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Clinical Specimens (clinical + specimen)
Selected AbstractsEvaluation and application of reverse transcription loop-mediated isothermal amplification for detection of noroviruses,JOURNAL OF MEDICAL VIROLOGY, Issue 3 2007Tomoko Yoda Abstract A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of norovirus (NV) was developed. In order to design primer sets for the detection of a wide range of NVs, NVs were categorized into three groups, that is, genogroup I (GI), prevalent GII, and minor GII; three sets of primers were developed for each group. Clinical specimens of patients suffering from enteric RNA viruses, such as NV, group A and C rotavirus, and sapovirus were examined using these primer sets. Various genotypes of NVs were detected in clinical specimens from patients infected with NV where no false positive reaction was observed with other enteric RNA viruses. Additionally, 88 samples of acute gastroenteritis outbreaks were analyzed by an RT-LAMP assay and compared with the results of routine RT-PCR. The results of the RT-LAMP assay corresponded well to that of RT-PCR. These findings suggest the practical application of the RT-LAMP assay for the detection of NVs in clinical specimens. Consequently, the RT-LAMP system and conventional detection kits (NVGI and NVGII detection kits; Eiken Chemical Co., Ltd., Japan) were compared. The detection rate of the prevalent and minor GII primer sets was similar to that of the conventional NVGII kit, while the detection rate of the GI primer set is different because it can detect several genotypes better than the conventional NVGI kit. This is an initial report that the RT-LAMP system is able to detect NVs in clinical specimens within a wide range. J. Med. Virol. 79:326,334, 2007. © 2007 Wiley-Liss, Inc. [source] Hepatitis E antibody profiles in serum and urineJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 3 2002M.S. Joshi Abstract The aim of this study was to evaluate anti-HEV antibody profiles in urine specimens in comparison to corresponding serum samples to assess the utility of urine as a clinical specimen. Paired serum and urine specimens from 71 hepatitis E patients, 33 non-E hepatitis patients, 63 patients with nonhepatic diseases, and 26 healthy individuals were tested by recombinant HEV protein (55 kD)-based indirect enzyme-linked immunosorbent assay (ELISA). Uronegativity for anti-HEV IgM was noted in 71 (100%) serologically confirmed patients with hepatitis E. Hepatitis E patients (10/10) showed urinary absence or very low levels of total IgM by capture ELISA, suggesting absence or low levels of filtration, and/or local synthesis, and/or transudation of IgM in urine during infection. When these patients were tested for total IgG and IgA, microquantities of immunoglobulins were noted in all urine samples (10/10 for each). However, the proportions of uropositivity for anti-HEV IgG and IgA in hepatitis E patients were low and indicated only 21.42% and 49.33% concordance with seropositivity, respectively. Control groups also showed low and variable uropositivity for anti-HEV IgG and IgA. Overall, HEV-specific antibodies exhibited by serum in recent and past infections were not found in urine. The study demonstrated the inadequacy of urine specimens for detection of hepatitis E antibodies. J. Clin. Lab. Anal. 16:137,142, 2002. © 2002 Wiley-Liss, Inc. [source] Linking Pneumocystis jiroveci sulfamethoxazole resistance to the alleles of the DHPS gene using functional complementation in Saccharomyces cerevisiaeCLINICAL MICROBIOLOGY AND INFECTION, Issue 5 2010R. Moukhlis Clin Microbiol Infect 2010; 16: 501,507 Abstract Curative and prophylactic therapy for Pneumocystis jiroveci pneumonia relies mainly on cotrimoxazole, an association of trimethoprim and sulfamethoxazole (SMX). SMX inhibits the folic acid pathway through competition with para-aminobenzoic acid (pABA), one of the two substrates of the dihydropteroate synthase (DHPS), a key enzyme in de novo folic acid synthesis. The most frequent non-synonymous single nucleotide polymorphisms (SNPs) in P. jiroveci DHPS are seen at positions 165 and 171, the combination leading to four possible different genetic alleles. A number of reports correlate prophylaxis failure and mutation in the P. jiroveci DHPS but, because of the impossibility of reliably cultivating P. jiroveci, the link between DHPS mutation(s) and SMX susceptibility is not definitively proven. To circumvent this limitation, the yeast Saccharomyces cerevisiae was used as a model. The introduction of the P. jiroveci DHPS gene, with or without point mutations, directly amplified from a clinical specimen and cloned in a centromeric plasmid into a DHPS-deleted yeast strain, allowed a fully effective complementation. However, in the presence of SMX at concentrations >250 mg/L, yeasts complemented with the double mutated allele showed a lower susceptibility compared with strains complemented with either a single mutated allele or wild-type alleles. These results confirm the need for prospective study of pneumocystosis, including systematic determination of the DHPS genotype, to clarify further the impact of mutations on clinical outcome. Additionally, the S. cerevisiae model proves to be useful for the study of still uninvestigated biological properties of P. jiroveci. [source] Detection of bacterial DNA by PCR and reverse hybridization in the 16S rRNA gene with particular reference to neonatal septicemiaACTA PAEDIATRICA, Issue 2 2001S Shang Aim: The clinical diagnosis of sepsis is difficult, particularly in neonates. It is necessary to develop a rapid and reliable method for detecting bacteria in blood and cerebrospinal fluid (CSF) Polymerase chain reaction (PCR) and reverse hybridization of the 16S rRNA gene would permit fast and sensitive determination of the presence of bacteria and differentiate gram-positive bacteria from gram-negative ones in clinical specimens. Methods: We developed a pair of primers according to the gene encoding 16SrRNA found in all bacteria. DNA fragments from different bacterial species and from clinical samples were detected with PCR, and with reverse hybridization using a universal