Multiplex Kit Research Articles

Inhibition of tumor necrosis factor improves conventional steroid therapy for Stevens-Johnson syndrome/toxic epidermal necrolysis in a cohort of patients

BackgroundSystemic steroid therapies for Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) have been challenged because of their limited benefits. Whether additional tumor necrosis factor (TNF) α inhibition provides an optimized approach remains unexplored. ObjectiveTo investigate the efficacy of TNF-α inhibition combined with a steroid to treat SJS/TEN and to identify potential biomarkers. MethodsTwenty-five patients with SJS/TEN were recruited and divided into 2 groups: 10 patients received methylprednisolone and 15 patients received etanercept plus methylprednisolone. Serum levels of granzyme B, perforin, interferon-γ, interleukin (IL) 6, IL-15, IL-18, macrophage inflammatory protein 1α, macrophage inflammatory protein 1β, and TNF-α were measured by multiplex cytokine analysis kits during the acute and resolution phases. ResultsCompared with the steroid monotherapy, the combination therapy significantly shortened the course of the initial steroid treatment and the duration of the acute stage, hospitalization stay, and skin re-epithelialization. Although both therapies significantly reduced IL-15 levels; the combination therapy also decreased IL-6 and IL-18 levels. While the level of IL-15 was positively correlated with skin re-epithelialization time in both groups, the level of IL-6 served as an additional marker for the course of the disease in the combination therapy group. LimitationsThe cohort size is relatively small. ConclusionAdditional TNF-α inhibition to steroid treatment appeared to improve outcomes for SJS/TEN.

Cytokine signature for predicting new-onset prediabetes after acute pancreatitis: A prospective longitudinal cohort study.

Acute inflammation of the pancreas often leads to metabolic sequelae, the most common of which is new-onset prediabetes (and, ultimately, diabetes). However, there is a lack of studies on predictors of this sequela. The aim was to investigate whether cytokines/chemokines measured at baseline are predictive of new-onset prediabetes after acute pancreatitis (NOPAP). This was a prospective longitudinal cohort study (as part of the LACERTA project) that included 68 individuals with non-necrotising acute pancreatitis who had no diabetes mellitus. Of them, 17 individuals had prediabetes at baseline and during follow-up, 37 individuals had normoglycaemia at baseline and during follow-up, and 14 individuals had normoglycaemia at baseline and developed NOPAP during follow-up. A commercially available human cytokine/chemokine multiplex kit was used to measure a total of 28 analytes at baseline. Multinomial regression analyses were conducted to investigate the associations between the cytokines/chemokines and the three study groups. Interleukin-1β and interferon γ significantly predicted progression to NOPAP with an odds ratio (95% confidence interval) of 1.097 (1.002, 1.201) and 1.094 (1.003, 1.192), respectively (after accounting for age, sex, body mass index, and aetiology of acute pancreatitis). None of the studied cytokines/chemokines showed statistically significant associations with the antecedent prediabetes group (after accounting for the above covariates). Elevated levels of interleukin-1β and interferon γ in acute pancreatitis individuals with normoglycaemia at baseline may predict progression to NOPAP during follow-up.

Clinical Comparative Evaluation of the LabTurboTM AIO® Reverse Transcription-Polymerase Chain Reaction and World Health Organization-Recommended Assays for the Detection of Emerging SARS-CoV-2 Variants of Concern.

PurposeSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent behind coronavirus disease-2019 (COVID-19). Single-plex reverse transcription-polymerase chain reaction (RT-PCR)-based assays are widely used for COVID-19 detection but exhibit decreased sensitivity and specificity in detecting the rapidly spreading SARS-CoV-2 variants; in contrast, multiplex RT-PCR reportedly yields better results. Here, we aimed at comparatively analyzing the clinical performance of the LabTurboTM AIO COVID-19 RNA testing kit, a multiplex quantitative RT-PCR kit, including a three-target (E, N1, and RNase P), single-reaction, triplex assay used for SARS-CoV-2 detection, with that of the WHO-recommended RT-PCR assay.Materials and MethodsResidual, natural, nasopharyngeal swabs obtained from universal transport medium specimens at SARS-CoV-2 testing centers (n = 414) were collected from May to October 2021. For SARS-CoV-2 qRT-PCR, total viral nucleic acid was extracted. The limit of detection (LOD) and the comparative clinical performances of the LabTurboTM AIO COVID-19 RNA kit and the WHO-recommended RT-PCR assay were assessed. Statistical analysis of the correlation was performed and results with R2 values >0.9 were considered to be highly correlated.ResultsThe LOD of the LabTurboTM AIO COVID-19 RNA kit was 9.4 copies/reaction for the target genes N1 and E. The results obtained from 102 SARS-CoV-2-positive and 312 SARS-CoV-2-negative samples showed 100% correlation with previous WHO-recommended RT-PCR assay results.ConclusionMultiplex qRT-PCR is a critical tool for detecting unknown pathogens and employs multiple target genes. The LabTurboTM AIO COVID-19 RNA testing kit provides an effective and efficient assay for SARS-CoV-2 detection and is highly compatible with SARS-CoV-2 variants.

Роль матриксной металлопротеиназы-2 и альфа-субъединицы рецептора интерлейкина-2 в развитии критического состояния у больных пневмонией при гриппе A/H1N1

The aim of the research. To assess the role of matrix metalloproteinase-2 (MMP-2) and the alpha subunit of the interleukin-2 receptor (IL-2 Ra) in development of critical illness in pneumonia patients with underlying influenza A (H1N1). Material and methods. A total of 85 patients with pneumonia associated with influenza A (H1N1) were examined. Among them, 30 patients suffered from severe pneumonia while 55 had non-severe pneumonia. Th e mean age of the patients amounted to 52 [38; 67]. Th e group was constituted by 48 % male and 52 % female patients. Th e diagnosis of infl uenza A / H1N1 was confirmed by positive PCR test results. Th e CURB / CRB-65 scales were used to diagnose and assess the severity of pneumonia; this was also performed using SMART-COP, the Federal Clinical Guidelines of the Ministry of Health of the Russian Federation “Community-acquired pneumonia in adults”, 2019 and the IDSA / ATS criteria (pneumonia was considered severe in presence of one major or three minor criteria). Plasma concentration of MMP-2 and IL-2 Ra molecules (sCD25) was measured through flow cytometry with the Beckman Coulter analyser (USA) using LEGENDplex™ HU Immune Checkpoint Panel 1 and Human Vascular Inflammation Panel 1 Beckman Coulter (USA) multiplex assay kits. Results. It was established that patients with severe pneumonia and underlying influenza A (H1N1) had plasma concentration of MMP-2 and sCD25 increased 2-fold and 2.5-fold, respectively. Conclusion. Statistically signifi cant increase in the concentration of MMP-2 and IL-2 Ra in pneumonia patients with underlying influenza A (H1N1) indicates the occurrence of proinflammatory conditions for the development of a critical state, as well as the involvement of negative regulation components of the T-cell response into the cascade of adaptive immune responses, which is associated with severity of the condition.

A novel multiplex PCR assay to detect and distinguish between different types of Paenibacillus larvae and Melissococcus plutonius, and a survey of foulbrood pathogen contamination in Japanese honey.

Paenibacillus larvae and Melissococcus plutonius are the causative agents of American and European foulbroods of honey bees, respectively. Since their virulence and resistance to disinfectants differ depending on the genotypes/phenotypes of the strains, the discrimination of strain types is important for the effective control of these diseases. Methods to detect and differentiate pathogens in honey are useful for surveying the contamination status of beehives/apiaries. In the present study, we selected a sequence (GenBank accession no. FI763267) as the specific target for enterobacterial repetitive intergenic consensus (ERIC) II-type P. larvae strains for the first time and developed a novel multiplex PCR assay that precisely distinguishes between the major types of foulbrood pathogens (ERIC I and II P. larvae and typical and atypical M. plutonius) in one reaction. In addition, we found that commercially available kits designed for DNA extraction from Mycobacterium in feces efficiently extracted DNA from foulbrood pathogens in honey. Using the multiplex PCR assay and DNA extraction kits, all the targeted types of P. larvae and M. plutonius were detected in honey spiked with the pathogens at a concentration of 100 bacterial cells/strain/ml. Moreover, 94% of the Japanese honey samples examined in the present study were contaminated with one or more types of the foulbrood pathogens. These results indicate that the newly developed methods are useful for detecting foulbrood pathogens in honey. The epidemiological information obtained by these methods will contribute to the effective control of foulbroods in apiaries.

