Types Of Clinical Samples Research Articles

PREVALENCE OF EXFOLIATIVE TOXIN GENES AMONG CLINICAL ISOLATES OF STAPHYLOCOCCUS AUREUS IN IRAQ

The aim of this study is to determine the prevalence of eta, etb and etd genes among clinical isolates of S. aureus. 91 isolates of the bacterium were isolated from different clinical sites during the period from 2019 to 2020 from Baghdad hospitals, all the isolates were diagnosed by different biochemical tests and molecular method (PCR) using nuc gene. the PCR technique was used to detect eta, etb and etd genes among the isolates, the results showed that 91(100%) of the isolates were have nuc gene. while, 83 ( 91.2%) of the isolates at least carrying one of the ET genes; 20 (22%) , 0 (0%) and 63 (69.2%) of the 91 isolates expressed eta, etb and etd genes, respectively. While, 8 (8.8%) of isolates were lacking of these genes. In addition, 14 (15.4%) of isolates were carrying both eta and etd genes. Although the etd gene was found in all sample types, but eta gene was found only in wound, ear, throat and nose while, etb gene was not found in all types of clinical samples.

A Performance Assessment Study of Different Clinical Samples for Rapid COVID-19 Antigen Diagnosis Tests.

Accurate diagnosis to limit the spread of SARS-CoV-2 is crucial for the clinical management of this lethal infection. Recently, many low-cost and easy-to-use rapid test kits (RTK) have been developed in many countries for the massive screening of SARS-CoV-2. Thus, evaluating the accuracy and reliability of an RTK is critical. The current study was conducted on 157 individuals to evaluate the performance accuracy of rapid SARS-CoV-2 antigen detection kits using different clinical samples compared with qRT-PCR results. Nasopharyngeal swabs were collected from patients for qRT-PCR and RTK tests, and then buccal and nasal, and nasal swabs were collected for RTK tests separately. The nasal and buccal swabs showed high sensitivity (98%) and specificity (100%) compared with the qRT-PCR results. Meanwhile, for nasal, the sensitivity was 96% with 98% specificity, and nasopharyngeal swabs showed 98% sensitivity and 94% specificity. Fisher’s exact test revealed statistical significance (p < 0.05) between nasopharyngeal, nasal and buccal, and nasal swabs compared with qRT-PCR results. The study concludes that different clinical samples used for the rapid diagnosis of SARS-CoV-2 showed high sensitivities and specificities compared with qRT-PCR. The RTKs using nasal and buccal, nasopharyngeal, and nasal swabs are valuable tools for the early detection of SARS-CoV-2, especially when molecular detections are available with limited access and a high infectivity rate, when the timely detection of virus cases is urgently needed. These types of clinical samples are effective to be used by RTKs for surveillance among community and healthcare workers.

Comparison of Carbapenem Resistance Detected by the BD Phoenix Automated System in Enterobacteriaceae Isolates with E-Test Method

Objective: Automatic identification and antimicrobial susceptibility systems are frequently used to identify clinical isolates in hospitalized patients, but mistakes in these systems can lead to potentially devastating treatment failures for patients. Therefore, the ‘‘Centers for Disease Control and Prevention (CDC)’’ recommends confirming all Carbapenem-resistant and low-susceptibility isolates with a different method. The aim of this study is to compare the Carbapenem susceptibility results of isolates reported as Carbapenem-resistant Enterobacteriaceae according to the BD Phoenix 100 automated system with the E-test method. Materials and Methods: The study included 70 strains of Carbapenem-resistant Enterobacteriaceae members which were isolated and grown from several types of clinical samples in the Medical Microbiology Laboratory. Conventional methods (Gram stain, negative oxidase test) and the BD Phoenix 100 automated system were used to identify the isolates. The susceptibility of all strains to imipenem, ertapenem and meropenem was investigated by E-test method. Automated system results and E-test results were compared. Results: The frequency distribution of all isolated bacterial strains comprised K. pneumoniae in 56 (80%) of the samples included in the study. The automated system test results were correlated with the results of the E-test at a rate of 96.1 % for the imipenem-resistant strains, 84.3% for the meropenem-resistant strains, 84.1% for the ertapenem-resistant strains Conclusions: Automated systems are frequently used in microbiology laboratories to identify isolates. However, automated systems can show a high error rate against some antimicrobials. For this reason, comparing the results of automated system test results with tests such as E-test is very important to prevent both treatment failures and inappropriate antibiotic use that may occur on a patient basis.