bacterial probe, a gram-positive probe and a gram-negative probe. Results: A 371 bp DNA fragment was amplified from 20 different bacterial species. No signal was observed when human DNA and viruses were used as templates. The sensitivity could be improved to 10T -12 g. All 26 culture-positive clinical samples (22 blood samples and 4 CSF samples) were positive with PCR. The gram-negative and gram-positive probes hybridized to clinical samples and to known bacterial controls, as predicted by Gram's stain characteristics. Conclusions: Our results suggest that the method of PCR and reverse hybridization is rapid, sensitive and specific in detecting bacterial infections. This finding may be significant in the clinical diagnosis of sepsis in neonates. [source] Validated protocol for FoxP3 reveals increased expression in type 1 diabetes patients,CYTOMETRY, Issue 2 2009Jean Grant Abstract Background FoxP3 has become a key identifier of regulatory T cells. Investigators have used a variety of antibodies and methods for detecting FoxP3 by flow cytometry. To standardize FoxP3 antibody staining for use in clinical trial samples, we tested various antibodies from different vendors, cell preparation protocols and fix/perm reagents, and cell isolation procedures. Using this optimized staining protocol, we evaluated clinical specimens from patients with multiple sclerosis (MS) or type 1 diabetes. Methods FoxP3 antibodies from eBioscience (236A/E7 and PCH101) and BioLegend (206D) were evaluated along with their respective methods and fix/perm reagents for preparation and staining of FoxP3 for flow cytometry. Fresh washed blood and frozen or fresh PBMC were evaluated. Upon optimization of the protocol, clinical samples (frozen PBMC) from patients with MS or type 1 diabetes and healthy control donors were evaluated with the BioLegend antibody. Results Clone 206D from BioLegend yielded optimal staining and the fix/perm reagents from both eBioscience and BioLegend were comparable. Data were also comparable between cells separated by Ficoll (fresh or frozen) and washed blood samples, allowing this protocol to be applicable to different types of samples. We validated this protocol using clinical samples and saw a significant increase in FoxP3 expression in the patients with type 1 diabetes but not in the MS. Conclusions The results from this study will allow the assessment of FoxP3 by flow cytometry on samples from clinical sites that are analyzed in real time on fresh blood or frozen PBMC. © 2008 Clinical Cytometry Society [source] Application of flow cytometry for biomarker-based cervical cancer cells detectionDIAGNOSTIC CYTOPATHOLOGY, Issue 2 2008Jian Ling Ph.D. Abstract The Pap test used for cervical cancer screening is subjective, labor-intensive, and has relatively low sensitivity and specificity for the detection of underlying clinically significant lesions. The objective of this study is to develop a biomarker/flow cytometry-based approach for cervical cancer screening. Immunofluorescence technology to quantify cervical cell expression of two biomarkers p16INK4A and Mcm5 was developed and evaluated by both microcopy and flow cytometry. The capability of using biomarker/flow cytometry approach to detect rare-event dysplastic cells in a large background of benign epithelial and inflammatory cells was evaluated. The results indicate that flow cytometry could detect 0.01% dysplastic cells in a background of normal cervical epithelial cells with the combination of the two biomarkers. Thirty-two clinical specimens were used to test the biomarker/flow cytometry-based approach and the results were compared with the liquid-based cervical cytology. The experiment yielded 100% sensitivity and 93% specificity with reference to the liquid-based cervical cytology. This study indicates the promise of using multi-color fluorescence flow cytometry for biomarker-based cervical cancer screening. This molecular-based, potentially high-throughput and automated method is expected to provide an alternative/auxiliary means of cervical cancer screening. Diagn. Cytopathol. 2008;36:76,84. © 2008 Wiley-Liss, Inc. [source] Colony blot assay: a useful method to detect multiple pneumococcal serotypes within clinical specimensFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 3 2004D Bogaert Abstract The efficacy of pneumococal conjugate vaccines in young children may be complicated by serotype replacement. We developed a colony blot assay which enables the identification of re-colonization with novel serotypes (replacement), overgrowth by minor co-colonizing serotypes or suppression of previously predominant vaccine serotype strains as a result of vaccination. This method allows the identification of multiple serotypes in a single specimen in a ratio of 1:1000. In order to demonstrate the potential of our method, we investigated the consecutive nasopharyngeal samples of 26 children who had shown a shift in pneumococcal colonization after conjugate vaccination. Mixed colonization was found once in 15 pre-vaccination samples and four times in 26 post-vaccination samples. In the remaining children ,true replacement' had presumably occurred. Hence, we conclude that the colony blot assay is an easy to apply method, which allows the identification of different pneumococcal serotypes within single clinical specimens. [source] Molecular typing of meningococci: recommendations for target choice and nomenclatureFEMS MICROBIOLOGY REVIEWS, Issue 1 2007Keith A. Jolley Abstract The diversity and dynamics of Neisseria meningitidis populations generate a requirement for high resolution, comprehensive, and portable typing schemes for meningococcal disease surveillance. Molecular approaches, specifically DNA amplification and sequencing, are the methods of choice for various reasons, including: their generic nature and portability, comprehensive coverage, and ready implementation to culture negative clinical specimens. The following target genes are recommended: (1) the variable regions of the antigen-encoding genes porA and fetA and, if additional resolution is required, the porB gene for rapid investigation of disease outbreaks and investigating the distribution of antigenic variants; (2) the