The Role of IL-5, IL-33 and Total IgE in a Sample of Workers Suffering from Paints and Mills Asthmatic Patients

A case-control retrospective study was conducted during November 2020 on 88 asthmatic patients (38 male mill and 50 male paints workers)with age ranged between (15-25),and 44 control with age ranged between (15-25)years to assess the role of total IgE ,interleukine5(IL-5),and interleukin33(IL-33)serum levels in pathophysiology of disease using ELISA and multiplex immunoblot kits. Some hematological changes such as(Eosinophil and Basophil%)by Beckman coulter. The patients’ samples were obtained from Nature Dyes Company Ltd in the industrial region of Aweerij Industrial, as well as Al-Dora flour mill in Baghdad, which is part of the general company for grain processing. The tests were carried out at the Specialized Center of Allergy in Baghdad/Al-Rusafa from October 2020 to April 2021. Specialized respiratory disorders and Asthma diagnose these cases, which are clinically diagnosed according to international guide lines. The present study confirms the highly significant role of total IgE and IL5,IL-33 levels compared with control in pathophysiology of bronchial asthma and it correlation with disease severity and allergen type in adults. Results showed that there were highly significant increase in Eosinophil and basophil % in cases asthma of paints and mills patients with control group. In addition, there were a positive correlation between Eosinophil and Basophil in asthma of paints, and in asthma of mills. There was positive correlation between total IgE with IL-5 and IL-33 in asthmatic group.

Assessment of Genetic Polymorphisms in the Rewa Population of Central India Using Y-Chromosomal STR Markers.

The aim of the study was to analyze unrelated samples from the Rewa male population of Central India by targeting Y-short tandem repeats (STR) and then compare the results to previously published Y-STR haplotype data.Materials and MethodsA total of 181 unrelated male subjects from the Rewa population were genotyped for seventeen Y-STRs (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and Y-GATA-H4) by using an AmpFlSTR® Yfiler™ multiplex kit (Thermo Fisher Scientific, USA). The allele frequencies and forensic parameters were evaluated.ResultsA total of 111 distinct Y-STR alleles with corresponding frequencies ranging from 0.006 to 0.829 were identified. The gene diversity values ranged from 0.3092 at DYS437 to 0.8188 at DYS385b. The studied population showed a high level of haplotype diversity (0.9985) and discrimination capacity (0.927). A haplotype analysis was also conducted. Among the 181 unrelated male samples, 165 haplotypes and 153 unique haplotypes were found. Additionally, Rst (genetic distance) values were calculated using the analysis of molecular variance (AMOVA) for the studied population and for other 18 populations described in the literature. The Rst provides a convenient parameter for estimating the level of genetic differentiation from the microsatellite data. Based on these Rst values and using the multidimensional scaling plot, a neighbor-joining tree was constructed.ConclusionThe high values of haplotype diversity and discrimination capacity indicate a great potential for distinguishing between male individuals in the studied population. The present population data are expected to find their use in forensic caseworks and population genetics.

Evaluation of the Multiplex Real-Time PCR DermaGenius® Assay for the Detection of Dermatophytes in Hair Samples from Senegal.

For the successful treatment of dermatophytoses, especially tinea capitis, there is a need for accurate and rapid diagnostic methods. A lot of recent literature has focused on the detection of dermatophytes directly on sample material such as nails, hair and skin scrapings. Molecular tools offer the ability to rapidly diagnose dermatophytosis within 48 h. This study aimed to compare the results of a commercial real-time PCR (real-time PCR) assay DermaGenius®(DG) 2.0 complete multiplex kit with those of conventional diagnostic methods (direct microscopy and culture). A total of 129 hair samples were collected in Dakar (Senegal) from patients suspected of dermatophytosis. DG was applied for the molecular detection of Candida albicans, Trichophyton rubrum/soudanense, T. interdigitale, T. tonsurans, T. mentagrophytes, T. violaceum, Microsporum canis, M. audouinii, Epidermophyton floccosum, T. benhamiae and T. verrucosum. Dermatophytes species and C. albicans were differentiated by melting curve analysis. The sensitivity and specificity of the PCR assay were 89.3% and 75.3%, respectively. DG PCR was significantly more sensitive than culture (p < 0.001). DG PCR is fast and robust to contamination. In this paper, the main questions discussed were the replacement of culture by a broad-spectrum fungal real-time PCR and the implementation of DG PCR into a routine laboratory in Senegal.

