Different Types Of Clinical Samples Research Articles

Abstract 5164: Deployment of ASPYRE-Lung targeted variant panel across three sites and testing with FFPE tissue and cytology-derived nucleic acid samples

Abstract While there is no shortage of approved targeted therapeutics to address driver mutations in NSCLC, many patients are not tested for these alterations due to gaps in biomarker testing access. Challenges in NSCLC molecular testing include the need for assessment of multiple variants in both DNA and RNA, samples are often small specimens, cost, and clinical need for rapid turn-around time when patients are deteriorating and require timely treatment initiation. ASPYRE-Lung detects a targeted panel of 114 actionable genomic variants across 11 genes, covering NCCN treatment guideline-recommended biomarkers for samples that are associated with NSCLC. The assay simultaneously analyzes DNA and RNA with inputs of only 20 ng and 6 ng respectively. ASPYRE-Lung is easily adoptable by standard clinical laboratories, requiring only PCR and qPCR thermocyclers, with no specialist bioinformatics skills required for analysis and a run time of 2 days from specimen to result. We established the assay at two external sites and Biofidelity laboratories, with multiple operators at each site. We first determined that the assay had acceptable performance at each site, using a panel of contrived control samples. We then examined 56 matched DNA and RNA samples extracted from clinical FFPE lung tissue specimens. We also tested 29 unmatched samples derived from FFPE tissue (13), fine needle aspirate (FNA, 4), FNA rinse (5), peritoneal fluid (1), pleural effusion (1), pleural fluid (1) and fresh tissue (4), at all 3 sites. Data were analyzed using a simple cloud-based turnkey analysis solution. All samples yielded valid results across all runs. We assessed reproducibility (inter-run precision) using all 85 clinical samples, across new and experienced users, achieving a positive percent agreement (PPA) of 100% and negative percent agreement (NPA) of 99.99%. We then assessed concordance with NGS results, investigating any discrepancies. The 69 FFPE tissue-derived samples (56 matched DNA/RNA, 7 unmatched DNA, and 6 TNA) had PPA 96.55% and NPA 99.98%. The 16 cytopathology-derived samples had PPA 100% and NPA 99.96%. Together, these results demonstrate high concordance of ASPYRE-Lung and NGS across different types of clinical samples, alongside ease of adoption of the ASPYRE-Lung assay with simple training and no specialist expertise required. ASPYRE-Lung promises to enable all patients with NSCLC to start appropriate treatment in a cost-effective and timely manner. Citation Format: Sarah E. Herlihy, Caren Gentile, Samantha J. Scott, Brandon A. Smith, Kathryn A. Stoll, Kala F. Schilter, Justyna Mordaka, Rebecca Palmer, Christina Xyrafaki, Elizabeth Gillon-Zhang, Candace King, Ryan Evans, Amanda Green, Ana-Luisa Silva, Magdalena Stolarek-Januszkiewicz, Kristine von Bargen, Iyelola Turner, Chau Ha Ho, Alejandra Collazos, Nicola Potts, Dilyara Nugent, Jinsy Jose, Eleanor Gray, Elyse Shapiro, Wendy J. Levin, Aishling Cooke, Barnaby Balmforth, Robert Osborne, Honey V. Reddi, Vivianna M. Van Deerlin. Deployment of ASPYRE-Lung targeted variant panel across three sites and testing with FFPE tissue and cytology-derived nucleic acid samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5164.

Next-Generation Sequencing for the Detection of Microbial Agents in Avian Clinical Samples.

Direct-targeted next-generation sequencing (tNGS), with its undoubtedly superior diagnostic capacity over real-time PCR (RT-PCR), and direct-non-targeted NGS (ntNGS), with its higher capacity to identify and characterize multiple agents, are both likely to become diagnostic methods of choice in the future. tNGS is a rapid and sensitive method for precise characterization of suspected agents. ntNGS, also known as agnostic diagnosis, does not require a hypothesis and has been used to identify unsuspected infections in clinical samples. Implemented in the form of multiplexed total DNA metagenomics or as total RNA sequencing, the approach produces comprehensive and actionable reports that allow semi-quantitative identification of most of the agents present in respiratory, cloacal, and tissue samples. The diagnostic benefits of the use of direct tNGS and ntNGS are high specificity, compatibility with different types of clinical samples (fresh, frozen, FTA cards, and paraffin-embedded), production of nearly complete infection profiles (viruses, bacteria, fungus, and parasites), production of "semi-quantitative" information, direct agent genotyping, and infectious agent mutational information. The achievements of NGS in terms of diagnosing poultry problems are described here, along with future applications. Multiplexing, development of standard operating procedures, robotics, sequencing kits, automated bioinformatics, cloud computing, and artificial intelligence (AI) are disciplines converging toward the use of this technology for active surveillance in poultry farms. Other advances in human and veterinary NGS sequencing are likely to be adaptable to avian species in the future.

