Types Of Clinical Samples Research Articles

The Reliability and Validity of the Three Modified Versions of the Children's Fear Survey Schedule-Dental Subscale of 9-12 Year Old Children in a Clinical Setting in Bosnia and Herzegovina.

This study sought to obtain a comprehensive, reliable and valid instrument for evaluation of the presence of dental fear and anxiety (DFA) in children, through evaluation of the reliability and validity of three modified versions of the Children's Fear Survey Schedule-Dental Subscale (CFSS-DS). The study sample comprised children aged 9, 10, 11 and 12 years. The first sample group (200 patients) filled in a modified version of the CFSSDS scale, the second sample group (100 patients) filled in a short form of the Dental Fear Survey Schedule, and the third sample group (100 patients) filled in a short version of the CFSS-DS scale, prior to dental treatment, respectively. In order to determine test-retest reliability, the 184 patients from the first sample group filled in the modified version of the CFSS-DS scale again, prior to their next scheduled dental appointment. The modified version of the CFSS-DS scale had the best internal consistency reliability (α=0.907), as well as validity results, compared to the other two instruments used. Test-retest reliability was moderate (Intraclass correlation coefficient: 0.58). Of the three psychometric instruments used for evaluation of DFA presence in children, the modified version of the CFSS-DS scale showed the most clinically adequate reliability and validity values. This study thus provides a new psychometric instrument that should be considered for clinical use in evaluation of DFA presence in 9-12 year-olds, in a clinical setting sample type of children.

Molecular Diagnosis of Visceral Leishmaniasis.

Visceral leishmaniasis (VL), a deadly parasitic disease, is a major public health concern globally. Countries affected by VL have signed the London Declaration on Neglected Tropical Diseases and committed to eliminate VL as a public health problem by 2020. To achieve and sustain VL elimination, it will become progressively important not to miss any remaining cases in the community who can maintain transmission. This requires accurate identification of symptomatic and asymptomatic carriers using highly sensitive diagnostic tools at theprimary health service setting. The rK39 rapid diagnostic test (RDT) is the most widely used tool and with its good sensitivity and specificity is the first choice for decentralized diagnosis of VL in endemic areas. However, this test cannot discriminate between current, subclinical, or past infections and is useless for diagnosis of relapses and as a prognostic (cure) test. Importantly, as the goal of elimination of VL as a public health problem is approaching, the number of people susceptible to infection will increase. Therefore, correct diagnosis using a highly sensitive diagnostic test is crucial for applying appropriate treatment and management of cases. Recent advances in molecular techniques have improved Leishmania detection and quantification, and therefore this technology has become increasingly relevant due to its possible application in a variety of clinical sample types. Most importantly, given current problems in identifying asymptomatic individuals because of poor correlation between the main methods of detection, molecular tests are valuable for VL elimination programs, especially to monitor changes in burden of infection in specific communities. This review provides a comprehensive overview of the available VL diagnostics and discusses the usefulness of molecular methods in the diagnosis, quantification, and species differentiation as well as their clinical applications.

A bench-top automated workstation for nucleic acid isolation from clinical sample types

Systems that automate extraction of nucleic acid from cells or viruses in complex clinical matrices have tremendous value even in the absence of an integrated downstream detector. We describe our bench-top automated workstation that integrates our previously-reported extraction method – TruTip – with our newly-developed mechanical lysis method. This is the first report of this method for homogenizing viscous and heterogeneous samples and lysing difficult-to-disrupt cells using "MagVor": a rotating magnet that rotates a miniature stir disk amidst glass beads confined inside of a disposable tube. Using this system, we demonstrate automated nucleic acid extraction from methicillin-resistant Staphylococcus aureus (MRSA) in nasopharyngeal aspirate (NPA), influenza A in nasopharyngeal swabs (NPS), human genomic DNA from whole blood, and Mycobacterium tuberculosis in NPA. The automated workstation yields nucleic acid with comparable extraction efficiency to manual protocols, which include commercially-available Qiagen spin column kits, across each of these sample types. This work expands the scope of applications beyond previous reports of TruTip to include difficult-to-disrupt cell types and automates the process, including a method for removal of organics, inside a compact bench-top workstation.

Advanced microRNA-based cancer diagnostics using amplified time-gated FRET.