seven multilocus sequence typing loci,these data are essential for the most effective national, and international management of meningococcal disease, as well as being invaluable in studies of meningococcal population biology and evolution. These targets have been employed extensively in reference laboratories throughout the world and validated protocols have been published. It is further recommended that a modified nomenclature be adopted of the form: serogroup: PorA type: FetA type: sequence type (clonal complex), thus: B: P1.19,15: F5-1: ST-33 (cc32). [source] Cocirculation of and coinfections with hepatitis A virus subgenotypes IIIA and IB in patients from Pune, western IndiaHEPATOLOGY RESEARCH, Issue 2 2007Shobha Chitambar Aim:, During the 1990s, a changing pattern of epidemiology of hepatitis A was reported in different populations of India. The present study was undertaken to investigate the molecular epidemiology of hepatitis A virus (HAV) strains over a period of 10 years. Methods:, Stool/serum samples were collected from hepatitis A patients clinically presenting acute viral hepatitis and hepatic encephalopathy. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to detect HAV-RNA. HAV genomes were examined by sequencing PCR products of VP1/2A junction (168 bp) and RNA polymerase (116 bp) regions. Results:, Subgenotype IIIA and IB were detected in 74.2% and 9.7% of specimens, respectively, while 16.1% of patients had mixed infections. Sewage samples also showed presence of both IIIA (9/10) and IB (1/10) subgenotypes. RNA polymerase region showed two clusters constituting 51.6% and 19.4% strains closer to Nor21 and HM175 strains, respectively, in clinical specimens. Three isolates appeared as discordant subgenotypes in VP1/2A and RNA polymerase regions. Conclusion:, The data revealed cocirculation of and coinfection with subgenotypes IIIA and IB, with predominance of IIIA and genetic heterogeneity of HAV strains in western India. [source] Detection of drug-resistant HIV minorities in clinical specimens and therapy failureHIV MEDICINE, Issue 3 2008S Louvel Objective Particularly for therapy-experienced patients, resistance assessment by genotypic or phenotypic methods produces discordances. This study seeks proof that differences may arise from the fact that genotyping produces a single summary sequence whereas replicative phenotyping (rPhenotyping) functionally detects and assigns resistances in mixed HIV populations. Methods For validation, defined mixes of wild-type and M184V mutant were analysed by rPhenotyping or standard genotyping. Allele-specific and quantitative polymerase chain reaction (PCR) set detection and quantification limits for minor virus populations in vitro and in authentic clinical samples showing geno-/pheno-discrepant lamivudine resistance. Results Allele-specific and real-time PCR methods detected down to 0.3% of mutant M184V. The functional assessment was sensitive enough to reveal <1% of mutant M184V in mixed samples. Also in discordant samples from the diagnostic routine, in which rPhenotyping had identified drug resistance, real-time PCR confirmed minute amounts of mutant M184V. Conclusion By utilizing the replication dynamics of HIV under drug pressure, a rPhenotyping format potently reveals relevant therapy-resistant minority species, even of HIV known to possess reduced replicative fitness. With its rapid turnaround of 8 days and its high sensitivity, our rPhenotyping system may be a valuable diagnostic tool for detecting the early emergence of therapy-threatening HIV minorities or the persistence of residual resistant virus. [source] Prognostic evaluation of epidermal fatty acid-binding protein and calcyphosine, two proteins implicated in endometrial cancer using a proteomic approachINTERNATIONAL JOURNAL OF CANCER, Issue 10 2008Zhengyu Li Abstract With the aim to translate the discovery from proteomic research into clinical applications, we identified epidermal fatty acid-binding protein (E-FABP) and calcyphosine (CAPS) by MALDI-Q-TOF MS and validated their overexpressions by immunoblotting. Their expression statuses were examined by immunohistochemistry in 39 normal endometrium, 29 endometrial intraepithelial neoplasia (EIN) and 84 endometrial cancer (EC) cases. We evaluated the correlations to the clinicopathologic characteristics and determined whether these proteins had prognostic significance. Expressions of E-FABP and CAPS were increased 2.64- and 2.18-fold in EC by immunoblotting. Immunoreactivity of both E-FABP and CAPS were stronger in EC than in EIN or normal tissues (p < 0.001 and < 0.001). Stronger immunoreactivity of E-FABP and CAPS were shown to present with poor differentiation (p = 0.032 and 0.001), but no relevance was observed with staging (p = 1.368 and 4.306). Survival analysis indicated that immunoreactivity of CAPS was correlated to poor survival (p = 0.018), but E-FABP status appeared to be no correlation to the clinical outcome of patients (p = 0.865). Multivariate analysis indicated that CAPS might be an independent prognostic factor for survival in patients with EC (p = 0.008). Results demonstrated the ubiquitous overexpressions of E-FABP and CAPS in EC and the correlations to the clinicopathologic parameters. CAPS might be a potential prognostic factor for survival in patients with EC. The research pattern from proteomics to clinical specimens would have widespread applications. © 2008 Wiley-Liss, Inc. [source] Extraintestinal manifestations of Edwardsiella tarda infectionINTERNATIONAL JOURNAL OF CLINICAL PRACTICE, Issue 8 2005I-K Wang Summary Edwardsiella tarda, a member of the family Enterobacteriaceae, is a rare human pathogen. Gastroenteritis is the most frequently reported manifestation of E. tarda infection. In contrast, extraintestinal infection with E. tarda has rarely been reported. This study made a retrospective case and microbiological data review of patients with extraintestinal E. tarda infections to further understand this disease. This study retrospectively reviewed the charts of all isolates of E. tarda cultures from clinical specimens other than faeces at Chang Gung Memorial Hospital, Taoyuan, Taiwan from October 1998 through December 