Bocaparvovirus in infants hospitalized in a neonatal intensive care unit in Midwest Brazil during the COVID-19 pandemic (2021-2022).

Human bocaparvoviruses (HBoVs) belong to the Parvoviridae family, being currently classified into four species (HBoV1-4). These viruses have been found in association with respiratory and gastroenteric symptoms, as well as in asymptomatic individuals. This study aimed to investigate the occurrence of HBoVs in infants under 5 months old admitted to a Neonatal Intensive Care Unit (NICU) during the COVID-19 pandemic (between March 2021 and March 2022). Clinical samples (nasopharyngeal swab, serum, stool, and urine) were screened by qPCR TaqMan. The HBoV was detected in samples of 31.6% (12/38) of participants. The most frequent alteration among the HBoV-positive neonates was the chest X-ray with interstitial infiltrate, followed by tachycardia and vomiting. Viral DNA was detected in more than one type of clinical sample in three of the participants in association with respiratory symptoms. Two participants had positive stool samples with or without enteric symptoms. HBoV intermittent and continuous positivity patterns were observed. The present study stands out for the prospective evaluation of positivity for HBoV in different types of clinical samples from a population of hospitalized infants. Our data supports circulation of HBoV in nosocomial environment during the COVID-19 pandemic.

P395 Clinical utility of semi-nested conventional PCR for diagnosis of mucormycosis in fresh clinical samples

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesWe aim to assess the clinical utility of the semi-nested conventional PCR in smear positive, culture-negative clinical samples for diagnosis of mucormycosis.MethodsThis prospective study was conducted for a period of 3 months (April-June 2021). A total of 218 clinical samples were included from patients in whom smear was positive for aseptate hyphae, but the culture failed to grow within 2 days of incubation or smear was negative but had high suspicion of mucormycosis. Molecular diagnosis was attempted using semi-nested PCR with Mucorales-specific primers targeting 18S region, described previously by Bialek et al.1. Phenol-chloroform based manual DNA extraction protocol was optimized in the mycology laboratory of the department of medical microbiology, PGIMER, Chandigarh. This method was applicable to different types of clinical samples to yield good quality DNA with minimal chances of extraneous contamination (Fig. 1). Amplified PCR products were further sequenced to identify the causative species (Fig. 2).ResultsAmong 218 patients with suspected mucormycosis included in this study, the majority were rhino-orbito-cerebral mucormycosis (ROCM), (77.7%, n = 169), followed by pulmonary mucormycosis (19.2%, n = 42), cutaneous (0.02%, n = 4), and gastro-intestinal (GI) mucormycosis (0.01%, n = 3). In 24 samples, the presence of both septate and aseptate hyphae was seen under microscopic examination raising the possibility of mixed infection. On microscopic examination, 90.3% samples (197/218) had aseptate hyphae while the remaining samples were smear-negative but had strong clinical suspicion of mucormycosis. The molecular technique was able to identify causative agent in 154 culture-negative samples (81.9%, 154/188) and 52.4% (11/21) in smear-negative cases. Among 218 patients, only 20 samples show delayed growth of Mucorales, and on comparison with molecular results 100% concordance was observed. However, 10 patients with strong clinical suspicion for mucormycosis were negative by both conventional and molecular methods. The low culture positivity necessities the molecular diagnosis based on in-house semi-nested PCR using above-mentioned primers followed by Sanger sequencing. In the case of 24 mixed infections with aseptate-septate hyphae, Mucorales-specific PCR correctly identified 23/24 (95.8%) Mucorales as a causative agent. The overall turn-around-time from the sample receiving to diagnosis was ˂48 h. Overall, R. arrhizus (85/143, 59.4%) was most commonly associated with ROCM, while R. microsporus (13/38, 34.2%), and R. homothallicus (5/38, 13.1%) were seen mainly with pulmonary mucormycosis. Apophysomyces and Saksenaea genus were associated with GI and cutaneous mucormycosis.ConclusionsThe molecular technique utilizing semi-nested PCR, followed by Sanger sequencing was able to identify Mucorales species 81.9% of culture-negative cases. Optimized manual DNA extraction protocol is suitable for different sample types, with minimum chances of extraneous contamination and offers low cost with a shorter turnaround time (TAT).