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.

Indirect Immunoperoxidase Test (IPT) for Detection of Antibodies Against African Swine Fever Virus (ASFV) on African Green Monkey Cell Lines (Vero, MS).

The immunoperoxidase technique (IPT) is an immunocytochemistry technique on fixed infected cells to determine the antibody-antigen complex formation through the action of the peroxidase enzyme. In this procedure, African green monkey established cell lines, such as Vero or MS, are infected with African swine fever virus (ASFV)-adapted isolates to these cell cultures. The infected cells are fixed and then used as antigens to determine the presence of the specific antibodies against ASFV in samples of swine origin. IPT has many advantages for the serological diagnosis of ASF. Firstly, IPT has a higher sensitivity than ELISA or immunoblot assay (IB) for the detection of antibodies against ASFV. Secondly, it has greater versatility to analyze any type of clinical sample of porcine origin and to conduct research studies on the epidemiological situation, through the presence of antibody titers in exudates from organs and tissues. Thirdly, the preparation of IPT plates with cells infected with ASFV is simple, although laborious, that can be stored for a long time and allow the analysis of a large number of samples.

Evaluating sensitivity and specificity of the Biomeme Franklin™ three9 real-time PCR device and SARS-CoV-2 go-strips assay using clinical samples.

We evaluated the sensitivity and specificity of the Biomeme Franklin™ three9 Real-Time PCR Thermocycler and Biomeme SARS-CoV-2 Go-Strips in the detection of SARS-CoV-2. The Biomeme Franklin™ three9 platform is a portable, battery-operated system that could be used in remote settings. We assessed performance of the Biomeme SARS-CoV-2 detection system at a wide range of viral concentrations, examined cross-reactivity of the SARS-CoV-2 Go-Strips against several near-neighbor respiratory pathogens, and evaluated agreement against the BioFire® Respiratory Panel 2.1 in four clinical sample types. Our data indicate the Biomeme Go-Strips can reliably detect SARS-CoV-2 at a concentration of 4.2 × 103 copies/mL. No cross reactivity of the Go-Strips targets was detected against any of the tested near-neighbor respiratory pathogens. Cohen's kappa statistics ranged from 0.68 to 0.92 between results from the Biomeme SARS-CoV-2 Go-Strips and the BioFire® Respiratory Panel 2.1 in all the different sample types. Compared to the BioFire® Respiratory Panel 2.1, the Biomeme SARS-CoV-2 Go-Strips demonstrated statistically significantly lower sensitivity in 3 out of 5 sample types. Overall, our study demonstrates the Biomeme Franklin™ three9 used with the SARS-CoV-2 Go-Strips is an effective system for the detection of SARS-CoV-2 that could potentially be used in a remote or austere environment.

NR4A1 Affects Endometrial Receptivity by Participating in Mesenchymal-Epithelial Transition of Endometrial Stromal Cells.

Decidualization is a substantive differentiation process experienced by endometrium to prepare for pregnancy. During this process, the endometrial stromal cells are transformed to endometrial epithelial cells. The receptivity of endometrium is necessary for the decidualization and successful implantation of endometrium, while the main hormones coordinating this process are estrogen and progesterone (P). In our study, the immunofluorescence, qPCR, and western blot experiments were conducted on different types of clinical endometrial tissue samples. The experimental results show that in the endometrium of normal subjects during the luteal phase, the protein level and serum P4 level of the orphan nuclear receptor NR4A1 messenger RNA were all significantly higher than those of patients with endometriosis or primary infertility, and the two levels presented positive correlation. Through decidualization induction of the human endometrial stromal cells (hESCs) cultured in vitro and additional P treatment, the results of chromatin immunoprecipitation and other experiments show that the P treatment could upregulate the expression of NR4A1 in hESCs, and this process was mediated under the direct effect of progesterone receptor (PR) and NR4A1. When the NR4A1 in hESCs was silenced, the promotion of hESC proliferation by P was inhibited. P and overexpressed NR4A1 increased the expression of epithelial cell marker in decidual hESCs, and qPCR showed that NR4A1's response to P was earlier than that of the epithelial cell marker. The results of spheroid adhesion assay show that the silent NR4A1 had reduced the adhesion of decidual hESCs induced in vitro to embryo. To sum it up, NR4A1 participated in the decidualization process by responding to the P regulation via and by promoting the hESCs' mesenchymal-epithelial transition, so as to further influence the receptivity of endometrium.