MicroRNAs (miRNAs) play an important role in cellular functions and in the development and progression of cancer. Precise quantification of endogenous miRNAs from different clinical patient and control samples combined with a one-to-one comparison to standard technologies is a challenging but necessary endeavor that is largely neglected by many emerging fluorescence technologies. Here, we present a simple, precise, sensitive, and specific ratiometric assay for absolute quantification of miRNAs. Isothermally amplified time-gated Förster resonance energy transfer (TG-FRET) between Tb donors and dye acceptors resulted in miRNA assays with single-nucleotide variant specificity and detection limits down to 4.2 ± 0.5 attomoles. Quantification of miR-21 from human tissues and plasma samples revealed the relevance for breast and ovarian cancer diagnostics. Analysis of miR-132 and miR-146a from acute monocytic leukemia cells (THP-1) demonstrated the broad applicability to different miRNAs and other types of clinical samples. Direct comparison to the gold standard RT-qPCR showed advantages of amplified TG-FRET concerning precision and specificity when quantifying low concentrations of miRNAs as required for diagnostic applications. Our results demonstrate that a careful implementation of rolling circle amplification and TG-FRET into one straightforward nucleic acid detection method can significantly advance the possibilities of miRNA-based cancer diagnostics and research.

The development of laboratory diagnostic of leprosy using polymerase chain reaction

The purpose of study is to develop techniques of laboratory diagnostic of leprosy on the basis of polymerase chain reaction. The techniques were worked through such as extraction of DNA from various clinical material (bioptates and scarifcate of skin, scrapes from mucous surface of nose and trophic ulcers, blood serum). The systems of oligonucleotides were constructed to 16S pRNA and probe for identifcation of Mycobacterium leprae in real-time format for amplifer DT-96 ("The DNA-Technology", Russia). The optimal regimen of amplifcation was worked out. To control polymerase chain reaction processing a system of internal control was introduced. The evaluation of specifcity of test-system was implemented using 17 strains of various types of mycobacteria and clinical samples from 32 patients with leprosy and 15 healthy individuals being in family contact with patients with leprosy. The 100% sensitivity was established concerning detection of Mycobacterium leprae in bioptates of skin using polymerase chain reaction technique as compared with standard bacterioscopic technique (88.9%). In all other clinical samples, a higher sensitivity of developed test-system on the basis of polymerase chain reaction was detected too. The proposed test-system has higher sensitivity and specifcity that permits to resolve an issue of fast identifcation of Mycobacterium leprae. It can be applied in epidemiological studies at investigation of prevalence of agent of disease.

Colorimetric assay of rare disseminated tumor cells in real sample by aptamer-induced rolling circle amplification on cell surface

The identification of tumor cells disseminating in body fluids plays a critical role in the accurate diagnosis of cancer. Herein, a simple and pragmatic colorimetric strategy has been developed for ultrasensitive detection of tumor cells based on aptamer-induced rolling circle amplification (RCA) on cell surface. An Epithelial Cell Adhesion Molecule (EpCAM)-targeted aptamer was selected as recognition agent to specifically recognize and bind with tumor cells. Following, the aptamer-induced RCA performed in situ, presenting numerous G-quadruplexes sequences on the cell surface. G-quadruplex/hemin DNAzyme then formed and catalyzed colorimetric reaction for tumor cells detection. The proposed aptamer-induced RCA strategy displayed remarkable signal amplification performance and high specificity. The designed colorimetric assay could distinguish as low as 10 cancer cells in 10000 times of benign cells mixture, showing very high sensitivity and selectivity. Moreover, it has been successfully verified that the established strategy could directly identify tumor cells disseminated from various primary sites in multiple types of clinical body fluid samples. Thus this proposed strategy provided a simple, handy and powerful platform for ultrasensitive detection of tumor cells and had great potential for clinical application in convenient assay of rare disseminated tumor cells in body fluid samples for early cancer screening.

DIGE Saturation Labeling for Scarce Amounts of Protein from Formalin-Fixed Paraffin-Embedded (FFPE) Tissue.