Urinary Exosomal Long Noncoding RNA TERC as a Noninvasive Diagnostic and Prognostic Biomarker for Bladder Urothelial Carcinoma.

Purpose Bladder cancer is one of the most common urological malignancies worldwide, and approximately 90% of bladder cancer cases are histologically typed as bladder urothelial carcinoma (BLCA). Exosomes are 30 to 200 nm extracellular vesicles that transport microRNAs, long noncoding RNAs (lncRNAs), mRNAs, circular RNAs, and proteins across tissues and through circulation. Urinary exosomes may contain genetic information from tumor cells. Herein, we explored the clinical significance of urinary exosomal lncRNA telomerase RNA component (TERC) levels to provide an urgently needed diagnostic and prognostic biomarker for BLCA. Materials and Methods In this study, we used RNA-sequencing of samples from four BLCA patients and three healthy controls to identify that TERC was differentially expressed in urinary exosomes. We then used quantitative PCR in different types of clinical samples to validate the biomarker and analyzed results using receiver operating characteristic curves. Results We found that TERC was significantly upregulated in urinary exosomes from BLCA patients compared with those from healthy controls (P < 0.0001). Urinary exosomal TERC showed higher sensitivity (78.65%) and accuracy (77.78%) than existing indicators including nuclear matrix protein-22 and urine cytometry. Using the cut-off value 4.302, the area under the curve for urinary exosomal TERC was 0.836 (95% confidence interval: 0.768–0.891, P < 0.0001). Furthermore, this noninvasive assay could distinguish low-grade and high-grade tumors (P = 0.0153). Conclusions TERC is enriched in urinary exosomes from BLCA patients. Urinary exosomal TERC could become a diagnostic and prognostic biomarker for BLCA that allows clinicians to realize noninvasive detection of BLCA.

Profile of Bacteria with ARGs Among Real-World Samples from ICU Admission Patients with Pulmonary Infection Revealed by Metagenomic NGS.

BackgroundTreatment of pulmonary infections in the intensive care unit (ICU) represents a great challenge, especially infections caused by antibiotic resistance pathogens. A thorough and up-to-date knowledge of the local spectrum of antibiotic resistant bacteria can improve the antibiotic treatment efficiency. In this study, we aimed to reveal the profile of bacteria with antibiotic resistance genes (ARGs) in real-world samples from ICU admission patients with pulmonary infection in Mainland, China, by metagenomic next-generation sequencing (mNGS).MethodsA total of 504 different types of clinical samples from 452 ICU admission patients with pulmonary infection were detected by mNGS analysis.ResultsA total of 485 samples from 434 patients got successful mNGS results. Among 434 patients, one or more bacteria with ARGs were detected in 192 patients (44.24%, 192/434), and ≥2 bacteria with ARGs were detected in 85 (19.59%, 85/434) patients. The predominant detected bacteria were Corynebacterium striatum (C. striatum) (11.76%, 51/434), Acinetobacter baumannii (A. baumannii) (11.52%, 50/434) and Enterococcus faecium (E. faecium) (8.99%, 39/434). ermX conferred resistance to MSLB and cmx to phenicol were the only two ARGs detected in C. striatum; in A. baumannii, most of ARGs were resistance-nodulation-division (RND)-type efflux pumps genes, which conferred resistance to multi-drug; ermB conferred resistance to MSLB and efmA to multi-drug were the predominant ARGs in E. faecium. Bacteria with ARGs were detected in 50% (140/280) bronchoalveolar lavage fluid (BALF) and 50.5% (48/95) sputum samples, which were significantly higher than in blood and cerebrospinal fluid (CSF) samples.ConclusionHigh level of bacteria with ARGs was observed in clinical samples, especially BALF and sputum samples from ICU admission patients with pulmonary infection in Mainland, China. And C. striatum resistant to MSLB and/or phenicol, multi-drug resistance A. baumannii and E. faecium were the lead bacteria.