2001. Edwardsiella tarda was isolated from 22 clinical specimens from 22 hospitalised patients (13 females and nine males). The extraintestinal manifestations of E. tarda infection included biliary tract infection, bacteraemia, skin and soft tissue infection, liver abscess, peritonitis, intra-abdominal abscess, and tubo-ovarian abscess. The major underlying diseases predisposing to E. tarda extraintestinal infection were hepatobiliary diseases, malignancy and diabetes mellitus. The overall mortality rate of E. tarda extraintestinal infection in the present series was 22.7% (5/22), and four (40%) of 10 patients with bacteraemia expired. Although rare, human E. tarda extraintestinal infections can have diverse clinical manifestations and moreover may cause severe and life-threatening infections. Consequently, E. tarda should be considered a potentially important pathogen. [source] Antisense oligodeoxynucleotide therapy targeting clusterin gene for prostate cancer: Vancouver experience from discovery to clinicINTERNATIONAL JOURNAL OF UROLOGY, Issue 9 2005HIDEAKI MIYAKE Abstract Background The objective of this study was to review our experience in the development of antisense (AS) oligodeoxynucleotide (ODN) therapy for prostate cancer targeting antiapoptotic gene, clusterin. Methods We initially summarized our data demonstrating that clusterin could be an optimal therapeutic target for prostate cancer, then presented the process of developing AS ODN therapy using several preclinical animal models. Finally, the preliminary data of the recently completed phase I clinical trial using AS clusterin ODN as well as the future prospects of this therapy are discussed. Results Expression of clusterin was highly up-regulated after androgen withdrawal and during progression to androgen-independence, but low or absent in untreated tissues in both prostate cancer animal model systems and human clinical specimens. Introduction of the clusterin gene into human prostate cancer cells confers resistance to several therapeutic stimuli, including androgen ablation, chemotherapy and radiation. AS ODN targeting the translation initiation site of the clusterin gene markedly inhibited clusterin expression in prostate cancer cells in a dose-dependent and sequence-specific manner. Systemic treatment with AS clusterin ODN enhanced the effects of several conventional therapies through the effective induction of apoptosis in prostate cancer xenograft models. Based on these findings, a phase I clinical trial was completed using AS clusterin ODN incorporating 2,-O-(2-methoxy)ethyl-gapmer backbone (OGX-011), showing up to 90% suppression of clusterin in prostate cancer. Conclusions The data described above identified clusterin as an antiapoptotic gene up-regulated in an adaptive cell survival manner following various cell death triggers that helps confer a phenotype resistant to therapeutic stimuli. Inhibition of clusterin expression using AS ODN technology enhances apoptosis induced by several conventional treatments, resulting in the delay of AI progression and improved survival. Clinical trials using AS ODN confirm potent suppression of clusterin expression and phase II studies will begin in early 2005. [source] Identification of Escherichia coli O172 O-antigen gene cluster and development of a serogroup-specific PCR assayJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2004H. Guo Abstract Aim:, To characterize the locus for O-antigen biosynthesis from Escherichia coli O172 type strain and to develop a rapid, specific and sensitive PCR-based method for identification and detection of E. coli O172. Methods and Results:, DNA of O-antigen gene cluster of E. coli O172 was amplified by long-range PCR method using primers based on housekeeping genes galF and gnd Shot gun bank was constructed and high quality sequencing was performed. The putative genes for synthesis of UDP-FucNAc, O-unit flippase, O-antigen polymerase and glycosyltransferases were assigned by the homology search. The evolutionary relationship between O-antigen gene clusters of E. coli O172 and E. coli O26 is shown by sequence comparison. Genes specific to E. coli O172 strains were identified by PCR assays using primers based on genes for O-unit flippase, O-antigen polymerase and glycosyltransferases. The specificity of PCR assays was tested using all E. coli and Shigella O-antigen type strains, as well as 24 clinical E. coli isolates. The sensitivity of PCR assays was determined, and the detection limits were 1 pg ,l,1 chromosomal DNA, 0·2 CFU g,1 pork and 0·2 CFU ml,1 water. The total time required from beginning to end of the procedure was within 16 h. Conclusion:, The O-antigen gene cluster of E. coli O172 was identified and PCR assays based on O-antigen specific genes showed high specificity and sensitivity. Significance and Impact of the Study:, An O-antigen gene cluster was identified by sequencing. The specific genes were determined for E. coli O172. The sensitivity of O-antigen specific PCR assay was tested. Although Shiga toxin-producing O172 strains were not yet isolated from clinical specimens, they may emerge as pathogens. [source] Evaluation of extracts of Jatropha curcas and Moringa oleifera in culture media for selective inhibition of saprophytic fungal contaminantsJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 3 2009Grace Mebi Ayanbimpe Abstract Most fungi occur in nature and utilize simple sources of carbohydrates and nitrogen for growth. Sabouraud's dextrose agar has been an ideal medium for primary isolation of fungi from clinical specimens, but for specimens from nonsterile sites or heavily contaminated ones, it has been necessary to include inhibitory substances such as antibiotics like chloramphenicol (antibacterial) and cycloheximide (antifungal). The problems we have in the our laboratory owing to frequent contamination of cultures and the delays in the procurement of cycloheximide have stimulated a search for alternatives in our local environment to enhance effective laboratory diagnoses of fungal infections. Purified extracts of the leaves and bark of Jatropha curcas and Moringa oleifera (common plants in our locality) were tested against clinical isolates of