In this chapter, we describe the utility of difference gel electrophoresis (DIGE) as a proteomics platform for the global detection of expressed proteins in formalin-fixed paraffin-embedded (FFPE) tissues and its use for biomarker discovery/identification of proteins that may contribute to cancer development and progression. Formalin fixation and paraffin embedding of tissue is the standard processing methodology practiced in pathology laboratories worldwide, resulting in a highly stable form of tissue that is easily stored due to its inherent stability at room temperature. Consequently, FFPE tissues represent an attractive reservoir of clinical material for conducting retrospective protein biomarker analysis. A limitation for proteomics research in this type of clinical sample is the amount of viable protein that can be obtained from fixed tissues. Tissue biopsies are precious samples that can generally be acquired in very small amounts due to the invasive nature of the sample collection, mainly during surgery or biopsy. Subsequently, the amount of extracted protein can be, in many cases, very limited. The saturation DIGE technology has emerged as a useful method for protein analysis where only scarce amounts of protein are available. This approach can be adapted successfully to label low-level protein isolated from FFPE tissue.

Direct identification of clinical pathogens from liquid culture media by MALDI-TOF MS analysis

ObjectivesWe propose using MALDI-TOF MS as a tool for identifying microorganisms directly from liquid cultures after enrichment of the clinical sample in the media, to obtain a rapid microbiological diagnosis and an adequate administration of the antibiotic therapy in a clinical setting. MethodsTo evaluate this approach, a series of quality control isolates were grown in thioglycollate (TG) broth and brain heart infusion (BHI) broth and extracted under four different protocols before finally being identified by MALDI-TOF MS. After establishing the best extraction protocol, we validated the method in a total of 300 liquid cultures (150 in TG broth and 150 in BHI broth) of different types of clinical samples obtained from two tertiary Spanish hospitals. ResultsThe initial evaluation showed that the extraction protocol including a 5 minute sonication step yielded 100% valid identifications, with an average score value of 2.305. In the clinical validation of the procedure, 98% of the microorganisms identified from the TG broth were correctly identified relative to 97% of those identified from the BHI broth. In 24% of the samples analysed, growth by direct sowing was only successful in the liquid medium, and no growth was observed in the direct solid agar cultures. ConclusionsUse of MALDI-TOF MS plus the sonication-based extraction method enabled direct and accurate identification of microorganisms in liquid culture media in 15 minutes, in contrast to the 24 hours of subculture required for conventional identification, allowing the administration of a targeted antimicrobial therapy.

Duplex Quantitative Polymerase Chain Reaction Assay for Detection of Adenoviral and Lentiviral Vectors

The ability to detect and quantify viral vector sequences from a variety of clinical sample types is crucial to performing biosafety risk assessments. Viral vector-mediated gene transfer studies ar...

Novel Modulators of Asthma and Allergy: Exosomes and MicroRNAs.

Intercellular communication is crucial to the immune system response. In the recent years, the discovery of exosomes has changed the way immune response orchestration was understood. Exosomes are able to operate as independent units that act as mediators in both physiological and pathological conditions. These structures contain proteins, lipidic mediators, and nucleic acids and notoriously include microRNAs (miRNAs). miRNAs are short RNA sequences (around 19–22 nucleotides) with a high phylogenetic conservation and can partially or totally regulate multiple mRNAs, inhibiting protein synthesis. In respiratory diseases such as asthma and allergic sensitization, exosomes released by several cell types and their specific content perform crucial functions in the development and continuation of the pathogenic mechanisms. Released exosomes and miRNAs inside them have been found in different types of clinical samples, such as bronchoalveolar lavage fluids and sputum supernatants, providing new data about the environmental factors and mediators that participate in the inflammatory responses that lead to the exacerbation of asthma. In this review, we summarize our current knowledge of the role of exosomes and miRNAs in asthma and allergic sensitization, paying attention to the functions that both exosomes and miRNAs are described to perform through the literature. We review the effect of exosomes and miRNAs in cells implicated in asthma pathology and the genes and pathways that they modify in them, depicting how their behavior is altered in disease status. We also describe their possible repercussion in asthma diagnosis through their possible role as biomarkers. Therefore, both exosomes and miRNAs can be viewed as potential tools to be added to the arsenal of therapeutics to treat this disease.