Label-Free Mass Spectrometry-Based Quantification of Linker Histone H1 Variants in Clinical Samples

Epigenetic aberrations have been recognized as important contributors to cancer onset and development, and increasing evidence suggests that linker histone H1 variants may serve as biomarkers useful for patient stratification, as well as play an important role as drivers in cancer. Although traditionally histone H1 levels have been studied using antibody-based methods and RNA expression, these approaches suffer from limitations. Mass spectrometry (MS)-based proteomics represents the ideal tool to accurately quantify relative changes in protein abundance within complex samples. In this study, we used a label-free quantification approach to simultaneously analyze all somatic histone H1 variants in clinical samples and verified its applicability to laser micro-dissected tissue areas containing as low as 1000 cells. We then applied it to breast cancer patient samples, identifying differences in linker histone variants patters in primary triple-negative breast tumors with and without relapse after chemotherapy. This study highlights how label-free quantitation by MS is a valuable option to accurately quantitate histone H1 levels in different types of clinical samples, including very low-abundance patient tissues.

Foldscope as a primary diagnostic tool for oral and urinary tract infections and its effectiveness in oral health education.

Due to its speed, accuracy and cost-effectiveness, microscopy has become an integral part of clinical examination for disease diagnosis. However, modern microscopes are very costly and require skilled personnel for their operation and maintenance, and specimen processing and analysis is labour-intensive. Further, lack of such expensive diagnostic tools in remote areas is a serious concern. Affordable point-of-care diagnostic tools are the most useful for timely disease diagnosis and management. The Foldscope is an affordable origami-based microscopy device composed of a series of paper clippings, which, upon assembly, can hold a specimen slide for observation, and this specimen can be viewed via a mobile phone camera attached to it. The present study evaluated the use of the Foldscope in the clinical diagnosis of oral and urinary tract infections and evaluated its efficacy as a motivational tool for improving oral health among school children in India. We qualitatively compared the Foldscope to a clinical microscope by examining five different types of clinical samples. Of the different types of clinical samples, the Foldscope was effective in detecting infection in dental plaque samples and urine samples. Thus, we further analysed 31 dental plaque samples of patients aged 3-13 years and 25 urine samples of patients aged 11-62 years. We also evaluated the use of the Foldscope as an educational tool for motivating oral hygiene among 80 school children aged 12 years and found that students in the Foldscope intervention group had better measures of oral hygiene than did students in the nonintervention group. In summary, our study indicated that the Foldscope is useful in detecting urinary tract infections and kidney stones in urine samples and is a useful motivational tool for oral health education among school-aged children. Furthermore, it may also be useful in oral health monitoring in resource poor settings. LAY DESCRIPTION: Poor and remote population often suffers due to lack of capacity to buy products or avail services which are expensive. In such a society people are not able to afford even the basic detection of diseases. Foldscope may come to rescue here! Foldscope is a paper-based, use-and-throw, affordable microscope. This study explores the use of Foldscope in clinics and diseases diagnosis using patient samples. Preliminary detection of disease associated symptoms in dental and urinary infections and digital record keeping via storage in mobile phone memory and hospital OPD records for monitoring patient's health are some of the advantages of Foldscope for clinical use in resource poor settings. It identifies that Foldscope not only can be used by students or teachers for educational purposes but it can also pave a path for promotion of dental hygiene among young children. The study also suggests that further improvement in design or resolution of Foldscope will broaden the scope of its application, in future.

