Routine Diagnostic Laboratory Research Articles

Low-cost conventional PCR techniques enable simultaneous detection of bacterial sexually transmitted infections with enhanced sensitivity and specificity

PurposeNeisseria gonorrhoeae (NG), Chlamydia trachomatis (CT) and Mycoplasma hominis (MH), the three most common treatable bacterial sexually transmitted infections (STIs) worldwide can lead to many complications if remain untreated. Screening of high-risk population with highly sensitive methods will lead to significant improvement in patient outcomes and will prevent downward transmission. The advantages of Polymerase chain reaction (PCR) based assay are not only high sensitivity and specificity, but also detection of multiple organisms in a single reaction which reduce the result turn-around time. The aim of the present study was to evaluate the feasibility of a multiplex PCR assay method targeting 16S rRNA gene for simultaneous detection of NG, CT and MH infection along with their trend and occurrence among high-risk population in Assam, Northeast India. MethodsA cross-sectional study was undertaken, where a total of 200 randomly selected patients from high-risk population were included. After validation of singleplex PCR, Multiplex PCR (M-PCR) was performed along with the traditional culture method for NG. Results & conclusionThe overall agreement of M-PCR with singleplex PCR was very high (100%). The occurrence of STI was found to be very high (101/200; 50.5%). Furthermore, co-infection was detected in 10/200; 5%) individuals. Infection was more common among young individuals (p < 0.05) and males out-numbered females (p < 0.05). The most common organism detected was CT (42/200; 21%) followed by NG (41/200; 20.5%) and MH (20/200; 10%). The M-PCR assay workflow is simple, cost effective and can be used in routine diagnostic laboratories with basic molecular facilities.

Potential application of body fluids autofluorescence in the non-invasive diagnosis of endometrial cancer.

Endometrial carcinoma (EC) is the most common cancer of the female reproductive tract in developed countries. The prognosis and 5-year survival rates are closely tied to the stage diagnosis. Current routine diagnostic methods of EC are either lacking specificity or are uncomfortable, invasive and painful for the patient. As of now, the gold diagnostic standard is endometrial biopsy. Early and non-invasive diagnosis of EC requires the identification of new biomarkers of disease and a screening test applicable to routine laboratory diagnostics. The application of untargeted metabolomics combined with artificial intelligence and biostatistics tools has the potential to qualitatively and quantitatively represent the metabolome, but its introduction into routine diagnostics is currently unrealistic due to the financial, time and interpretation challenges. Fluorescence spectral analysis of body fluids utilizes autofluorescence of certain metabolites to define the composition of the metabolome under physiological conditions. This review highlights the potential of fluorescence spectroscopy in the early detection of EC. Data obtained by three-dimensional fluorescence spectroscopy define the quantitative and qualitative composition of the complex fluorescent metabolome and are useful for identifying biochemical metabolic changes associated with endometrial carcinogenesis. Autofluorescence of biological fluids has the prospect of providing new molecular markers of EC. By integrating machine learning and artificial intelligence algorithms in the data analysis of the fluorescent metabolome, this technique has great potential to be implemented in routine laboratory diagnostics.

EpiDiP/NanoDiP: a versatile unsupervised machine learning edge computing platform for epigenomic tumour diagnostics

DNA methylation analysis based on supervised machine learning algorithms with static reference data, allowing diagnostic tumour typing with unprecedented precision, has quickly become a new standard of care. Whereas genome-wide diagnostic methylation profiling is mostly performed on microarrays, an increasing number of institutions additionally employ nanopore sequencing as a faster alternative. In addition, methylation-specific parallel sequencing can generate methylation and genomic copy number data. Given these diverse approaches to methylation profiling, to date, there is no single tool that allows (1) classification and interpretation of microarray, nanopore and parallel sequencing data, (2) direct control of nanopore sequencers, and (3) the integration of microarray-based methylation reference data. Furthermore, no software capable of entirely running in routine diagnostic laboratory environments lacking high-performance computing and network infrastructure exists. To overcome these shortcomings, we present EpiDiP/NanoDiP as an open-source DNA methylation and copy number profiling suite, which has been benchmarked against an established supervised machine learning approach using in-house routine diagnostics data obtained between 2019 and 2021. Running locally on portable, cost- and energy-saving system-on-chip as well as gpGPU-augmented edge computing devices, NanoDiP works in offline mode, ensuring data privacy. It does not require the rigid training data annotation of supervised approaches. Furthermore, NanoDiP is the core of our public, free-of-charge EpiDiP web service which enables comparative methylation data analysis against an extensive reference data collection. We envision this versatile platform as a useful resource not only for neuropathologists and surgical pathologists but also for the tumour epigenetics research community. In daily diagnostic routine, analysis of native, unfixed biopsies by NanoDiP delivers molecular tumour classification in an intraoperative time frame.