fungi at various concentrations to determine the minimum inhibitory concentration at which common fungal contaminants are inhibited, without affecting the growth of the pathogenic fungi sought for. At a concentration of 0.75,mg,ml,1 contaminants were totally inhibited by the leaf extracts. The bark extracts did not inhibit any fungus even at higher concentrations. From the results it was evident that the leaf extracts of both plants have potentials for use as inhibitory substances in culture media against contaminant fungi including Aspergillus spp., Penicillium spp., etc. J. curcas and M. oleifera are very common plants in our locality. They can be obtained at almost no cost and at any time needed. The benefits of these findings to mycology laboratories in a developing country are enormous. J. Clin. Lab. Anal. 23:161,164, 2009. © 2009 Wiley-Liss, Inc. [source] Development of real-time detection direct test for hepatitis B virus and comparison with two commercial tests using the WHO international standardJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 11 2003MOTOKAZU MUKAIDE Abstract Aims:, A highly reproducible and sensitive hepatitis B virus real-time detection direct (HBV RTD-direct) test using DNA extraction by magnetic beads coated with polyclonal anti-HBsAg, followed by the real-time detection polymerase chain reaction (PCR) method, was developed for the detection of HBV DNA. Methods:, The HBV DNA could be extracted from the HBsAg positive viral particles without resulting in viral DNA fragmentation. The HBV RTD-direct test was validated using a serial dilution panel of the WHO standard HBV DNA 97/746 I. Results:, The test had a dynamic range of 0.7,8.0 log10 international units (IU) per mL and the results were shown to be comparable to those obtained with two commercially available tests: the HBV DNA transcription-mediated amplification-hybridization protection assay and the Amplicor HBV Monitor test. In addition, the HBV RTD-direct test, based on magnetic extraction, successfully eliminated PCR inhibitors in clinical specimens. Conclusion:, We conclude that the HBV RTD-direct test is an excellent alternative for monitoring patients undergoing antiviral treatment or for screening various clinical specimens. [source] Viral loads of herpes simplex virus in clinical samples,A 5-year retrospective analysis,JOURNAL OF MEDICAL VIROLOGY, Issue 11 2010Julian W. Tang Abstract Viral loads of herpes simplex virus (HSV) are not monitored usually for the effective clinical management of HSV-related diseases. However, recently, there has been more interest about the typical HSV levels in clinical specimens, and how such data may improve understanding of the behavior of this virus in such clinical presentations, particularly in immunocompromised patients, where more prolonged therapy using higher doses of antiviral drugs may be required. Using an in-house quantitative HSV-1/HSV-2 polymerase chain reaction assay, an observational, retrospective 5-year analysis of diagnostic, quantitative HSV-1 and HSV-2 DNA levels was conducted. The results (all in median log10 DNA copies/ml), including perhaps the first quantitative comparison of cerebrospinal fluid (CSF) HSV viral loads, were as follows: CSF: HSV-1, 3.40 (range 2.30,8.98) versus HSV-2, 3.60 (range 2.31,6.86) (P,=,0.559); plasma: HSV-1, 3.20 (range 2.23,5.51) versus HSV-2, 3.20 (range 3.18,3.41) (P,=,0.905); genital swabs: HSV-1, 6.79 (range 2.28,8.48) versus HSV-2, 6.97 (range 3.40,9.66) (P,=,0.810); oral swabs: HSV-1, 7.28 (range 2.46,10.04) versus HSV-2, 5.62 (range 4.60,6.63) (P,=,0.529). Note that with the samples usually collected for HSV testing (i.e., CSF, plasma, oral, and genital swabs) there was no significant difference in the viral loads between HSV-1 and HSV-2 types, nor between immunocompetent and immunocompromised patients for each of these different HSV types. Indeed, even between immunocompromised patients with similar diseases, for these samples, the HSV loads were found to vary considerably. These findings may therefore limit the usefulness of monitoring HSV loads in everyday clinical practice. J. Med. Virol. 82:1911,1916, 2010. © 2010 Wiley-Liss, Inc. [source] Analysis of mixed infections by multiple genotypes of human cytomegalovirus in immunocompromised patientsJOURNAL OF MEDICAL VIROLOGY, Issue 5 2009P. Sowmya Abstract Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in immunocompromised patients. The present study was carried out to determine the frequency of occurrence of multiple genotypes of HCMV in immunocompromised patients, to determine if there is any discrepancy in identification of mixed infections by multiple genotypes in paired clinical specimens obtained from patients and to determine the significance of viral load differences between patients infected with single and multiple genotypes. One hundred clinical specimens from 75 patients were included in the study. Real-time PCR; Multiplex PCR and PCR-based RFLP were applied for the determination of viral load and genotyping of HCMV, respectively. Out of the 75 patients, 36 (48%) carried multiple genotypes. Discrepancy with regard to detection of genotypes were found in 17/25 patients whose paired clinical specimens were analyzed. Mixed genotypes were found more often in peripheral blood than urine or intraocular fluids collected from the same patient. There was a statistically significant difference between the median viral loads of clinical specimens carrying single genotypes and multiple genotypes. Mixed infections with multiple genotypes were found predominantly in the leukocyte fraction of peripheral blood specimens. The detection of mixed infections by multiple genotypes in the hypervariable regions of HCMV can be a surrogate marker of an increase in viral load. J. Med. Virol. 