Clinical spectrum of endemic leptospirosis in relation to cytokine response.

To describe the clinical spectrum and the cytokine response of leptospirosis patients in an endemic setting of Sri Lanka. Patients presenting to the university teaching hospital, Anuradhapura, Sri Lanka with a leptospirosis-compatible illness were recruited over a period of 12 months starting from June 2012. Daily clinical and biochemical parameters of the patients were prospectively assessed with a follow-up of 14 days after discharge. A magnetic bead-based multiplex cytokine kit was used to detect 17 cytokines. Of the 142 clinically suspected leptospirosis patients recruited, 47 were confirmed and, 29 cases were labeled as "probable." Thrombocytopenia and leukocytosis were observed at least once during the hospital stay among 76(54%) and 39(28%) patients, respectively. Acute kidney injury was observed in 31 patients (22%) and it was significantly higher among confirmed and probable cases. Hu TNF-α and IL-1β were detected only in patients without complications. Hu MIP-1b levels were significantly higher among patients with complications. During the convalescence period, all tested serum cytokine levels were lower compared to the acute sample, except for IL-8. The cytokine response during the acute phase clustered in four different groups. High serum creatinine was associated GM-CSF, high IL-5 and IL-6 level were correlates with lung involvement and saturation drop. The patients with high billirubin (direct)>7 mmol/l had high IL-13 levels. Results of this study confirms that the knowledge on cytokine response in leptospirosis could be more complex than other similar tropical disease, and biosignatures that provide diagnostic and prognostic information for human leptospirosis remain to be discovered.

POSSIBLE VIRAL ETIOLOGY OF GLIOBLASTOMA MULTIFORME

Objective: Recently, CMV, EBV, BKV and JC virus have been suggested to contribute to gliomagenesis, but evidence is largely contradictory. The aim of this study was to test 50 tissue samples from patients with GBM and 21 normal brain tissues obtained from autopsy material of patients without brain tumors in order to investigate the presence of possible oncogenic microorganisms, including EBV, JCV, BKV and Toxoplasma gondii, and to evaluate patient clinical characteristics of patients with respect to microorganism findings.&#x0D; Material and Method: Fifty formalin-fixed paraffin-embedded specimens obtained from glioblastoma tissue and 21 normal brain tissues obtained in the autopsy of individuals without brain tumor were retrospectively analyzed. After de-paraffinization of tissue samples, DNA extraction was performed for real-time polymerase chain reaction (RT-PCR) analysis to detect BKV, JCV, EBV and Toxoplasma gondii via commercially-available multiplex kits.&#x0D; Results: Strikingly, viral DNA was detected in 12 specimens (24%) of the GBM group and in none of the non-tumor brain specimens (p=0.014). BKV was detected in 4, EBV was detected in 3 (of these, 2 were alive during the study), Toxoplasma gondii was detected in 5 and JCV genotype was detected in 1 of the total 50 GBM tissue specimens. &#x0D; Conclusion: Our results suggest that viruses may be associated with the development or progression of GBM. Understanding the role of BKV, JCV, EBV and other oncoviruses in the etiology of gliomas would likely open up new avenues for the treatment and management of this highly fatal central nervous system tumor.