Preliminary study on buffy coat smear and molecular detection of microfilaria in domestic chickens (Gallus gallus domesticus) raised in Southern Thailand

Background and Aim: Filarial nematode typically produces a larval stage (microfilariae) in the bloodstream of vertebrate hosts, where microfilariae reside in the blood or subcutaneous tissues. Filarial nematodes cause human diseases, such as river blindness and elephantiasis, which are widely studied. However, in avian species, they are overlooked because they are nonpathogenic. In Thailand, microfilaria can be found in wild birds and domestic chickens. Recently, an increase in the number of blood samples submitted to veterinary diagnostic laboratories may have increased the number of microfilariae. Therefore, knowledge about filarial species and reliable detection methods are important. Therefore, this study aimed to investigate the efficacy of buffy coat smear and polymerase chain reaction (PCR)-based methods for the detection of microfilaria in domestic chickens. In addition, parasites were identified using the sequence of the cytochrome c oxidase subunit 1 (COX1) gene. Materials and Methods: Giemsa-stained buffy coat smears from a previous study were reanalyzed. These available buffy coat smears were prepared from 55 domestic chickens raised as backyard free-ranging in Southern Thailand. Fifty-seven frozen genomic DNA extracted from chicken blood were used to detect the presence of the COX1 gene in Onchocercidae nematodes. The nested PCR protocol for amplification of the OnchoCOI_ R2-OnchoCOI_ R2 fragment of the COX1 gene was applied from a previous report. Sequences of COX1 were analyzed to identify Onchocercidae nematodes and if they were single or mixed infections. We constructed Bayesian phylogenetics to identify parasites and assessment of the relationship between filarial nematodes in avian species and other vertebrate hosts. Results: Buffy coat smears from 15 samples revealed microfilaria. Of these 15 samples, only eight were positive for COX1 nested-PCR amplification. The other two buffy coat-negative samples were also positive for nested-PCR. Sequencing of these 11 nested PCR-positive samples revealed that almost all of them were Onchocercidae nematodes. Bayesian phylogenetic analysis showed that chicken Onchocercidae spp. were grouped with other avian filarial nematodes. However, all chickens Onchocercidae spp. showed a double peak in the sequencing chromatogram, indicating mixed filarial infection (species or haplotypes). Therefore, no chicken Onchocercidae sequence was deposited on National Center for Biotechnology Information, GenBank. Conclusion: Giemsa-stained buffy coat smear was a reliable method for the detection of chicken microfilaria in routine veterinary diagnostic laboratories. Development of a new PCR-based method is necessary. This method may provide greater sensitivity and specificity of detection. In addition, the PCR method allowed us to access the genetic characteristics of nematodes, which helped us maximize our knowledge of nematodes. Further investigations, such as the pathogenicity of filarial nematodes in chickens and their potential vectors, are required. Keywords: Buffy coat smear, Chickens, Cytochrome c oxidase I, Microfilaria, Nested-polymerase chain reaction.

Development of an agar-based screening method for terbinafine, itraconazole, and amorolfine susceptibility testing of Trichophyton spp.

Resistance in dermatophytes is an emerging global public health issue. We, therefore, developed an agar-based method for screening Trichophyton spp. susceptibility to terbinafine (TRB), itraconazole (ITC), and amorolfine (AMF) and validated it using molecularly characterized isolates. Α total of 40 Trichophyton spp. isolates, 28 TRB wild type (WT) (13 T. rubrum, 10 T. mentagrophytes, 5 T. interdigitale) and 12 TRB non-WT (7 T. rubrum, 5 T. indotineae) with different alterations in the squalene epoxidase (SQLE) gene, were used. The optimal test conditions (inoculum and drug concentrations, incubation time, and temperature) and stability over time were evaluated. The method was then applied for 86 WT Trichophyton spp. clinical isolates (68 T. rubrum, 7 T. interdigitale, 6 T. tonsurans, 5 T. mentagrophytes) and 4 non-WT T. indotineae. Optimal growth of drug-free controls was observed using an inoculum of 20 µL 0.5 McFarland after 5-7 days of incubation at 30°C. The optimal concentrations that prevented the growth of WT isolates were 0.016 mg/L of TRB, 1 mg/L of ITC, and 0.25 mg/L of AMF, whereas 0.125 mg/L of TRB was used for the detection of Trichophyton strong SQLE mutants (MIC ≥0.25 mg/L). The agar plates were stable up to 4 months. Inter-observer and inter-experimental agreement were 100%, and the method successfully detected TRB non-WT Trichophyton spp. strains showing 100% agreement with the reference EUCAST methodology. An agar-based method was developed for screening Trichophyton spp. in order to detect TRB non-WT weak and strong mutant isolates facilitating their detection in non-expert routine diagnostic laboratories.