Abstract 742: Comprehensive detection of all major classes of MET deregulation by Anchored Multiplex PCR and next-generation sequencing

Abstract Introduction: Deregulation of the proto-oncogene, MET, confers an aggressive phenotype in a variety of human cancers, promoting proliferation, invasive growth and angiogenesis. MET deregulation can be driven by gene amplification, overexpression, exon 14 skipping, gene fusions and single nucleotide variants (SNVs), such as kinase-activating point mutations. MET is a target of intensive drug development efforts, although the various mutated forms of MET exhibit unique drug sensitivities. Therefore, detection of these mutations has an important role in the development of drugs targeting MET, and has the potential to guide treatments for cancers driven by MET deregulation. Next-generation sequencing (NGS) enables comprehensive detection of all mutation types from whole genomes and transcriptomes. However, low detection sensitivity, high input requirement and high costs render these approaches impractical for routine detection of mutations from low-input clinical sample types. We developed a targeted NGS assay based on Anchored Multiplex PCR (AMP™) to detect all types of mutations driving MET deregulation from a single sample. Methods: AMP only requires a single gene-specific primer for amplification, enabling open-ended capture of DNA and cDNA fragments for NGS-based detection of known and unknown mutations. We developed AMP-based Archer® VariantPlex™ and FusionPlex® library preparation assays to detect mutations from DNA and RNA, respectively. AMP probes were designed to cover the MET gene for detection of copy numbers variants (CNVs) and SNVs from DNA, and known and novel fusions, exon skipping and expression levels from RNA. Results: We show that the VariantPlex assay enables NGS-based detection of MET amplifications from DNA in concordance with FISH results. Further NGS analysis of RNA from the same sample using the FusionPlex assay revealed the resulting overexpression of MET. We also demonstrate that AMP-enabled open-ended capture of cDNA fragments allows for reliable detection of exon 14 skipping in FFPE samples and in cells, consistent with RT-PCR results. Parallel analysis of DNA from the cell samples revealed splice site mutations that have been previously reported to drive exon 14 skipping. Furthermore, this open-ended capture also permitted identification of a novel GTF2I:MET gene fusion in a patient-derived xenograft model. Finally, we detected an kinase-activating point mutation in MET, p.Y1253D, by analysis of genomic DNA with the VariantPlex NGS assay. Conclusions: These results show that AMP-based VariantPlex and FusionPlex Assays enable comprehensive detection of multiple mutation types from low-input clinical sample types, such as FFPE specimens. As MET deregulation can be driven by many different genetic aberrations, this allows for NGS-based characterization of MET deregulation from a single sample. Citation Format: Brian A. Kudlow, Josh Haimes, Marc Bessette, Namitha Manoj, Laura M. Griffin, Danielle Murphy, Robert Shoemaker, Jason Amsbaugh, Joshua Stahl. Comprehensive detection of all major classes of MET deregulation by Anchored Multiplex PCR and next-generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 742. doi:10.1158/1538-7445.AM2017-742

El trastorno límite de personalidad en la producción científica publicada en revistas editadas en España

The objective of this work is to analyze the scientific literature production on borderline personality disorder in Spain over the 2010-2015 period, making a comparative study of articles collected from scientific journals in the field of psychology from several perspectives: year of publication, type of journal (indexing), type of study, clinical sample, and main topic. After the analysis of 80 articles obtained in WOS, Scopus, Psicodoc, Dialnet, and ISOC databases, it was found that most analysis units appear in type C and D publications in adult population and in theoretical studies, being the prevailing subject different psychotherapeutic strategies, especially Dialectic-Behavioral and Transference-Focused Therapy, although with increasing trends of new procedures applied to BPD population. The scarcity of publications on topics such as psychopharmacological and etiological studies, design of new measuring instruments, or differential diagnosis deserves special attention.

Optimization of CSF3R Mutation Detection in Severe Congenital Neutropenia and Cyclic Neutropenia Patients for Routine Diagnostics Using Next Generation Sequencing