CSF apolipoprotein B is associated with early tau pathology and selective activation of a cytokine cascade in cognitively unaffected subjects with a parental history of Alzheimer’s disease

AbstractBackgroundWe examine the role of CSF apolipoprotein B (apoB) as a putative indicator of early tau pathology in pre‐symptomatic Alzheimer’s disease (AD).MethodAmong 129 cognitively unimpaired elderly at increased risk of AD (PREVENT‐AD cohort), we assessed blood and cerebrospinal fluid apoB, apoC3 and apoE protein levels using the the Milliplex APOMAG‐62k human apolipoprotein cardiovascular disease multiplex kit (EMD‐Millipore, MA, USA) with those of amyloid β42, total tau and phosphorylated tau (the Innotest enzyme‐linked immunosorbent assay kit (Fujirebio, Ghent, Belgium). We contrasted results with alterations in several inflammation biomarkers using Luminex’s Milliplex HCYTMAG60PMX29BK xMap technology ((EMD‐Millipore, USA).ResultAmong cognitively unimpaired participants, we observed significant correlations between baseline CSF, but not blood, apoB levels and both CSF t‐tau (R2 = 0.23, P &lt; 0.0001) and P‐tau (R2 = 0.28, P &lt; 0.0001). No association was detected between CSF apoB and CSF Aβ42 levels. Using multiple brain blood barrier permeability assays, we found that the contribution from peripheral apoB to be negligible. Concomitant analyses of several key cytokines and chemokines in the CSF failed to show any association following FDR correction, except for IL‐15 (R2: 0.38, p &lt; 0.001). IL‐15, a potent inducer of reactive gliosis in both astrocytes and microglia, is normally synthesized by compromised neurons in the CNS.ConclusionCSF apoB, which is normally produced by microglia, markedly associates with early tau dysregulation and the activation of select cytokines in asymptomatic subjects, years before the onset of symptoms.

Time-Dependent Changes in Hepatic Sphingolipid Accumulation and PI3K/Akt/mTOR Signaling Pathway in a Rat Model of NAFLD.

Increased lipid bioavailability in a diet favors lipid accumulation, enhancing hepatic lipotoxicity and contributing to insulin resistance (IR) development. The aim of our study was to examine time-dependent alterations in the intrahepatic content of sphingolipids and insulin signaling pathway in rats fed a high-fat diet (HFD). The experiment was conducted on male Wistar rats receiving a standard diet or HFD for five weeks. At the end of each experimental feeding week, liver sphingolipids were determined using high-performance liquid chromatography. The expression of proteins from the sphingolipid pathway and glucose transporter expression were assessed by Western blot. The content of phosphorylated form of proteins from the insulin pathway was detected by a multiplex assay kit. Our results revealed that HFD enhanced hepatic ceramide deposition by increasing the expression of selected proteins from sphingomyelin and salvage pathways in the last two weeks. Importantly, we observed a significant inhibition of Akt phosphorylation in the first week of HFD and stimulation of PTEN and mTOR phosphorylation at the end of HFD. These changes worsened the PI3K/Akt/mTOR signaling pathway. We may postulate that HFD-induced reduction in the insulin action in the time-dependent matter was exerted by excessive accumulation of sphingosine and sphinganine rather than ceramide.

Establishing an integrated pipeline for automatic and efficient detection of trace DNA encountered in forensic applications

The analysis of trace DNA is a crucial component in forensic applications. Biological materials containing low-level DNA collected at crime scenes, such as fingerprints, can be valuable as evidence. Automatic detection of biological samples has been largely embraced in forensic applications to meet the increasing throughput requirements. However, the amount of DNA automatically retrieved from trace evidence often tends to be small and unstable, ultimately resulting in poor detection of DNA profiles. Thus, in this work, we introduced a robust DNA extraction and purification platform named Bionewtech® BN3200 (Bionewtech®, Shanghai, China) with the goal of constructing a rapid automatic detection system for trace DNA. The establishment of automatic detection system for trace DNA mainly encompassed two parts: assessing the sensitivity of automatic extraction platform and screening the optimal short tandem repeat (STR) typing kit. The sensitivity of Bionewtech® BN3200 platform based on Ultra-sensitive DNA Extraction kit was initially estimated, demonstrating that this extraction platform might contain large potential in the trace DNA extraction. For the amplification part, three sets of commercial multiplex STR typing kits were selected as candidates, and the amplified products were further genotyped on the Applied Biosystems 3500xl Genetic Analyzer. After comparation, SiFa™ 23 Plex Kit was determined as the most suitable amplification system for trace DNA. Eventually, the newly exploited trace DNA detection system was successfully implemented in the detection of fingerprints derived from glass surfaces with the five-seconds contact time. As a result, the DNA recovered from the fingerprints fluctuated approximately from 57.60 pg to 18.05 ng, in addition, over 70% of the total STR loci were detected in 75% of the fingerprint samples.