Fatal Lodderomyces elongisporus Fungaemia in COVID-19 Patient

Lodderomyces elongisporus, an ascomycetous yeast, is often misidentified as Candida parapsilosis due to its physiologic similarity. Compared to Candida parapsilosis complex, L. elongisporus is still inferior in regard to incidence and virulence. We report a case of L. elongisporus fungaemia in a patient with diabetic ketoacidosis (DKA) precipitated by COVID-19 category 5 infection. A 52-year-old diabetic and hypertensive lady was brought in for fever, cough and lethargy for four days, with sudden onset of dyspnoea. Upon arrival, she was febrile, and in metabolic ketoacidosis. Her lung auscultation was clear. SARS-CoV-2 RNA was detected via real-time polymerase chain reaction (RT-PCR). Her blood culture grew L. elongisporus, identified via matrix-assisted laser desorption/ionization time of flight (MALDI-TOF). Although treated with intravenous (IV) amphotericin B for four days, she succumbed on the sixth day of admission. Accurate identification of this yeast, especially in differentiating it with Candida parapsilosis complex, remains a diagnostic challenge for routine diagnostic laboratories. MALDI-TOF offers a reliable alternative for accurate and prompt diagnosis. Co-infections with COVID-19 have never been recorded worldwide. We highlight the first case of L. elongisporus isolation co-existing with SARS-CoV-2 infection during this pandemic.

Can C-reactive proteinbe used as a surrogate marker of IL-6 in a broad array of clinical entities?

Background: C-reactive protein (CRP) is commonly performed, whereascytokine testing is limited to research. Aims: To determine CRP correlation to cytokinesIL-6, IL-1β and TNF-α. Results:Consecutive samples (n =307)were collected over 24 h. Ninety-six patients (31%) had acute infections, and 23 patients (7.5%) had autoimmune or inflammatory disease presentations. A strong correlation between CRP and two IL-6 assays (r=0.74 and r=0.71; p<0.001) was present. CRP did not correlate with IL-1β and TNF-α across the data set. Bacterial infection had a significantly higher CRP and IL-6 (p<0.001), while only CRP was elevated in inflammatory and autoimmune diseases (p<0.001). Discussion: CRP may be used as a surrogate marker of IL-6 levels in the routine diagnostic laboratories.

Utility of Flowcytometry in the Diagnosis of Myelodysplasia in Routine Laboratories