81:861,869, 2009. © 2009 Wiley-Liss, Inc. [source] Evaluation and application of reverse transcription loop-mediated isothermal amplification for detection of noroviruses,JOURNAL OF MEDICAL VIROLOGY, Issue 3 2007Tomoko Yoda Abstract A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of norovirus (NV) was developed. In order to design primer sets for the detection of a wide range of NVs, NVs were categorized into three groups, that is, genogroup I (GI), prevalent GII, and minor GII; three sets of primers were developed for each group. Clinical specimens of patients suffering from enteric RNA viruses, such as NV, group A and C rotavirus, and sapovirus were examined using these primer sets. Various genotypes of NVs were detected in clinical specimens from patients infected with NV where no false positive reaction was observed with other enteric RNA viruses. Additionally, 88 samples of acute gastroenteritis outbreaks were analyzed by an RT-LAMP assay and compared with the results of routine RT-PCR. The results of the RT-LAMP assay corresponded well to that of RT-PCR. These findings suggest the practical application of the RT-LAMP assay for the detection of NVs in clinical specimens. Consequently, the RT-LAMP system and conventional detection kits (NVGI and NVGII detection kits; Eiken Chemical Co., Ltd., Japan) were compared. The detection rate of the prevalent and minor GII primer sets was similar to that of the conventional NVGII kit, while the detection rate of the GI primer set is different because it can detect several genotypes better than the conventional NVGI kit. This is an initial report that the RT-LAMP system is able to detect NVs in clinical specimens within a wide range. J. Med. Virol. 79:326,334, 2007. © 2007 Wiley-Liss, Inc. [source] Detection and genotyping of human papillomavirus in cervical samples from Italian patientsJOURNAL OF MEDICAL VIROLOGY, Issue 4 2005M.A. De Francesco Abstract Human papillomaviruses (HPVs) are etiological agents of cervical cancer. In order to assess the epidemiological incidence and frequency of different HPV types, we applied a polymerase chain reaction (PCR)-direct sequencing approach based on the use of MY09/MY11 primers as compared to Hybrid Capture assay. Cervical samples were taken from 1,500 women, both with normal and abnormal cytological smears, and we found an incidence of 6.6% of HPV infection in Brescia. Overall, 97 samples tested HPV-positive, yielding 18 HPV types. The four most frequent HPV types were: HPV 16, -31, -6, and -58. This approach could be used in ordinary laboratory settings for quick and reliable typing of known and novel HPVs from clinical specimens and it could also be applied to anti-cancer vaccine development. J. Med. Virol. 75:588,592, 2005. © 2005 Wiley-Liss, Inc. [source] Rapid and sensitive detection of mumps virus RNA directly from clinical samples by real-time PCRJOURNAL OF MEDICAL VIROLOGY, Issue 3 2005Kazue Uchida Abstract A rapid, sensitive, and specific assay to detect mumps virus RNA directly from clinical specimens using a real-time PCR assay was developed. The assay was capable of detecting five copies of standard plasmid containing cDNA from the mumps virus F gene. No cross-reactions were observed with other members of Paramyxoviridae, or with viruses or bacteria known to be meningitis pathogens. Seventy-three clinical samples consisting of throat swabs collected from patients with parotitis, and cerebrospinal fluid (CSF) collected from patients with aseptic meningitis, were examined with a real-time PCR assay developed by the authors, reverse-transcription nested-PCR (RT-n-PCR), and virus isolation using cell culture. Like the RT-n-PCR assay, the real-time PCR assay could detect mumps virus RNA in approximately 70% of both throat swabs and CSF samples, while, by tissue culture, mumps virus was isolated from only approximately 20% of CSF and 50% of throat swab samples. In addition, the real-time PCR assay could be developed easily into a quantitative assay for clinical specimens containing more than 1,800 copies of mumps virus RNA/ml by using serial dilutions of the standard plasmid. The results suggest that the real-time PCR assay is useful for identification of mumps virus infections, not only in typical cases, but also in suspected cases, which show only symptoms of meningitis or encephalitis. J. Med. Virol. 75:470,474, 2005. © 2005 Wiley-Liss, Inc. [source] Future Prospects for Biomarkers of Alcohol Consumption and Alcohol-Induced DisordersALCOHOLISM, Issue 6 2010Willard M. Freeman The lack of reliable measures of alcohol intake is a major obstacle to the diagnosis, treatment, and research of alcohol abuse and alcoholism. Successful development of a biomarker that allows for accurate assessment of alcohol intake and drinking patterns would not only be a major advance in clinical care but also a valuable research tool. A number of advances have been made in testing the validity of proposed biomarkers as well as in identifying potential new biomarkers through systems biology approaches. This commentary will examine the definition of a biomarker of heavy drinking, the types of potential biomarkers, the steps in biomarker development, the current state of biomarker development, and critical obstacles for the field. The challenges in developing biomarkers for alcohol treatment and research are similar to those found in other fields. However, the alcohol research field must reach a competitive level of rigor and organization. We recommend that NIAAA consider taking a leadership role in organizing investigators in the field and providing a common set of clinical specimens for biomarker validation studies. [source] Simultaneous detection of two verotoxin genes using dual-label time-resolved fluorescence immunoassay with duplex PCRLUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, Issue 2 2002Kazuyuki Watanabe Abstract Verotoxin (VT) produced by several Escherichia coli serotypes causes haemorrhagic colitis and has been associated with haemolytic uraemic syndrome in humans. Two types of verotoxin are known. Conventional diagnosis of verotoxin-producing Escherichia coli (VTEC) is conducted after isolation of bacteria from clinical specimens, followed by serological determination and identification of VTs. This method is complicated and time-consuming. Recently, rapid, direct immunological methods for identification of VTEC, i.e. immunochromatography and latex agglutination, have been developed. However, these techniques continue to suffer from limited sensitivity and a lack of specificity. These difficulties arise from the fact that the antibody used