Severe congenital neutropenia (CN) is a group of bone marrow failure syndromes characterized by absolute neutrophil counts below 0.5x109/L, susceptibility to bacterial infections and frequently associated with maturation arrest at promyelocyte stage in the bone marrow (BM).There is a high incidence of malignant transformation among CN patients with a cumulative rate of MDS/AML 22 % after 15 years of G-CSF treatment. The acquisition of G-CSFR truncating mutations is a risk factor for leukemic transformation in CN patients. Therefore, annual monitoring of CSF3Rmutations by means of next generation sequencing (NGS) is required for identification of CN patients with high risk of MDS/AML development.Since CSF3R mutations usually occur at low frequency without additional clinical features, it is important to carefully select suitable clinical sample type and methods for mutation detection. Next, it remains to be evaluated which CN genetic subgroups should be considered for annual screening of CSF3Rmutations.We performed CSF3R mutational screening in DNA and/or cDNA in 101 patients (ELANE, n = 42; HAX1, n = 16; G6PC3, n = 7; JAGN1, n = 2; WASP, n = 1; digenic ELANE, HAX1, n =1; digenic HAX1 and G6PC3, n =1; inherited mutations in CSF3R, n = 2; genetically unclassified CN, n = 9; cyclic neutropenia (CyN), n = 20) from the European Branch of the Severe Congenital Neutropenia Registry (SCNIR).Using DNA deep sequencing we screened 63 of 81 CN-patients and 20 CyN patients. Using this method, we identified CSF3R mutations in 22.2% (14/63) of CN patients and 10% (2/20) of CyN patients. The frequency of CSF3R mutations in CN patients with known inherited mutations was 20% (11/55): 30 % (3/10) in CN-HAX1 patients, and 22.9 % (8/35) in CN-ELANE patients. Interestingly, 3/8 (37.5 %) patients harbouring CSF3R mutations were observed in genetically unclassified CN. We did not detect any acquired CSF3R mutations in the small groups of CN patients (n=10) harbouring inherited G6PC3, JAGN1, CSF3Ror digenic mutations.In order to increase the sensitivity of mutation detection we performed cDNA deep sequencing of the critical region of G-CSFR. We sequenced 38 CN patients (ELANE, n = 15; HAX1, n = 11; JAGN1, n = 2; G6PC3, n = 2; WASP, n = 1; germline CSF3R, n = 1; genetically unclassified, n = 6). We found 13% (2/15) CN-ELANE, 27% (3/11) CN-HAX1 and 33% (2/6) genetically unclassified CN patients to be positive for acquired mutations in the critical region of G-CSFR. One CN patient with WASP mutation also acquired CSF3R mutation. Based on our sequencing data we would suggest CSF3Rmutation sequencing in all studied groups of patients regardless of mutations in ELANE and HAX1 genes.Intriguingly, 3 out of 5 CN patients with CSF3R mutations detected by cDNA deep sequencing were negative based on results of previous DNA deep sequencing. All of them were found to acquire low frequency CSF3R mutant clones (ELANE pos. patient with 0.3% of p.Q739* clone; genetically unclassified CN patient with 2% of p.Q749*clone; HAX1pos. patient with 0.9% of p.Q749* clone) in cDNA deep sequencing.In 2 patients (one CyN-ELANE and one CN-HAX1) with multiple acquired CSF3R mutations we compared mutant clone enrichment in different cell types (BM MNC; BM PMN; PB MNC and PB PMN) by means of cDNA and DNA deep sequencing. In the CyN-ELANE patient with 2 CSF3R mutant clones, the highest mutant allele frequency (MAF) was detected in the cDNA sample of PB PMN (11% of p.Q749* clone and 0.44% of p.Q739* clone), whereas in the PB MNC cDNA sample clone p.Q749* had only 2.5% MAF and clone P.Q739 was not detectable. Similar to that, in the CN-HAX1 patient the highest MAFs for all 3 CSF3R mutant clones were in PB PMN cDNA and the lowest in PB MNC DNA sample. Frequency of mutated CSF3Rclones in BM PMNs of both patients was comparable to PB PMN samples.Taken together, sequencing of cDNA extracted from peripheral blood or bone marrow PMN samples may provide better results than from MNC in terms of frequency of CSF3R mutation detection in CN and CyN patients. Sequencing of cDNA extracted from BM or PB samples allows enrichment of G-CSFR expressing mutant cells, but due to intrinsic low fidelity of reverse transcriptase the threshold level for positive calls could not be improved significantly (current threshold for candidate calls is 0.2-0.5%).We would suggest CSF3R mutation screening using deep-sequencing of cDNA from peripheral blood PMN in all patient groups (CN and CyN) for routine diagnostics. DisclosuresNo relevant conflicts of interest to declare.

Prevalence and molecular characterization of human rhinovirus in stool samples of individuals with and without acute gastroenteritis.