Development of a standardized multiplex SSR kit for genotyping both goats and sheep

In this technical note, we report the development of a reliable standardized protocol based on the multiplex SSR-PCR approach for genotyping the two small ruminant species, goats and sheep. The method is based on PCR conditions optimization allowing 12 informative microsatellites (SSR) markers to be co-amplified in two multiplex sets of 6 SSR markers, followed by a co-migration into one run onto a genetic analyzer. The performance of the assay was tested on a total of 207 goats and 197 sheep, originating from different breeds and localities sampled mainly in Benin, but also to lesser extent in Belgium. The optimized assay showed that the 12 selected markers were suited to amplify all the loci distributed between the two species with valuable genetic information, specific-species allelic patterns and similar polymorphism information content. The validated loci were polymorphic with high allele numbers for the two species, ranging from 6 to 21 for goats and 7 to 15 for sheep, with average 10.58 alleles per locus for goats and 11.33 for sheep, respectively. The Polymorphic Information Content (PIC) of selected markers averaged a value of 0.712 for goats and 0.751 for sheep. The differentiation of SSR alleles at an intraspecific resolution level allowed different heterotic groups to be detected, such as geographical or breeding groups. The newly developed genotyping kit could be successfully applied in diverse genetic variability studies and relatedness analyses, as well as for population structure assessment, parentage determination and traceability concerns, within and among sheep and goat species.

Validation and forensic application of a new 19 X-STR loci multiplex system.

The Microreader™ 19X Direct ID System was a newly developed multiplex PCR kit, which could detect 19 X-chromosomal STR loci (DXS6795, DXS9907, DXS6803, GATA172D05, DXS6807, GATA31E08, DXS7423, DXS6810, DXS101, DXS9902, DXS7133, DXS6800, DXS981, DXS10162, DXS6809, DXS10135, HPRTB, GATA165B12, DXS10079) and the sex determination locus of AMEL simultaneously. Different from other X-STR multiplex PCR kits, no linkage groups are included in this system, so the likelihood ratios could be calculated without the consideration of linkage groups. In this study, PCR conditions, sensitivity, species specificity, stability, DNA mixtures, concordance, stutter, sizing precision and population studies were conducted according to the SWGDAM developmental validation guidelines. The results indicated that this new X-STRs multiplex system was an efficient and reliable detection system, which could facilitate human kinship analysis and identification testing, as a powerful supplementary to autosomal STR kits.

A Ge.F.I. – ISFG European collaborative study on DNA identification of Cannabis sativa samples using a 13-locus multiplex STR method

Cannabis sativa is the most used controlled substance in Europe. With the advent of new and less restrictive European laws on cannabis sale for recreational use (including in Italy), an increase in indoor cannabis crops were observed. This increase was possible due to the availability of cannabis seeds through the internet market. Genetic identification of cannabis can link seizures and if in possession then might aid in an investigation. A 13-locus multiplex STR method was previously developed and validated by Houston et al. A collaborative exercise was organized by the Italian Forensic Geneticists – International Society of Forensic Genetics (Ge.F.I. – ISFG) Working Group with the aim to test the reproducibility, reliability and robustness of this multiplex cannabis STR kit. Twenty-one laboratories from three European countries participated in the collaborative exercise and were asked to perform STR typing of two cannabis samples. Cannabis DNA samples and the multiplex STR kit were provided by the University of Barcelona and Sam Houston State University. Different platforms for PCR amplification, capillary electrophoresis (CE) and genotyping software were selected at the discretion of the participating laboratories. Although the participating laboratories used different PCR equipment, CE platforms and genotyping software, concordant results were obtained from the majority of the samples. The overall genotyping success ratio was 96%. Only minor artifacts were observed. The mean peak height ratio was estimated to be 76.3% and 78.1% for sample 1 and sample 2, respectively. The lowest amount of −1 / + 1 stutter percentage produced, when the height of the parent allele was higher than 8000 RFU, resulted to be less than 10% of the parent allele height. Few common issues were observed such as a minor peak imbalance in some heterozygous loci, some artifact peaks and few instances of allelic drop-out. The results of this collaborative exercise demonstrated the robustness and applicability of the 13-locus system for cannabis DNA profiling for forensic purposes.