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.

Strategies for Engineering Affordable Technologies for Point-of-Care Diagnostics of Infectious Diseases.

Disease prevalence is highest in low-resource settings (LRS) due to the lack of funds, infrastructure, and personnel required to carry out laboratory-based molecular tests. In high-resource settings, gold-standard molecular tests for diseases consist of nucleic acid amplification tests (NAATs) due to their excellent sensitivity and specificity. These tests require the extraction, amplification, and detection of nucleic acids from clinical samples. In high-resource settings, all three of these steps require highly specialized, costly, and onerous equipment that cannot be used in LRS. Nucleic acid extraction involves multiple centrifugation steps. Amplification consists of the polymerase chain reaction (PCR), which requires thermal cyclers. The detection of amplified DNA is typically done with specialized thermal cyclers that are capable of fluorescence detection. Traditional methods used to extract, amplify, and detect nucleic acids cannot be used outside of a laboratory in LRS. Thus, there is a need for affordable point-of-care devices to ease the high burden of disease in LRS.The past decade of work on paper-based fluidic devices has resulted in the invention of many paper-based biosensors for disease detection as well as isothermal amplification techniques that replace PCR. However, a challenge still remains in detecting pathogenic biomarkers from complex human samples without specialized laboratory equipment. Our research has focused on the development of affordable technologies to extract and detect nucleic acids in clinical samples with minimal equipment. Here we describe methods for the paper-based extraction, amplification, and detection of nucleic acids. This Account provides an overview of our latest technologies developed to detect an array of diseases in low-resource settings. We focus on detecting nucleic acids of H1N1, human papillomavirus (HPV), Neisseria gonorrheae (NG), Chlamydia trachomatis (CT), Trichomonas vaginalis (TV), and malaria from a variety of clinical sample types. H1N1 RNA was extracted from nasopharyngeal swabs; HPV, NG, and CT DNA were extracted from either cervical, urethral, or vaginal swabs; TV DNA was extracted from urine; and malaria DNA was extracted from whole blood. Different sample types necessitate different nucleic extraction protocols; we provide guidelines for assay design based on the clinical sample type used. We compare the pros and cons of different isothermal amplification techniques, namely, helicase-dependent amplification (HDA), loop-mediated isothermal amplification (LAMP), and a novel isothermal amplification technique that we developed: isothermal-identical multirepeat sequences (iso-IMRS). Finally, we compare various detection mechanisms, including lateral-flow and electrochemical readouts. Electrochemical readouts frequently employ gold electrodes due to strong gold-thiol coupling. However, the high cost of gold precludes their use in LRS. We discuss our development of novel gold leaf electrodes that can be made without specialized equipment for a fraction of the cost of commercially available gold electrodes.

Real-Time Recombinase Polymerase Amplification (RPA) Detection of Pseudomonas aeruginosa Using Magnetic Nano-Beads for DNA Extraction

Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous nonfermentative gram-negative bacillus, residing in nature widely as a conditional pathogen that is commonly isolated from nosocomial infection cases, having a larger genome (5.5–7 Mbp). P. aeruginosa possesses great environmental adaptability and higher mutation rates, which accounts for its ability to resist antibiotics. Furthermore, multi-antibiotics-resistant P. aeruginosa has recently been established to be responsible for increased nosocomial infection incidence. Therefore, to detect, diagnose, and treat this life-threatening infection early enough, we introduced a real-time recombinase polymerase amplification (RPA) method of rapid P. aeruginosa detection, which is time-saving, convenient, highly sensitive, and more specific than the traditional bacterial culture method. System effectiveness and efficiency were then determined by a series of experiments, where specific RPA primers and probes accurately distinguished P. aeruginosa from other common pathogenic bacteria with optimal RPA reaction procedures set at 39 °C for 20 min. Additionally, specific primers and probes were designed based on the oprF gene sequence of P. aeruginosa. Subsequently, a real-time RPA system was used to analyze 20 cases of different clinical samples for P. aeruginosa detection in blood, open wound swab, and sputum. Results indicated that this system was suitable for bacterial analysis of common clinical sample types. The DNA extraction method used in this study was based on the magnetic beads method. In conclusion, this study established that the real-time RPA system was a rapid, specific, and sensitive method for clinical P. aeruginosa detection, which can be integrated into automatic high-throughput detection machines.