INTRODUCTION Myelodysplastic syndromes (MDSs) cover a wide range of abnormalities, ranging from subtle morphological changes to overt findings such as prominently skewed morphology. The clinical picture is dominated by cytopenia of one or more lineages, defined by Haemoglobin&amp;lt;100g/L, absolute neutrophil count &amp;lt;1.8 x 10^9/L and platelets less than 100 x 10^9/L. The aberrant features, suggestive of MDS that can be picked up by flow cytometry can occasionally be missed on morphological features. In the presence of clonal haematopoiesis, these features detected by flow cytometry may help us to confidentially apply a label of MDS to an individual. MATERIALS AND METHODS All patients with suspected myelodysplastic syndrome and patients with acute myeloid leukemia (AML) &amp;lt;30% blasts. Profound dominance of myeloid blasts in a patient with AML automatically earns the patient the 2 points needed for the MDS label and the myeloblast dominance inevitably outnumbers B-cell progenitors, which will affect the Ogata scoring.Exclusion criteria: &amp;gt;2% monoclonal lymphocytes by flow cytometry&amp;lt;400 CD34+ events in the collected sample Parameters included: Ogata A simple 4-point scoring system by Ogata. Other features included as per European Leukemia Net guidelines on flow cytometry on MDS We included following nine parameters to be analysed in addition to the Ogata criteria: Myeloid progenitors: Increased expression of CD11bAbnormal expression of CD7 in progenitor cells (CD34/CD117 Positive cells) Monocytic Lineage: Decreased expression of CD11bDecreased expression of HLA-DRDecreased expression of CD14 Myeloid Lineage: Diminished expression of CD45Decreased expression of CD11bDecreased expression of CD13 or CD33 (Any one of them)Decreased expression of CD10 RESULTS In our analysis of normal patients, Ogata score was frequently positive in the category lymphocyte/ myeloblast CD45 ratio. This obviously may affect the sensitivity. In the series of 20 patients we analysed, we had only one patient with combined score of four or less (patient no: 12). Two patients with Ogata score of less than two fulfilled the flow cytometric criteria using the combined criteria (Patient no: 3 and 5). Since our numbers were low, we do not know whether the combined scoring system would gain more significance in a large cohort of patients. In one of the normal patients who presented with cytopenia with high MCV (patient no: 10) the morphological and flow cytometry features were not consistent with myelodysplasia. Moreover, the patient did not have clonal abnormalities. This patient could have classified as myelodysplasia by flow cytomeric criteria if Ogata score alone had been used. The sensitivity of the combined parameters in detecting myelodysplasia is 89.4% and specificity is 90.9%. CONCLUSION We devised this system based on the principles that, the Ogata scoring system is simple and reproducible. Moreover, its sensitivity can be improved by using nine additional markers based on WELLS criteria, which can be analysed based on mean fluorescence intensity (MFI) alone without much complexity. Our methodology was not demanding and it did not involve usage of special fluorescent dyes and hence can be reproduced in any routine diagnostic laboratories. In addition, we found that both sensitivity and specificity of Ogata scoring system has been improved by inclusion of the other parameters in this study. Our scoring system did not show any correlation with reference to the R-IPSS (Revised International Scoring System in myelodysplasia) at diagnosis. Many attempts have been made so far with extensive markers and application of Automated Intelligence strategies for the analysis of more complex combinations of parameters. However, a simplified, user friendly and cost effective method is the need of the hour.

Novel Biomarkers for Assessing Clinical Severity in Hereditary Spherocytosis - Application of Routine and Advanced Diagnostic Tests

Background: Hereditary spherocytosis (HS) is caused by pathogenic variants in genes encoding red blood cell (RBC) cytoskeletal and (trans)membrane proteins. This ultimately leads to instability of the RBC membrane and subsequent membrane loss, resulting in the transformation of the biconcave RBC into a poorly deformable spherocyte, which is susceptible to premature splenic clearance. The diagnosis of HS has substantially improved over the last years and new advanced techniques have been established, including osmotic gradient ektacytometry, eosin-5'-maleimide (EMA) binding test and next-generation sequencing (NGS). However, there is little knowledge on the applicability of these diagnostic outcome parameters as biomarkers for clinical severity in HS. Aim: To explore possible correlations between established and novel diagnostic outcome parameters and clinical severity in a well-defined cohort of HS patients. Methods: This monocenter retrospective cohort study involves patients diagnosed with HS upon referral to our department between 2012 and 2023. Clinical characteristics, routine and advanced diagnostic laboratory parameters, and genotype were evaluated. Clinical severity was classified as previously described, based on parameters such as hemoglobin, reticulocyte percentage and bilirubin 1. The following laboratory tests were included: routine hematological parameters (Cel-Dyn Sapphire or Alinity hq), EMA binding test, osmotic fragility test (OFT) and osmotic gradient ektacytometry (osmoscan, Lorrca MaxSis). The latter technique was also explored for other parameters than the commonly used EI max, O hyper and O min. NGS gene panel analysis was used for molecular diagnosis. Differences between groups were determined using either Chi-squared test, unpaired t-test or Mann-Whitney U test and correlations using point-biserial, Pearson's or Spearman's test via GraphPad Prism. Results: Eighty-two non-splenectomized patients were included. Thirty-five of these (42.7%) were classified as mild, and forty-seven as moderate-severe (57.3%)). Demographic characteristics nor genotype differed significantly, albeit SPTB variants occurred more frequently in the moderate-severe group (15 vs. 4 in the mild group). Expectedly, both reticulocyte count and EI max correlated with clinical severity. RBC distribution width (RDW) correlated moderately, suggesting greater RBC heterogeneity (r=0.606, p&amp;lt;0.0001) is associated with a more severe clinical phenotype. Similarly, EMA binding test results correlated with clinical severity (r=0.390, p&amp;lt;0.001), indicating more pronounced membrane loss in clinically more severe patients. We further evaluated three novel osmoscan parameters ( Figure 1): EI O290, the elongation index (EI) at physiologic isotonicity, which correlated with clinical severity (r=-0.500, p&amp;lt;0.0001), and also with EI max (r=0.856, p&amp;lt;0.0001). The O min-width and O max-width, defined as the difference in osmolality between the intersection points of y=EI min or EI max +/- 0.05 and the osmoscan curve. Thisassesses the effect of changes in osmolality on EI min or EI max, respectively. O min-width correlated with RDW (r-0.439, p&amp;lt;0.0001), OFT results (N=30, r=0.671, p&amp;lt;0.0001) and clinical severity (r=0.390, p&amp;lt;0.001). O max-width correlated with clinical severity (r=-0.375, p&amp;lt;0.001) and strongly with percentage of hyperchromic cells (N=76, r=-0.751, p&amp;lt;0.0001), yet weakly with RDW (-0.273, p&amp;lt;0.05). All significant correlations with clinical severity are displayed in Figure 2. Conclusion: In this study, a number of routine and advanceddiagnostic laboratory parameters were found to correlate with clinical severity in HS. O min-width and O max-width are novel osmoscan-derived biomarkers that correlated with RDW, hyperchromic cells (O max-width), OFT results (O min-width) and clinical severity. EI O290, another novel osmoscan-derived biomarker, also correlated with clinical severity, although not stronger than EI max and AUC. The biomarkers identified in this study, together with patient-reported outcome measures (PROMs), could be used to revise the previously established severity classification system and thereby aid in stratifying clinical severity and subsequent clinical decision-making.