Clinical decision making is improved by BioFire Pneumonia Plus in suspected lower respiratory tract infection after lung transplantation: Results of the prospective DBATE-IT study.

Lower respiratory tract infections (LRTIs) are a significant cause of morbidity and mortality in lung transplant (LTx) recipients. Timely and precise pathogen detection is vital to successful treatment. Multiplex PCR kits with short turnover times like the BioFire Pneumonia Plus (BFPPp) (manufactured by bioMérieux) may be a valuable addition to conventional tests. We performed a prospective observational cohort study in 60 LTx recipients with suspected LRTI. All patients received BFPPp testing of bronchoalveolar lavage fluid in addition to conventional tests including microbiological cultures and conventional diagnostics for respiratory viruses. Primary outcome was time-to-test-result; secondary outcomes included time-to-clinical-decision and BFPPp test accuracy compared to conventional tests. BFPPp provided results faster than conventional tests (2.3h [2-2.8] vs. 23.4h [21-62], p<0.001), allowing for faster clinical decisions (2.8 [2.2-44] vs. virology 28.1h [23.1-70.6] and microbiology 32.6h [4.6-70.9], both p<0.001). Based on all available diagnostic modalities, 26 (43%) patients were diagnosed with viral LRTI, nine (15 %) with non-viral LRTI, and five (8 %) with combined viral and non-viral LRTI. These diagnoses were established by BFPPp in 92%, 78%, and 100%, respectively. The remaining 20 patients (33 %) received a diagnosis other than LRTI. Preliminary therapies based on BFPPp results were upheld in 90% of cases. There were six treatment modifications based on pathogen-isolation by conventional testing missed by BFPPp, including three due to fungal pathogens not covered by the BFPPp. BFPPp offered faster test results compared to conventional tests with good concordance. The absence of fungal pathogens from the panel is a potential weakness in a severely immunosuppressed population.

Clinical performance of four multiplex real-time PCR kits detecting urogenital and sexually transmitted pathogens

ObjectivesWe evaluated the clinical performances of four multiplex real-time PCR commercial kits for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Trichomonas vaginalis: the STI PLUS ELITe MGB kit (ELITechGroup), N. gonorrhoeae/C. trachomatis/M. genitalium/T.vaginalis Real-TM kit (Sacace Biotechnologies), Allplex STI Essential kit (Seegene), and FTD Urethritis Plus kit (Fast-Track Diagnostics). MethodsThe kit performance for C. trachomatis, N. gonorrhoeae, M. genitalium and T. vaginalis detection was compared to that of the cobas CT/NG and TV/MG kits (Roche Diagnostics) using 425 samples, mainly urine and cervicovaginal, throat and rectal swabs. Detection of Ureaplasma parvum, U. urealyticum and Mycoplasma hominis were compared to that of in-house TaqMan PCRs. ResultsThe four kits showed good performances for the detection of C. trachomatis. They all presented a low positive agreement for the detection of M. genitalium and T. vaginalis (ranges 63.3–74.1% and 51.2–68.4%, respectively) compared to the cobas MG/TV kit. The Seegene and Sacace kits showed additional low positive agreement for the detection of N. gonorrhoeae (71.2%, 95%CI 61.8–79.0 and 63.1%, 95%CI 53.5–71.8, respectively). We observed a slight but significant lower negative agreement for N. gonorrhoeae detection using the ELITechGroup kit (92.5%, 89.1–94.9) and for M. genitalium detection using the Fast-Track kit (93.2%, 89.6–95.7) compared to other kits. ConclusionMultiplex real-time PCR kits are convenient methods for the detection of several pathogens associated with sexually transmitted infections (STIs) in a single step, but colonizing Ureaplasma spp. and M. hominis species should not be included in these kits. Users should be aware of the weak performance of some kits for the detection of M. genitalium and T. vaginalis.