in these procedures reacts exclusively with the O157 antigen; moreover, VTEC strains with non-O157 antigens, such as O26, O103 and O111 antigens, exist. These VTEC groups did not react with anti-O157 antibody. Consequently, it is necessary to diagnose the VT gene in these bacteria. Therefore, we have designed a sensitive and specific method for the detection of two VT genes simultaneously, utilizing duplex PCR with time-resolved fluorescence immunoassay (TRFIA). Copyright © 2002 John Wiley & Sons, Ltd. [source] Detection and quantitative analysis of human bocavirus associated with respiratory tract infection in Osaka City, JapanMICROBIOLOGY AND IMMUNOLOGY, Issue 5 2010Atsushi Kaida ABSTRACT HBoV was initially identified in patients with RTI in 2005. Since its discovery, there have been continual reports concerning HBoV detection and its prevalence. In this study of clinical specimens from young children, real-time PCR was undertaken to examine whether HBoV infection is associated with RTI and to support quantitative analysis of HBoV in these patients. In all, 376 specimens were collected from patients with RTI during April 2006,October 2008. Analyses revealed HBoV in 59 specimens (15.7%). Of HBoV-positive patients, children under the age of 3 years comprised 94.9%. Of the HBoV-positive samples, 47.5% were codetected with other respiratory viruses (dual infection, 27; triple infection, 1). During the study period, the numbers and rate of detection of HBoV were high mainly around May. Statistical analyses showed that the detection rate of HBoV during April,June was higher than during other months. Moreover, the viral load was greater in subjects with infection with HBoV alone than in subjects with mixed respiratory viral infections. Considering these results together, HBoV is probably associated with RTI in young children. However, the pathogenesis of this infection and the importance of the high rate of co-infection remain uncertain. Additional epidemiologic information and further analyses are necessary to clarify the virological characteristics and the linkage of HBoV to disease. [source] Androgen-independent expression of adrenomedullin and peptidylglycine ,-amidating monooxygenase in human prostatic carcinomaMOLECULAR CARCINOGENESIS, Issue 1 2003Nuria Jiménez Abstract Most of the locally advanced and metastatic prostate carcinomas (PCs) treated with antiandrogenic therapy eventually become refractory to this treatment. Locally produced factors may control prostate tumor biology after androgen withdrawal. Adrenomedullin (AM) is expressed in the prostate and could control cell growth in androgen-independent conditions. AM needs to be amidated by the enzyme peptidylglycine ,-amidating monooxygenase (PAM) to become fully active. The objective of the present study was to analyze whether the expression of preproadrenomedullin (preproAM) and PAM in PC is regulated by androgens. For this purpose, human in vitro and in vivo PC models were grown in the presence or absence of androgens, and the expression of AM and PAM was examined by immunohistochemistry, Western blotting, RT-PCR, and Northern blotting. Furthermore, immunohistochemical analysis of AM in clinical specimens was performed to test if its expression is related to Gleason score and antiandrogenic therapy. In PC cell lines and xenografts, mRNA and protein AM levels were similar in the presence or absence of androgens. PAM expression seemed to be induced by androgen-withdrawal. Our results in clinical samples showed no relationship between AM expression and Gleason score or antiandrogenic treatment. In conclusion, our results demonstrate that preproAM and PAM expression in the human prostate is androgen-independent. In addition, we also report for the first time the expression of a novel PAM transcript in PC, which has not been previously described in other tissues. © 2003 Wiley-Liss, Inc. [source] Isolation of Sporothrix schenckii from the environmental sources of cutaneous sporotrichosis patients in Himachal Pradesh, India: results of a pilot studyMYCOSES, Issue 6 2007Karan Inder Singh Mehta Summary Himachal Pradesh, India is a known endemic area for cutaneous sporotrichosis. No attempt has been made to isolate Sporothrix schenckii, the causative fungus, from environmental sources in this region or in India as such. This prospective study was carried out to isolate Sporothrix schenckii from different environmental samples collected from the vicinity of cutaneous sporotrichosis patients. All patients of cutaneous sporotrichosis diagnosed during March 2005,February 2006 were studied. Twenty-one biopsy specimens and 62 environmental samples of soil, various thorns, corn-stalk, grass-blades and sphagnum moss were subjected to mycologic culture on Sabouraud's glucose agar. Sporothrix schenckii was identified by colony characteristics, lacto-phenol cotton blue mounts and demonstration of temperature dimorphism. These patients (F : M 15 : 6) were between 12 and 72 years of age and had cutaneous lesions for 45 days to 4 years. Lymphocutaneous and fixed cutaneous sporotrichosis was seen in 14 (66.6%) and 7 (33.3%) patients respectively. Extremities were involved in 16 (76.2%); and 5 (23.8%) patients had facial lesions. Ten (47.4%) biopsy specimens and six environmental (three soil, three corn-stalk) samples were culture-positive, which showed morphological characteristics suggesting Sporothrix schenckii. No variation in colony characteristics and mycelial morphology was observed in growth isolates from clinical or environmental samples. Temperature dimorphism was observed in all the 10 isolates obtained from the clinical specimens and in two isolates cultured from corn-stalk. Corn-stalks are evidently important sources of Sporothrix schenckii infection although subsequent contamination of wounds appears more important for development of clinical disease. Culture of Sporothrix schenckii from environmental sources may not be always possible to correlate with profile of injuries. [source] Isolation of Exophiala dermatitidis from endotracheal aspirate of a cancer patientMYCOSES, Issue 6 2006S. J. Taj-Aldeen Summary Exophiala (Wangiella) dermatitidis is a melanised (darkly pigmented) yeast-like