Performance evaluation of fully automated cobas® 6800 CMV PCR for the detection and quantification of cytomegalovirus DNA in neonatal urine and saliva, and adult urine, saliva, and vaginal secretion.

Laboratory testing for cytomegalovirus (CMV) in bodily fluids is essential to manage congenital and prenatal CMV infection. The rapid and fully automated cobas® CMV PCR is approved only for the testing of plasma in transplant patients. To evaluate the performance of the cobas® CMV to detect and quantify CMV DNA in neonatal and adult female urine, saliva, and vaginal secretion, the limit of detection (LoD), limit of quantification (LoQ), imprecision, linearity, PCR efficiency, bias, analytical specificity, cross-reactivity, and cross-contamination of the cobas® CMV for urine, saliva, and vaginal secretion was determined. The performance of the assay was evaluated prospectively with two laboratory-developed PCR assays using neonatal and adult urine, saliva swabs, and vaginal swabs. The LoD and LoQ were 31 and 100 IU/mL, respectively, for urine, and 81 and 100 IU/mL, respectively, for vaginal secretion. The LoD and LoQ for saliva were the same (200 IU/mL). The cobas® CMV was precise (coefficient of variation ≤10%), linear (R2 ≥ 0.995), and efficient (1.07 and 1.09) between 100 and 250,000 IU/mL for the sample types. The bias and analytical specificity was <±0.30 log10 IU/mL and 100%, respectively. Cross-reactivity with non-CMV pathogens was not detected. Cross-contamination rate was 0.28%. The diagnostic accuracy, sensitivity, and specificity of the cobas® CMV for neonatal urine and saliva were ≥95.0%, ≥93.3%, and ≥90.4%, respectively. The overall percent agreement for adult urine, saliva, and vaginal secretion was 86.6%, 94.5%, and 89.4%, respectively. Taken together, the cobas® CMV demonstrated acceptable analytical and diagnostic performance, and is suitable for routine diagnostic laboratory investigation of CMV infection in neonates and adults.

Prevalence of Markers of Certain Blood-Borne Viral Infections in Pregnant Women and Their Partners in the Republic of Guinea

