clade in the 5′ NCR. The exact taxonomic position of this clade should be determined according to the clustering of these strains in the capsid region. Clearly, it is divergent from all known HRV and HEV clades in the 5′ NCR, but the decision on whether the strains represent HRV-C or some other picornavirus group cannot be made on the basis of the 5′ NCR alone. FIG. 1. Phylogenetic trees in the VP4/VP2 capsid coding region (A) and in the 5′ NCR (B) of different species of the enterovirus genus showing different clustering of the species in the two regions. Trees were constructed with MEGA4 using the neighbor-joining ... The area close to the beginning of the open reading frame in the 5′ NCR is known to be a recombination hot spot in HEV. Although frequent recombination has not yet been shown for HRV, the analysis of the complete genome sequence data of all HRV prototype strains has not yet been published. Furthermore, the number of completely sequenced genomes of circulating HRV strains has remained low and is too low to conclude that the evolution in the 5′ NCR is always congruent with that of the capsid. Therefore, we would see phylogenetic analysis of the 5′ NCR of HRV as a welcome addition to HRV research, but not a surrogate of capsid coding sequence-based typing.