Human rhinovirus (RV) most often causes mild upper respiratory tract infection. Although RV is routinely isolated from the respiratory tract, few studies have examined RV in other types of clinical samples. The prevalence of RV was examined in 1,294 stool samples collected mostly from children with acute gastroenteritis residing in Bangkok and Khon Kaen province of Thailand between January 2010 and October 2014. In addition, 591 samples from hand-foot-mouth disease (HFMD) or herpangina patients who do not have gastroenteritis served as a comparison group. Samples were initially screened by semi-nested PCR for the RV 5'UTR through the VP2 capsid region. RV genotyping and phylogenetic analysis were performed on the VP4/VP2 regions. Among children with acute gastroenteritis, RV was found in 2.3% (30/1,294) of stool samples, which comprised 47% (14/30) RV-A, 17% (5/30) RV-B, and 37% (11/30) RV-C. In the comparison group, 0.8% (5/591) was RV-positive and RV-C (3/5) was the major species found. Interestingly, RV was recovered more often from children with acute gastroenteritis than from those with HFMD or herpangina. As many as 31 RV types were present in the gastroenteritis stools, which were different than the types found in those with HFMD or herpangina. J. Med. Virol. 89:801-808, 2017. © 2016 Wiley Periodicals, Inc.

10 - Detection of Copy Number Variants by Next Generation Sequencing is Driven by Genomic DNA Integrity

Copy number variants (CNVs) are common oncogenic drivers, impacting more of the cancer genome than all other types of mutations combined. Next generation sequencing (NGS) of cancer genomes is a highly sensitive method to detect CNVs from clinical sample types. However, as FFPE storage of clinical specimens severely damages DNA and results in poor sequencing coverage, detection of low-level CNVs (<3-fold) and CNVs in samples with low tumor cellularity remains challenging. Anchored Multiplex PCR (AMP™) is a target enrichment strategy that increases the read depth of target sequences by NGS. Molecular barcoded adapters ligated to DNA fragments prior to PCR enables amplification of fragments as small as 50 bp. AMP therefore increases read depth and coverage of target regions, thus enhancing the detection sensitivity of CNVs by downstream NGS assays. Since DNA damage from FFPE storage limits the number of molecules available for library preparation, the amount of amplifiable DNA fragments, rather than the total mass of DNA, drives library complexity, sequencing coverage, and detection sensitivity. To demonstrate the effect of input genomic DNA integrity on CNV detection sensitivity, we used the Archer PreSeq™ DNA QC Assay to determine the amounts of amplifiable genomes from over 150 FFPE tumor samples. AMP-based NGS using Archer VariantPlex assays in conjunction with Archer Analysis detected CNVs down to 2-fold and demonstrated that input genomic DNA integrity predicts the detection limit of CNVs. The accuracy of low-level CNV detection was also confirmed by serial dilution of CNV-positive samples.

VIP: an integrated pipeline for metagenomics of virus identification and discovery.

Identification and discovery of viruses using next-generation sequencing technology is a fast-developing area with potential wide application in clinical diagnostics, public health monitoring and novel virus discovery. However, tremendous sequence data from NGS study has posed great challenge both in accuracy and velocity for application of NGS study. Here we describe VIP (“Virus Identification Pipeline”), a one-touch computational pipeline for virus identification and discovery from metagenomic NGS data. VIP performs the following steps to achieve its goal: (i) map and filter out background-related reads, (ii) extensive classification of reads on the basis of nucleotide and remote amino acid homology, (iii) multiple k-mer based de novo assembly and phylogenetic analysis to provide evolutionary insight. We validated the feasibility and veracity of this pipeline with sequencing results of various types of clinical samples and public datasets. VIP has also contributed to timely virus diagnosis (~10 min) in acutely ill patients, demonstrating its potential in the performance of unbiased NGS-based clinical studies with demand of short turnaround time. VIP is released under GPLv3 and is available for free download at: https://github.com/keylabivdc/VIP.