The aim of the work was to estimate the prevalence of HIV, HBV and HCV markers among pregnant women and their male partners in the Republic of Guinea.Materials and methods. The material of the study was blood plasma samples from 140 pregnant women living in Kindia prefecture and N’Zerekore prefecture, as well as 60 male partners who reported sexual contact with HIV-infected persons. The samples were examined for the presence of serological (HBsAg, HBeAg, antibodies anti-HBs IgG, anti-HBcore IgG, anti-HBe IgG, anti-HCV IgG, Ag/Ab-HIV) and molecular (HBV DNA, HCV RNA, HIV RNA) markers.Results and discussion. The age of the examined pregnant women ranged from 13 to 55 years and was on average (26.29±9.67) years. The age of men varied from 15 to 60 years, on average – (29.05±11.99) years. When assessing the prevalence of serological markers, antibodies to HCV were detected in 2.14 % cases in women and in 3.33 % cases in men. Antibodies to HIV were found in 6.43 % and 6.67 % women and men, respectively. Serological markers associated with HBV were detected in 80.71 % (HBsAg – 13.57 %) of women and 81.67 % (15 %) of men. In the pregnant women, HCV RNA was not detected, HIV RNA was revealed in 1 case, HBV DNA was identified in 26 cases (18.57 %), including 5 % HBsAg-negative hepatitis B cases. In the men group, HCV RNA and HIV RNA were detected in 3.33 % and 6.67 % cases, respectively. HBV DNA was determined in 16.67 % of men, including latent hepatitis B in one person. A significantly higher incidence of HIV in men compared to women is shown (χ2=3.907 at p&lt;0.05). The relative risk of HIV infection in men is nine times higher than in women: RR=9.333; p=0.0291; 95 % CI: 1.065–81.815 %. Four out of five identified HIV infection cases were co-infected with HBV and/or HCV. There is an obvious need to introduce screening for HIV, HCV, HBV, including latent hepatitis B, into routine laboratory diagnostics during examination of pregnant women and their partners, followed by couples counseling and vaccination against hepatitis B virus.

Rapid identification of Salmonella serovars Enteritidis and Typhimurium using whole cell matrix assisted laser desorption ionization – Time of flight mass spectrometry (MALDI-TOF MS) coupled with multivariate analysis and artificial intelligence

Salmonella is a common food-borne pathogen with Enteritidis and Typhimurium being among the most important serovars causing numerous outbreaks. A rapid method was investigated to identify these serovars using whole-cell MALDI-TOF MS coupled with multivariate analysis and artificial intelligence and 113 Salmonella strains, including 38 Enteritidis (SE), 38 Typhimurium (ST) and 37 strains from 32 other Salmonella serovars (SG). Datasets of ions (presence/absence) with high discriminative power were created using newly developed criteria and subject to multivariate analyses and eight artificial intelligence (AI) tools. Principal Component Analysis based on 55 or 88 selected ions separated SE, ST and SG without overlap on the first three principal components. Datasets were partitioned using five partitioning methods with 70% of samples for AI model training and 30% for validation. Of the eight AI models evaluated, high performance (HP) SVM and HP Neural were the top performers, identified three serovar groups 97% correctly on average (range 82%–100%) according to the validation results. Selection of serovar specific ions facilitated differentiation of serotypes using unsupervised model PCA and improved the accuracy of classification using AI significantly (p < 0.01). MALDI-TOF MS incorporated with advanced data processing and classification tools is a promising method to allow rapid identification of Salmonella serovars of concern in routine diagnostic laboratories.

Evaluation of six commercial and in-house phenotypic tests for detection of AmpC β-lactamases: is routine detection possible?

Phenotypic characterization of the prevalent AmpC β-lactamases in clinical isolates is essential for making informed empirical decisions and critical for strengthening antimicrobial stewardship programmes. This study focused on assessing six assays, two in-house and four commercial phenotypic tests for detection of AmpC, to study the feasibility of making its detection a routine diagnostic microbiology laboratory activity. A total of 116 non-duplicate Gram-negative bacteria that were resistant to third-generation cephalosporins and amoxicillin/clavulanic acid, and resistant or susceptible to piperacillin/tazobactam and carbapenems, were screened by cefoxitin discs for AmpC. These isolates were subjected to two in-house (AmpC Tris-EDTA and disc approximation) methods and four commercial tests: D69C AmpC Detection Set; D72C ESBL, AmpC & Carbapenemase Detection Set; combination disc test: ESBL + AmpC Screen Disc Kit; and AmpC MIC Test Strip for confirmation of AmpC production. Ten whole-genome-sequenced AmpC-confirmed Gram-negative isolates were used as positive controls and one as a negative control. The prevalence of AmpC β-lactamases was 16%. Escherichia coli was a major carrier of plasmid-mediated AmpC (26.5%), followed by Klebsiella pneumoniae (23.4%). Phenotypically, 61% of AmpCs were detected by Tris-EDTA (accuracy: 73.8%), 76% by disc approximation (accuracy: 89.2%), 75% by the D69C AmpC Detection Set (accuracy: 95.4%), 74% by the D72C AmpC, ESBL & Carbapenemase Detection Set (accuracy: 95.4%), 76% by the combination disc test (accuracy: 95.4%) and 63% by AmpC MIC Test Strip (accuracy: 87.7%). The sensitivity and specificity of D69C were 97.9% and 88.2%, respectively, and 95.9% and 93.8% for the combination disc test, while for the disc approximation test and D72C they were 93.9% and 75%, and 93.9% and 100%, respectively. Screening by cefoxitin screening was less sensitive (75%) and specific (25%). Disc approximation and the combination disc test detect AmpC in Enterobacterales and also Pseudomonas aeruginosa and Acinetobacter species. We recommend the in-house disc approximation test and the commercial D69C, as well as the combination disc test, as excellent tools for detection of AmpC. The cefoxitin test overcalls AmpC and cannot be considered a good stand-alone test for AmpC detection.