Multiplex PCR system for fungal pathogen detection

Introduction: The rapid and correct identification of pathogenic species is crucial for several reasons that differ according to the area of interest. In clinical laboratories it is important since knowing the pathogen guides appropriate treatment, dose and duration of therapy. In clinical microbiology laboratories culture and microscopic examination remain the “gold standard”. Biochemical and mass spectometry identification systems, such as the Vitek systems (BioMerieux®) or MALDI-TOF are examples of commercial systems for microbial identification with the advantage of being semi automatic. However, these methods are based on the regrowth and isolation of the microorganism from samples, which in some cases, such as from blood, is time consuming and the success rate of cultivation can be as low as 20%. Recently, several techniques based on the polymerase chain reaction (PCR) have been developed for microbial pathogenic detection and identification particularly Real-Time PCR methods. In this study we developed an alternative PCR based method for the detection of fungal pathogens involved in systemic infections. This is a multiplex PCR method that attributes to each species specific amplicon lengths and a fluoresce dye, according to previously design panels, enabling a fast and reproducible co-amplifying of several loci in a single PCR reaction. Materials and methods: First a panel for species identification, combining fluorescence with molecular weight of specific PCR fragments for Candida and Asprgillus was designed. The PCR fragments obtained are then analysed by capillary electrophoresis and GeneScan fragment analysis. This methodology was optimized using DNA extracted from strains previously identified and, in order to optimize the methodology to clinical samples, we also used serum from healthy donors spiked with different concentrations of fungal DNA. Then the method was tested in DNA extracted from different types of clinical samples, including blood, biopsies and bronchoalveolar lavages of patients with invasive fungal infections (IFI). Results: The optimization of the method, by using DNA from known strains belonging to the target species and strains form other species, showed 100% of specificity. The calculated yield from DNA extracted from serum spiked with fungal DNA was of around 80%. This DNA was then used to determine the sensitivity of the technique and results showed that we were able to obtain amplification products within a range of 1 to 10 pg of the total DNA extracted. Results obtained with DNA extracted from samples of IFI patients, showed that we were able to detect the specific fungal species in 75% of the samples. Several optimizations are being performed. Discussion and conclusions: This new methodology is a promising method since interpretation of results is easy, based on presence/absence of a particular peak of the panel, it is fast, accurate and reproducible and due to the design of the identification panel it is able to identify the pathogenic fungal species involved in mixed infections. Several optimizations are being performed to enhance sensitivity of the method.

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.

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.

Rapid diagnostic tests for identifying avian influenza A(H7N9) virus in clinical samples.

To determine sensitivity of rapid diagnostic tests for detecting influenza A(H7N9) virus, we compared rapid tests with PCR results and tested different types of clinical samples. Usefulness of seasonal influenza rapid tests for A(H7N9) virus infections is limited because of their low sensitivity for detecting virus in upper respiratory tract specimens.

Interpretation of Fungal Culture Results

Classically, diagnosis of fungal infections is based on microscopic examination coupled with attempts to culture the responsible fungus from a clinical sample. For some fungal infections (such as dermatophyte infections, infections with dimorphic fungi, and blood stream infections with Fusarium, Acremonium and allied genera) recovery of the fungus in culture from a patient with clinical symptoms is sufficient for diagnosis. However, in many cases, obtaining a yeast or filamentous fungus in culture is not easily interpreted in isolation. In such circumstances, decisions regarding the clinical significance of an isolate must consider the nature of the organism and the quantity isolated, the likelihood of it accidentally contaminating the specimen, whether fungal elements were seen upon microscopic examination of the sample, the clinical status of the patient, and whether there are other clinical or biological markers suggesting infection. This review discusses these considerations for the different types of clinical samples encountered in a microbiology laboratory.

Microbial Susceptibility and Plasmid Profiles of Methicillin-Resistant Staphylococcus aureus and Methicillin-Susceptible S. aureus.

Background:Today, significant increase in the prevalence and emergence of methicillin-resistant Staphylococcus aureus (MRSA) is a serious public health concern and is likely to have a dramatic negative impact on many current medical practices. Therefore, identification of MRSA strains is important for both clinical and epidemiological implications.Objectives:The present study was carried out to determine the frequency of methicillin resistant; antibiotic susceptibility and plasmid profiles of S. aureus recovered from different types of clinical samples of patients in Zabol, Iran.Material and Methods:Clinical samples from 500 outpatient and hospitalized patients were tested for S. aureus. The susceptibility of 106 S. aureus to 11 antibiotics was evaluated by the disk diffusion method and Etest oxacillin strips. The presence of mecA gene was investigated by polymerase chain reaction (PCR). The plasmid profile patterns of all isolates were determined by a modified alkaline lysis method.Results:A total of 67 (63.20%) strains were found to be MRSA isolates. Most of MRSA isolates showed high level of resistance to ampicillin, erythromycin, nalidixic acid, penicillin, and tetracycline. Twenty-six percent of MRSA isolates showed high level of resistance to oxacillin (minimum inhibitory concentration [MIC] ≥ 256 μg/mL). mecA gene was detected among 62 MRSA isolates. Totally, 75 isolates of both strains harbored plasmid.Conclusions:Resistance to oxacillin and other antibiotics was high, and most of the isolates were found to be multi-drug resistance (MDR). Plasmid analysis of representative S. aureus isolates also demonstrates the presence of a wide range of plasmid sizes, with no consistent relationship between plasmid profiles and resistance phenotypes. Regular surveillance of hospital infections and monitoring of their antibiotic sensitivity patterns are required to reduce MRSA prevalence. High prevalence and multi-drug resistance of MRSA isolates in southeast of Iran could be considered as an urgent warning for public health.