A SEARCH FOR ASSOCIATION BETWEEN BIOFILM STRENGTH AND SITES OF INFECTION CAUSED BY ACINETOBACTER BAUMANNII COMPLEX

INTRODUCTION: In the world of infectious diseases, a group of bacteria known as ESKAPE pathogens, are responsible for most of the life threatening multidrug resistant (MDR) and extensively drug resistant (XDR) infections worldwide. The ESKAPE pathogens comprise of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. AIMS AND OBJECTIVES: The objective of this study is isolate and identify Acinetobactor baumannii complex from clinical samples. Measure the biolm forming capacity of the isolates. Possible association of this biolm strength with the type of clinical sample from which they are isolated. MATERIALS AND METHODS: The samples which were tested in the laboratory were the following: Sputum, End tracheal tube aspirates, Pus, Urine, Blood for culture, Central venous catheter tips &amp; Cerebrospinal uid. Following culture media were taken: MaCconkey agar &amp; Blood agar. The colonies suggestive of Acinetobacter baumannii were selected. On MaCconkey agar, non- pigmented NLF colonies with pink to light lavender hue; and on blood agar, opaque non-hemolytic colonies were obtained. Gram stains from selected colony showed gram negative coccobacilli. Hanging drop preparation showed non-motile bacteria. RESULTS AND ANALYSIS: We found maximum number of strong biolm producers were found among the isolates causing infection of CVC tips (70%). This was closely followed by ETtube isolates (69.5%). The isolates from blood stream, urine, and sputum samples showed all the three patterns, namely, no biolm producer, moderate biolm producer, and strong biolm producer. Our study showed interestingly, no strong biolm producer was found from pus samples, whereas from the CSF isolates no non-producer of biolm emerged. CONCLUSION: This study centers around strength or pattern of biolm formation by this bacteria. It was interesting to nd that there was a statistically signicant association between patterns of biolm produced by Acinetobacter baumannii and the various samples from which they are isolated. This made us to hint at a probable relation between biolm patterns and organotropism as an hypothesis. Only further investigations will tell if it can stand the test of time.

Ophthalmological aspects of coronavirus infections

The novel coronavirus infection (COVID-19) caused by SARS-CоV-2 virus, may lead to ocular diseases besides affecting the respiratory system. Cororonavirus infection may be transmitted not only through the respiratory system but also through the conjunctiva. The review article summarizes the data on the origin and variants of coronaviruses that infect humans, as well as on SARS-CоV-2 structures. Literature data on prospects of molecular diagnostics of the disease, coronavirus type detection methods, clinical sample types and duration of the disease before it is diagnosed are discussed. The data on the relationship between the coronavirus infection and conjunctivitis are given. Molecular tests of the tear and conjunctival swabs were used to diagnose the coronavirus infection independently of whether symptoms of ocular disorders were present. The real-time reverse-transcription of RNA polymerase chain reaction (PCR) was the most informative method for diagnosis of SARS-CоV-2 in the early stage of COVID-19 (until the 9th day). The presence of SARS-CоV-2 in the tear fluid and conjunctival swabs indicates the need for safety measures to prevent virus transmission through the ocular surface, including protective goggles to be worn by healthcare workers. Since side effects in the eye may appear, COVID-19 patients with visual impairment who received etiotropic or pathogenetic treatment are advised to consult an ophthalmologist.