organism that has been reported from the environment and wild animals. The organism is a frequent coloniser of lungs of patients with cystic fibrosis and causes occasional disseminated phaeohyphomycosis and fungaemia. Exophiala dermatitidis is distributed worldwide, but cerebral cases are restricted to East Asia. We report a case of 54-year-old Qatari female patient with a known history of cancer, suffering from pulmonary disorder. Culture of endotracheal aspirate revealed the growth of E. dermatitidis concomitant with Candida krusei. The final diagnosis of E. dermatitidis and attribution to genotype B was achieved by sequencing the rDNA internal transcribed spacer (ITS) region. The present case concerns a pulmonary colonisation by E. dermatitidis, similar to that commonly seen in cystic fibrosis patients. For the detection of E. dermatitidis in clinical specimens culturing techniques are required. The patient finally expired with persistent cancer and C. krusei fungaemia. Review of literature and listing of E. dermatitidis cases published after 1992 show a sharp increase in clinical cases during the 1990s. [source] Comparison of standard phenotypic assays with a PCR method to discriminate Candida albicans and C. dubliniensisMYCOSES, Issue 1 2005B. Mähnß Summary In 1995, Candida dubliniensis was described as a new species in the genus Candida. Its close relationship to C. albicans has proved problematic in the identification of C. dubliniensis in clinical specimens. The objective of this study was to determine if reproducible differentiation between both species can be obtained by phenotypic assays. Therefore, 100 strains from 86 patients with the ability to produce chlamydospores were examined with different methods including API ID 32 C, colour development on CHROMagar, chlamydospore formation on Staib agar, growth at different temperatures and germ tube formation at 39 °C. Additionally, polymerase chain reaction (PCR) was used as gold standard. Six of the investigated strains were C. dubliniensis. The results suggest that there is still no single phenotypic method satisfactory to distinguish between C. albicans and C. dubliniensis. [source] Comparative evaluation of Candi Select test and conventional methods for identification of Candida albicans in routine clinical isolatesMYCOSES, Issue 3-4 2002S. Foongladda Candida albicans; Identifizierung; Candi Select- Test; Bewertung. Summary. The Candi Select test (Sanofi Diagnostics, Pasteur, Marnes-La-Coquette, France) is a new yeast-selective medium for the identification of Candida albicans in the clinical laboratory. The performance of this test was compared with the conventional methods of chlamydospore formation, germ tube formation and carbohydrate fermentation. Four hundred and twenty clinical yeast isolates from 412 fresh clinical specimens, including 283 C. albicans, 59 C. tropicalis, 39 Trichosporon spp., 19 C. glabrata, 11 Cryptococcus neoformans and 9 other yeasts, were evaluated. Colonies of C. albicans produced a blue-green colour on the Candi Select media which could be distinguished from the other yeasts with the naked eye within 24,48 h. The sensitivity and specificity of the Candi Select test for the identification of C. albicans were 99.65% and 97.08%, respectively. The blue-green colonies of C. albicans were easy to identify and recognize in mixed cultures and did not need detailed microscopic examination. Zusammenfassung., Der Candi Select-Test (Sanofi Diagnostics Pasteur, Marnes-La-Coquette, Frankreich) ist ein neuer selektiver Nährboden für die Identifizierung von Candida albicans im klinischen Labor. Die neue Methode wurde mit den konventionellen Methoden der Chlamydosporenbildung, der Keimschlauchbildung und der Kohlenhydrat-Gärung verglichen. Vierhundertzwanzig Hefeisolate, nämlich 283 C. albicans, 59 Candida tropicalis, 39 Trichosporon spp., 19 Candida glabrata, 11 Cryptococcus neoformans und 9 andere Hefen, isoliert aus 412 frischen klinischen Untersuchungsproben, wurden mit allen Methoden untersucht. Mit blossem Auge erkennbar, unterschieden sich innerhalb von 24,48 Stunden die blau-grünen Farbkolonien von C. albicans von allen anderen Hefen auf dem Candi Select Nährboden. Sensitivität und Spezifizität des Candi Select Tests für die Identifizierung von C. albicans betrugen 99.65% und 97.08%. Die blau-grünen Farbkolonien von C. albicans waren in den Mischkulturen leicht zu identifizieren, eine mikroskopische Untersuchung erübrigt sich daher. [source] Comparative proteomic analysis of mouse livers from embryo to adult reveals an association with progression of hepatocellular carcinomaPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2008Nikki P. Y. Lee Abstract To identify potential oncofetal biomarkers that distinguish hepatocellular carcinoma (HCC) from healthy liver tissues, we compared and analyzed the proteomic profiles of mouse livers at different developmental stages. Fetal (E13.5, E16.5), newborn (NB), postnatal (3-week) and adult (3-month) livers were isolated and profiled by 2-D PAGE. Statistical analysis using linear regression and false discovery rate (FDR) revealed that 361 protein spots showed significant changes. Unsupervised hierarchical tree analysis segregated the proteins into fetal, NB, and postnatal-adult clusters. Distinctive protein markers were identified by MALDI-TOF/MS and the corresponding mRNA profiles were further determined by Q-PCR. Fetal markers (hPCNA, hHSP7C, hHEM6) and postnatal-adult markers (hARGI1, hASSY, hBHMT, hFABPL) were selected for testing against a panel of seven human hepatocyte/HCC cell lines and 59 clinical specimens. The fetal proteins were found to be overexpressed in the metastatic HCC cell lines and the tumor tissues, whereas the postnatal-adult proteins were expressed in non-tumor tissues and normal hepatocytes. This "Ying-Yang" pattern, as orchestrated by distinct fetal and adult markers, is hypothesized to indicate the progressive change of the liver from a growing, less-differentiated organ into a functional metabolic center. Thus, embryogenesis and tumorigenesis share certain oncofetal markers and adult "hepatic" phenotypes are lost in HCC. [source] | ||||||||||||||||||||||||||||||||||