Introducing “Rapid phage susceptibility testing” (RPST): an accelerated lytic phage activity test for routine diagnostic laboratories within eight hours

Phage therapy, particularly for infections due to multi-drug-resistant bacteria, is increasingly gaining in importance. Consecutively, there is a rising need for phage testing in routine diagnostic laboratories. The incubation time of phage susceptibility testing for detecting lytic phage activity on phage/host strain combinations was evaluated. A standardized approach for routine diagnostic laboratories provided reliably detectable lysis zones within 8 hours.

Disc Test for Detecting Staphylococcus aureus Strains Producing Type A and Type C β-Lactamases.

Staphylococcus aureus can produce β-lactamases capable of hydrolyzing penicillins and first-generation cephalosporins. The propensity of type A and type C β-lactamase-producing S. aureus (TAPSA and TCPSA) to hydrolyze cefazolin at a high inoculum is termed the cefazolin inoculum effect (CIE). Strains with a CIE have a theoretical risk of causing treatment failure and are unable to be detected routinely by most laboratories. We developed a high-performing yet straightforward β-lactamase disc test that identifies and differentiates both TAPSA and TCPSA and is suitable for routine diagnostic laboratory workflows. Clinical isolates of S. aureus resistant to penicillin were identified, and their blaZ genes were sequenced. MICs were determined at low and high inocula (5 × 105 CFU/mL and 5 × 107 CFU/mL), and isolates demonstrating a CIE were characterized. A semimechanistic model was established to describe differential hydrolysis patterns, and candidate models were iteratively assessed using area-under-the-curve analysis from competitor receiver operating characteristic (ROC) curves. Biomarker thresholds were derived from Youdon index-derived optimal cutoff values. Genetic analysis of 99 isolates identified 26 TAPSA isolates and 45 TCPSA isolates. The model best differentiating TAPSA from non-TAPSA utilized cefazolin-to-cephalothin ratio analysis (sensitivity, 96.2%; specificity, 98.6%). The model best differentiating TCPSA from non-TCPSA incorporated cefazolin, cephalothin, and oxacillin (sensitivity, 88.6%; specificity, 96.6%). TAPSA and TCPSA can be differentiated using three antibiotic discs on a single agar plate. The test has potential value in typing the β-lactamase type from isolates from patients that are candidates for or have failed cefazolin therapy. IMPORTANCE The key significance of this article is that it details a straightforward method of performing a disc test that can differentiate Staphylococcus aureus isolates that are likely to be associated with a cefazolin inoculum effect and theoretical risk of cefazolin treatment failure from isolates that are less likely to be associated with a cefazolin inoculum effect.

Accurate molecular identification of different meat adulterations without carryover contaminations on a microarray chip PCR-directed microfluidic lateral flow strip device

Meat adulteration-based food fraud has recently become one of the global major economical, illegal, religious, and public health concerns. In this work, we developed a microarray chip polymerase chain reaction (PCR)-directed microfluidic lateral flow strip (LFS) device that facilitates the accurate and simultaneous identification of beef adulterated with chicken, duck, and pork, especially in processed beef products. To realize this goal, four pairs of amplification primers were designed and applied for specifically amplifying genomic DNA extracted from mixed meat powders in microarray chip. With the prominent advantage of this device lies in the flexible combination and integration of sample loading, detection, and reporting in microstructures, all the DNA amplicons can be individually visualized on the LFS unit, leading to the appearance of test lines (TC line, TD line, TP line, or TB line) as well as the control line (C line) for the species identification and quantification in beef products. Based on this new method, the adulterants were successfully distinguished and identified in mixtures down to 0.01% (wt.%) while the carryover aerogel contamination in routine molecular diagnostic laboratories was effectively avoided. The practicability, accuracy, and reliability of the device were further confirmed by using real-time PCR as a gold standard control on the successful identification of 50 processed ground meat samples sourced from local markets. The method and device proposed herein could be a useful tool for on-site identification of food authentication.