Relationship between super antigenicity, antimicrobial resistance and origin of Staphylococcus aureus isolated

Staphylococcus aureus is a pathogen that causes food poisoning as well as hospital and community acquired infections. Establish the profile of superantigen genes among hospital isolates in relation to clinical specimen type, susceptibility to antibiotics and hospital or community acquisition. Eighty one isolates obtained from patients at Colombian hospital, were classified by antimicrobial susceptibility, specimen type and hospital or community acquired . The PCR uniplex and multiplex was used for detection of 22 superantigen genes (18 enterotoxins, tsst-1 and three exfoliative toxins). Ninety five point one percent of isolates harbored one or more of the genes with an average of 5.6 genes. Prevalence of individual genes was variable and the most prevalent was seg (51.9%). Thirty nine genotypes were obtained, and the genotype gimnou (complete egc cluster) was the most prevalent alone (16.0%) and in association with other genes (13.6%). The correlation between presence of superantigens and clinical specimen or antimicrobial susceptibility showed no significant difference. But there was significant difference between presence of superantigens and the origin of the isolates, hospital or community acquired (p= 0.049). The results show the variability of the superantigen genes profile in hospital isolates and shows no conclusive relationship with the clinical sample type and antimicrobial susceptibility, but there was correlation with community and hospital isolates. The analysis of the interplay between virulence, epidemic and antibiotic resistance of bacterial populations is needed to predict the future of infectious diseases.

Electrochemical telomerase assay for screening for oral cancer

Telomerase has long been known to be a marker for cancer. We have developed a new method of detecting it: the electrochemical telomerase assay (ECTA). We have previously confirmed that the assay is easier to do and more precise than the conventional telomeric repeat amplification protocol, which is currently the most widely used. Here we describe a pilot study made to establish a screening system for oral cancer using ECTA. We evaluated three types of clinical samples obtained from 44 patients with oral cancer and 26 healthy volunteers: exfoliated cells from the whole oral cavity, exfoliated cells from local lesions, and tissue from the lesion itself. The current increase ratio (Δi) obtained by ECTA was significantly higher in the oral cancer group for each type of sampling used. The threshold value for Δi was 19% when calculated by analysis of receiver-operating characteristic curves. Sensitivity and specificity values were 86% and 85% for cells from the oral cavity, 82% and 85% in cells from local lesions, and 95% and 92% in cells from the tumour itself, respectively. There were also no significant differences in sensitivity and specificity associated with age, size of tumour, site of lesion, or degree of malignancy. ECTA therefore seems to be a promising assay for screening for oral cancer.

Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine.

Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.

In Vitro Antimicrobial Susceptibility of Nontuberculous Mycobacteria in Iran.

Many species of nontuberculous mycobacteria (NTM) have long been identified as important causes of human disease, the incidence of which is rising. Several reports have suggested increasing trend of both in vitro and in vivo resistance to available treatment regimes. The aim of this study was to evaluate antibiotic susceptibility of clinically relevant NTM isolates using standard microbroth dilution test. Antimicrobial susceptibility testing was performed following National Committee for Clinical Laboratory Standards methods for NTM isolates, including 85 Mycobacterium fortuitum, 39 Mycobacterium chelonae, and 30 Mycobacterium abscessus subsp. abscessus as rapidly growing mycobacteria and 48 Mycobacterium simiae and 40 Mycobacterium kansasii as slowly growing mycobacteria. All isolates were recovered from various types of clinical samples and identified by multilocus sequence analysis. Trimethoprim-sulfamethoxazole (TMP-SMZ), amikacin, tobramycin, clarithromycin, moxifloxacin, linezolid, and imipenem showed better activity against M. fortuitum rather than meropenem, ciprofloxacin, cefoxitin, and doxycycline. Amikacin was active against 93% of M. abscessus subsp. abscessus. Linezolid, clarithromycin, cefoxitin, ciprofloxacin, imipenem, moxifloxacin, tobramycin, TMP-SMZ, doxycycline, and meropenem showed some activities on M. abscessus subsp. abscessus as well. The majority of M. abscessus subsp. abscessus and M. chelonae strains were multidrug resistant. Among the 40 isolates of M. kansasii, all were susceptible to ethambutol, isoniazid, clarithromycin, moxifloxacin, and linezolid. These isolates were also resistant to doxycycline and 50% were resistant to rifampicin and ciprofloxacin. M. simiae was resistant to clarithromycin, doxycycline, isoniazid, and TMP-SMZ, and the majority of isolates showed high levels of resistance to linezolid, ethambutol, ciprofloxacin, streptomycin, and rifampicin. The majority of M. simiae isolates were multidrug resistant. Our data confirm the need for performing of standard susceptibility testing of any clinically important NTM isolate.