Genetic Characterization of Methicillin Resistant and Sensitive, Vancomycin Intermediate Staphylococcus aureus Strains Isolated from Different Iranian Hospitals.

Background. Global concerns have been raised due to upward trend of Vancomycin Intermediate Staphylococcus aureus (VISA) and Vancomycin Resistant Staphylococcus aureus (VRSA) reports which mean casting doubt on the absolute effectiveness of the last line of antibiotic treatment for S. aureus, vancomycin. Hence, epidemiological evaluation can improve global health care policies. Methodology. 171 Isolates of Staphylococcus aureus were collected from different types of clinical samples in selected hospitals in Isfahan, Mashhad, and Tehran, Iran. Then, they were evaluated by agar screening, disk diffusion, and MIC method to determine their resistance to vancomycin and methicillin. The isolated VISA strains were then confirmed with genetic analysis by the evaluation of mecA and vanA genes, SCCmec, agr, and spa type, and also toxin profiles. MLST was also performed. Results and Conclusion. Our data indicated that 67% of isolated S. aureus strains were resistant to methicillin. Furthermore, five isolates (2.9%) had intermediate resistance to vancomycin (VISA). In contrast to usual association of VISA with MRSA strains, we found two isolates of MSSA-VISA. Therefore, our data suggests a probable parallel growing trend of VISA towards MSSA, along with MRSA strains. However, more samples are required to confirm these primarily data. Moreover, genetic analysis of the isolated VISA strains revealed that these strains are endemic Asian clones.

Interlaboratory Reproducibility of Selective Reaction Monitoring Assays Using Multiple Upfront Analyte Enrichment Strategies

Over the past few years, mass spectrometry has emerged as a technology to complement and potentially replace standard immunoassays in routine clinical core laboratories. Application of mass spectrometry to protein and peptide measurement can provide advantages including high sensitivity, the ability to multiplex analytes, and high specificity at the amino acid sequence level. In our previous study, we demonstrated excellent reproducibility of mass spectrometry-selective reaction monitoring (MS-SRM) assays when applying standardized standard operating procedures (SOPs) to measure synthetic peptides in a complex sample, as lack of reproducibility has been a frequent criticism leveled at the use of mass spectrometers in the clinical laboratory compared to immunoassays. Furthermore, an important caveat of SRM-based assays for proteins is that many low-abundance analytes require some type of enrichment before detection with MS. This adds a level of complexity to the procedure and the potential for irreproducibility increases, especially across different laboratories with different operators. The purpose of this study was to test the interlaboratory reproducibility of SRM assays with various upfront enrichment strategies and different types of clinical samples (representing real-world body fluids commonly encountered in routine clinical laboratories). Three different, previously published enrichment strategies for low-abundance analytes and a no-enrichment strategy for high-abundance analytes were tested across four different laboratories using different liquid chromatography-SRM (LC-SRM) platforms and previously developed SOPs. The results demonstrated that these assays were indeed reproducible with coefficients of variation of less than 30% for the measurement of important clinical proteins across all four laboratories in real world samples.