The Experience of Implementing a National Antimicrobial Resistance Surveillance System in Brazil

Antimicrobial resistance (AMR) is a major public health threat of global proportions, which has the potential to lead to approximately ten million deaths per year by 2050. Pressured by this wicked problem, in 2014, the World Health Organization launched a call for member states to share AMR data through the implementation of the Global Antimicrobial Resistance Surveillance System (GLASS), to appropriately scale and monitor the general situation world-widely. In 2017, Brazil joined GLASS and, in 2018, started its own national antimicrobial surveillance program (BR-GLASS) to understand the impact of resistance in the country. We compiled data obtained from the complete routine of three hospitals' microbiology labs during the year of 2018. This pilot data sums up to 200,874 antimicrobial susceptibility test results from 11,347 isolates. It represents 119 different microorganisms recovered from 44 distinct types of clinical samples. Specimens came from patients originating from 301 Brazilian cities, with 4,950 of these isolates from presumed Healthcare-Associated Infections (HAIs) and the other 6,397 community-acquired cases. The female population offered 58% of the collected samples, while the other 42% were of male origin. The urinary tract was the most common topography (6,372/11,347 isolates), followed by blood samples (2,072/11,347). Gram-negative predominated the bacterial isolates: Escherichia coli was the most prevalent in general, representing 4,030 isolates (89.0% of these from the urinary tract). Coagulase-negative Staphylococci were the most prevalent bacteria in blood samples. Besides these two species, the ESKAPE group have consolidated their prevalence. Regarding drug susceptibility results, 141,648 (70.5%) were susceptible, 9,950 (4.9%) intermediate, and 49,276 (24.5%) resistant. Acinetobacter baumannii was the most worrisome microorganism, with 65.3% of the overall antimicrobial susceptibility tests showing resistance, followed by ESBL-producing Klebsiella pneumoniae, with a global resistance rate of 59%. Although this is a pilot project (still limited to one state), this database shows the importance of a nation-wide surveillance program,[153mm][-12mm] Q14 especially considering it already had patients coming from 301 distinct counties and 18 different states. The BR-GLASS Program is an ongoing project that intends to encompass at least 95 hospitals distributed in all five geographical regions in Brazil within the next 5 years.

Imaging of Tumor Boundary Based on Multielements and Molecular Fragments Heterogeneity in Lung Cancer

Elucidating the boundary of tumors is significant for cancer diagnosis and cure. In this work, our group proposed an innovative method for the diagnostic analysis of clinical lung cancer tissues based on label-free imaging. The heterogeneity of multielements and molecular fragments was obtained simultaneously for three types of clinical samples containing different proportions of cancerous tissue by laser-induced breakdown spectroscopy (LIBS) imaging. The results of elemental imaging mainly reveal the differential distribution of Ca, Mg, Na, and Cu in cancer tissues, paracancer tissues, and normal tissues. In particular, for the first time, we discovered the spatial heterogeneity of molecular fragments between tumor and necrosis regions. The tumor regions produced more CN molecules during laser ablation, while almost no C2 molecules were produced. Further spatial segmentation shows the clearer boundary of the tumor and necrotic areas in cancer tissues and paracancer tissues. Our work indicates that the heterogeneity of multielements and molecular fragments has great potential as diagnostic biomarkers of tumors boundary.

Flow-FISH Using Nucleic Acid Mimic Probes for the Detection of Bacteria.

Flow-Fluorescence in situ hybridization (Flow-FISH) enables multiparametric high-throughput detection of target nucleic acid sequences at the single cell-level, allowing an accurate quantification of different cell populations by using a combination of flow cytometry and fluorescent in situ hybridization (FISH). In this chapter, a flow-FISH protocol is described with labeled nucleic acid mimics (NAMs) (e.g. LNA/2'OMe and PNA) acting as the reporter molecules. This protocol allows for the specific detection of bacterial cells. Hence, this protocol can be carried out with minor adjustments, in order to simultaneously detect different species of bacteria in different types of clinical, food, or environmental samples.

METHODOLOGY CONSIDERATIONS FOR THE MOLECULAR DETECTION OF TORCH ORGANISMS USING PCR APPROACHES

There is a need for a reliable and cost-effective molecular diagnostic assay for the diagnosis of TORCH [Toxoplasma gondii, Other (Varicella-Zoster virus and Parvovirus B19), Rubella virus, Cytomegalovirus, and Herpes Simplex virus] infections. This would enable early and precise detection of pathogens even in a very low copy number. However, the selection of genomic target, specimen matrix, and different PCR methods can significantly affect the sensitivity and specificity of TORCH molecular detection. This review aimed to provide a comparative analysis of clinical sample types, target nucleotide sequences, and PCR detection approaches for molecular detection of TORCH organisms. This review will aid in the development of a sensitive molecular assay for quick and precise detection of TORCH organisms.