Comparison of the PowerChek SARS-CoV-2, Influenza A&B, RSV Multiplex Real-time PCR Kit and BioFire Respiratory Panel 2.1 for simultaneous detection of SARS-CoV-2, influenza A and B, and respiratory syncytial virus

The potential co-circulation of SARS-CoV-2, influenza, and respiratory syncytial virus (RSV) could pose an unprecedented challenge to healthcare systems worldwide. Here, we compared the performance of the PowerChek SARS-CoV-2, Influenza A&B, RSV Multiplex Real-time PCR Kit (PowerChek) for simultaneous detection of SARS-CoV-2, influenza A and B, and respiratory syncytial virus with that of BioFire Respiratory Panel 2.1 (RP2.1) using 175 nasopharyngeal swab (NPS) specimens. Positive percent agreement and negative percent agreement of the PowerChek assay compared to RP2.1 were as follows: 100 % (40/40) and 100 % (135/135) for SARS-CoV-2; 100 % (39/39) and 100 % (136/136) for influenza A; 100 % (35/35) and 100 % (140/140) for influenza B; and 93.1 % (27/29) and 100 % (146/146) for RSV, respectively. The limit of detection (LOD) was accessed using RNA standards for each virus, and the LOD values of the PowerChek assay for SARS-CoV-2, influenza A and B, and RSV were 0.36, 1.24, 0.09, and 0.63 copies/μL, respectively. Our results demonstrate that the PowerChek assay is sensitive and accurate for detection of SARS-CoV-2, influenza A and B, and RSV, suggesting that this assay can be a valuable diagnostic tool when SARS-CoV-2, influenza, and RSV are co-circulating.

Distribution of clinical isolates obtained from sterile body fluids: a four-year retrospective data

Aim: Infections of sterile body fluids (SBFs) require rapid and accurate diagnosis and treatment, since their morbidities and mortalities are high. To achieve this goal, definite epidemiologic data is absolutely required, since empiric and preemptive treatments are mainly based on this. The aim of this study was to evaluate infectious agents isolated from SBFs, susceptibility results and molecular analysis (PCR) data, retrospectively.&#x0D; Material and Method: Clinical samples of SBFs (Cerebrospinal, pleural, peritoneal, pericardial and synovial fluids) obtained from January 2017 to December 2020 in Atatürk City Hospital (tertiary center) were included. Identification of bacterial and fungal agents and antibiotic susceptibility were done by conventional and automated system (BD Phoenix™, Becton Dickinson Co., Sparks, MD, USA). Löwenstein-Jensen media and BACTEC Mycobacteria Growth Indicator Tube 960 (Becton Dickinson Co., Sparks, MD, USA) were used for mycobacterial analysis. Bosphore Viral Meningitis Panel Multiplex PCR Kit (Anatolia Geneworks, İstanbul, Turkey) were applied to detect HSV-1, HSV-2, VZV, Enterovirus and/or Parechovirus.&#x0D; Results: A total of 221 (9.74%) organisms were detected among 2269 samples. Particularly common gram negative bacterial agents covered the top of the list (Escherichia coli, Pseudomonas spp., Klebsiella spp. and Acinetobacter baumannii-Acinetobacter calcoaceticus complex). Staphylococcus aureus was the most frequent gram positive strain, followed by enteroccocci. Most of the A. baumannii isolates were multidrug resistant, Pseudomonas spp. showed over than 20% resistance rate to ceftazidime, cefepime and piperacillin-tazobactam. All enterococci were vancomycin-susceptible, one S. aureus strain was methicillin-resistant. All Mycobacterium tuberculosis complex isolates were found to be susceptible to first-line anti-tuberculosis drugs.&#x0D; Conclusions: Continuous laboratory surveillance even in local phase is important to guide clinicians. Even though our data did not show significant changes, improvements on laboratory capabilities and clinical awareness must be done. Isolation rates might be underestimated due to requirement of improvements in our laboratory, especially about sampling, anaerobe transportation and usage of blood culture vials.