Brucella suis biovar 1 infection in a dog with orchitis in Germany.

In 2021, a case of canine brucellosis diagnosed in a dog with orchitis was presented to a veterinary practice in Germany. Serological testing excluded Brucella (B.) canis as a causative agent, but molecular analysis revealed the presence of B. suis biovar 1. Since biovar 1 is not endemic in Europe and the dog had no history of travel to endemic areas, a comprehensive epidemiological investigation was conducted using whole genome sequence data to determine the source of infection. We describe the clinical progress of the animal and the potential infection of a veterinary clinic employee. The findings highlight the importance of considering less common Brucella species as possible causes of canine brucellosis. The data also emphasize that it is quite challenging to identify Brucella species in a routine diagnostic laboratory and to conduct epidemiological investigations to unveil possible transmission routes.

Comparison of the Etest and Sensititre methods for anaerobe susceptibility testing

Background and Aims: Antimicrobial susceptibility testing of anaerobic clinical isolates is of paramount importance for patient therapy and resistance monitoring. In our laboratory the MIC gradient Etest method and broth microdilution with Sensititre trays are used for susceptibility testing of anaerobes and the aim of this study was to compare the two methods on a panel of anaerobes routinely isolated from patients in the province of Bolzano, Italy. Materials and Methods: Totally, 74 non-repetitive Gram-positive and Gram-negative patient isolates were tested with Etest strips on Fastidious Anaerobe Agar (F.A.A.) and with Sensititre trays, according to manufacturer’s instructions. Interpretation of MICs was by EUCAST or CLSI criteria, resistance percentages were calculated and Categorical Agreement (CA) and Essential Agreement (EA) between the two methods were determined. Results: Of the 74 isolates, 68 (91.9%) grew on both systems and agreement for these was compared in the study. CA for all isolates was ≥90% for all tested antibiotics except moxifloxacin, whereas EA was generally lower. Resistance was generally low, except for clindamycin in all isolates and tigecycline in Gram-negatives. In our study Etest was a superior and more handy method. Conclusions: To conclude, we believe the Etest method is more suitable for routine diagnostic laboratory usage. Nevertheless, multicenter studies are required to evaluate the two methods for anaerobic susceptibility testing.

ChatGPT, GPT-4, and Other Large Language Models: The Next Revolution for Clinical Microbiology?

ChatGPT, GPT-4, and Bard are highly advanced natural language process-based computer programs (chatbots) that simulate and process human conversation in written or spoken form. Recently released by the company OpenAI, ChatGPT was trained on billions of unknown text elements (tokens) and rapidly gained wide attention for its ability to respond to questions in an articulate manner across a wide range of knowledge domains. These potentially disruptive large language model (LLM) technologies have a broad range of conceivable applications in medicine and medical microbiology. In this opinion article, I describe how chatbot technologies work and discuss the strengths and weaknesses of ChatGPT, GPT-4, and other LLMs for applications in the routine diagnostic laboratory, focusing on various use cases for the pre- to post-analytical process.

Clinical Evaluation of Commercial HARDSON COVID-19 Antigen Rapid Test Kit for Routine COVID-19 Diagnosis.

This study aimed to evaluate the sensitivity, specificity, and accuracy of the commercial HARDSON COVID-19 Antigen Rapid Test Kit for diagnosing COVID-19 among the Iranian population by compared with the results of commercial RT-PCR. Two nasopharyngeal swabs were collected from each patient. One swab was tested with HARDSON COVID-19 Antigen Rapid Test Kit, and the second swab was placed in 3 mL of a virus-transmitted inactivated media for RT-PCR testing. Then, the results of both tests were compared to investigate the diagnostic accuracy of the rapid antigen test. A total of 275 suspected COVID-19 patients' samples were collected to investigate the diagnostic accuracy of HARDSON COVID-19 Antigen Rapid Test Kit. In this study, 162 positive and 113 negative samples were evaluated. As a result, the sensitivity, specificity, and accuracy of HARDSON COVID-19 Antigen Rapid Test Kit were 90%, 100%, and 94%, respectively. The diagnostic kit analyzed in this study indicated excellent specificity and a relatively good overall sensitivity for the diagnosis of COVID-19 when compared with the RT-PCR detection kit. Based on the result of this study, COVID-19 Antigen Rapid Test Kit indicated a good sensitivity (96%) in low cycle threshold (Ct) value, and it would be recommended to be integrated into routine diagnostic laboratories and used as an at-home rapid antigen test.