Production of an anticanine globulin (polyspecific) reagent for laboratory investigation

Canine allo- or autoantibodies are clinically important, but attachment of these immunoglobulin G (IgG) antibodies does not produce observable haemagglutination. Antibody to canine globulins is required to demonstrate sensitisation of red blood cells. Commercial reagents are available, but these often differ in sensitivity and specificity. Rabbit anticanine globulins (polyspecific) were produced for use in canine blood compatibility testing and in the investigation of immune-mediated haemolytic anaemia. Canine sera was pooled, IgG was purified and subsequently used to immunise rabbits. A rising titre of anticanine IgG was demonstrated by indirect enzyme-linked immunosorbent assay. Rabbit anticanine complement was isolated and investigated by agglutination of complement-coated canine red blood cells. Both antibodies were purified and checked for crossreactivity before being combined to polyspecific anticanine globulins. The obtained reagent was used to indicate sensitised canine red blood cells and free antibodies in three different types of clinical samples, including blood for compatibility testing and that for investigation of immune-mediated haemolytic anaemia and screening for post-transfusion alloantibodies and was also compared to commercial Coombs' reagent. The product provided results in accordance with those from commercial Coombs' reagent. The sensitivity for canine crossmatching was 100% and specificity for diagnosing immune-mediated haemolytic anaemia was 87%. This product is helpful for canine crossmatching purposes and in the investigation of immune-mediated haemolytic anaemia.

Compact optical microfluidic uric acid analysis system

We designed, fabricated and tested a novel compact fluorescence analysis system for quantification of uric acid (UA) in clinical samples at the point-of-care. To perform an analysis, diluted saliva, urine or blood samples are simply placed in a disposable thin-film sample holder using a dropper. A new enzyme immobilization technique was developed to retain within the sample holder two enzymes and a molecule, which transforms into a fluorescer in amounts depending on the UA concentration. The small instrument (7.5 cm × 5 cm × 5 cm) into which the sample holder is placed for analysis contains an LED, a narrow-band filter and an amplified photodiode. The analysis time is 30 s, and the dynamic range of the system is 4–400 μM of UA. The calibration curve for transparent saliva and urine was made using solutions of UA. The calibration curve for opaque blood was obtained with spiked samples of blood. The three different types of clinical samples were collected from three subjects and simply diluted before their measurements. Analysis with our instrument yielded UA concentrations within the expected concentration ranges. Development of instruments based on the current laboratory prototype is expected to result in products for clinical trials and point-of-care.

Comparative Evaluation of Three Culture Methods for the Isolation of Mycobacteria from Clinical Samples

We assessed the capacity of two liquid-medium culture methods with automated incubation and reading systems (MB/BacT ALERT 3D System and BACTEC MGIT 960 System) and one solid-medium culture method (Löwenstein- Jensen) to detect mycobacteria in different types of clinical samples. Out of 1,770 cultured clinical samples (1,519 of respiratory origin and 251 of nonrespiratory origin), mycobacteria were isolated in 156 samples (135 M. tuberculosis complex, 8 M. chelonae, 6 M. kansasii, 4 M. fortuitum, 2 M. gordonae, and 1 M. marinum) by at least one of the methods used. The BACTEC MGIT 960 System proved to be the most sensitive method (86.5%), especially in the detection of M. tuberculosis complex (89.1%). However, Löwenstein- Jensen culture was the most sensitive (76.2%) to detect nontuberculous mycobacteria. The BACTEC MGIT 960 System showed the lowest mean detection time for mycobacterial growth (15.3 days), significantly shorter than the other two methods. Highest sensitivity (95.5%) and specificity (99.6%) values were obtained using the BACTEC MGIT 960 System with the Löwenstein-Jensen culture method, which was also the only combination capable of detecting 100% of the nontuberculous mycobacteria.

Association of TGIFLX/Y mRNA expression with prostate cancer

Prostate cancer is the most common type of solid tumor and a leading cause of cancer-related death of men living in the developed world. In recent years, the molecular mechanisms involved in prostate cancer development and/or progression have been intensely studied and several genes have been identified. TGIFLX/Y (TGIFLX and TGIFLY) are members of the homeobox superfamily of genes whose function(s) is unknown. To investigate TGIFLX/Y mRNA expression in prostate cancer, we studied two different types of clinical samples, namely 60 prostate tumors and 15 cases of benign prostate hyperplasia (BPH), by RT-PCR. Our results revealed that most prostate tumors (73.5%) express at least one of these genes, although different patterns of TGIFLX/Y mRNA expression were observed. In some tumor samples the expression of both genes was detected, while in others no expression of either gene was observed. Notably, there was a significant correlation between expression of both TGIFLX and TGIFLY and a Gleason score of >or=6 (P = 0.038). By contrast, expression of TGIFLX/Y mRNA in BPH samples could not be detected. These results suggest an association of TGIFLX/Y expression with the progression of prostate cancer.