Challenges and considerations on quality control and evaluation of pathogen metagenomic next-generation sequencing

Metagenomic next-generation sequencing (mNGS) could be used for pathogen detection from nearly all types of clinical samples. Especially, the unique diagnostic capability of pathogen mNGS detecting unknown causative agent of infectious diseases makes this method become an importation complement and irreplaceable component for conventional routine laboratory test. However, the complexity of the testing process, the rapid product update, and the insufficiency in quality control and evaluation methods that all make clinical transformation, industry development, and regulation of this technology full of challenge and uncertainty. This review briefly introduces the technical advantages and challenges, and describes the general workflow and quality control steps in details. Finally, it focuses on current considerations regarding quality evaluation methods and standards for pathogen mNGS.

Non-antibiotic compounds affecting the growth of urinary pathogens during urine culture: a preliminary in vitro study

Introduction: Urine samples are one of the most frequently submitted samples for culture to clinical microbiology laboratories, exceeding the number of most of the other clinical sample types. Various non-antibiotic pharmaceutical compounds may have inhibitory properties on bacteria, as many of these agents accumlate in/eliminated through urine. Methods: Sixty (n=60) pharmacological agents were tested during our experiments. Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700603 (ESBL-producing) and Staphylococcus aureus ATCC 29213 were the bacterial strains utilized in this study. Detection of inhibitory activity among the tested compounds was performed on Mueller-Hinton plates, using disk diffusion method. Results: Nineteen (n=19) compounds presented with various levels of inhibitory activity on the tested bacterial strains (four compounds for K. pneumoniae, seven compounds on E. coli and sixteen compounds on S. aureus). The compounds showed the highest levels of inhibitory activity on B. subtilis ATCC 6633, which is one of the main bacterial strains used for the screening of the ’intrinsic’ antibacterial activity of urine. Conclusion: Our results suggest that further experiments, involving additional pharmacological agents is warranted, to establish the full extent of their influence on the appropriate culture of urine samples.

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.

Toward a comprehensive study for multielemental quantitative LA-ICP MS bioimaging in soft tissues

Quantitative localization of metals in biological tissue sections is critical to obtain insight into metal toxicity mechanisms or their beneficial characteristics. This study presents the development of a quantitative LA-ICP MS bioimaging methodology based on the polymer film strategy and internal standardization. To maximize the number of elements mapped, an aqueous soluble polymer (dextran) was selected. Among the elements studied, the great majority (eight out eleven), i.e., Co, Ni, Cu, Zn, Se, Mo, Cd and Pt, exhibited linear regression after LA-ICP MS analysis of metal-spiked polymer standards. Methodology performances were carefully assessed as a function of the three internal standards (In, Rh and Ir) considered, the analytical operational conditions (ICP power, addition of O2 to ICP, and laser fluency) and the thickness of the biological tissue section. The results indicated that three groups (Co, Mo; Ni, Cu, Pt; and Zn, Se, Cd) of elements could be distinguished from their analytical response as a function of analytical conditions and the internal standard. These different element behaviors appeared to be mainly First Ionization Potential dependent (FIP). For elements with lower FIP (Co, Ni, Cu, Mo and Pt), differential responses due to carbon load in the ICP MS plasma could be efficiently corrected as a function of analytical conditions. Matrix effects were more pronounced for higher FIP elements (i.e., Zn, Cd and Se), and analysis of <10-μm thin sections without the addition of O2 to ICP MS plasma is recommended. LODs are in the range of 0.1–0.5 μg g−1 for Co, Mo, Cu, Ni, Pt and Cd as well as 0.9 and 1 μg g−1 for Zn and Se, respectively. The methodology was validated by means of a homemade metal-spiked kidney homogenate analyzed by LA-ICP MS imaging, and Co, Ni, Cu, Mo, and Pt provided the closest concentrations (5–29% bias) to the target values determined by ICP MS after mineralization. The methodology was applied to two types of clinical human samples undergoing different sample preparation protocols that did not affect internal standard homogeneity in the polymer film. This methodology is the first reported for the quantitative bioimaging of eight elements simultaneously.