Laboratory Identification Research Articles

Optimized multilocus sequence analysis for laboratory identification of pathogens of ixodid tick-borne borreliosis

Introduction. The most common etiological agents of ixodid tick-borne borreliosis (ITBB) in Russia are Borrelia garinii, B. afzelii, B. bavariensis. Multilocus sequence typing and multilocus sequence analysis (MLSA) have been used in recent studies for Borrelia species identification. The results of using the MLSA scheme for identification of pathogens causing erythemic forms of ITBB have been presented earlier.
 The purpose of the study was to explore the possibility of MLSA optimization for laboratory identification of ITBB pathogens. Objectives: comparative analysis of nucleotide sequences of 6 conserved genes (rrs, hbb, fla, groEL, recA, ospA) and the rrfA-rrlB intergenic spacer, which are recommended by the MLSA protocol; identification of the minimum set of genes, the concatenated sequences of which are essential for species identification of Borrelia isolates.
 Materials and methods. The sequences of the above loci of 23 reference isolates collected from patients with ITBB and assigned, using MLSA, to B. bavariensis were compared with the sequences of similar genes of other Borrelia species available in international databases. The UPGMA method was used to build and analyze dendrograms based on the obtained data.
 Results. The sequences of ospA gene loci of reference species demonstrated the greatest difference (not less than 8.5%) from the sequences of the above gene in other analyzed species of Borrelia; approximately similar species-related differences (not less than 6.7%) were demonstrated by the comparison of recA gene sequences. The sequences of the identified variants of these two genes in B. bavariensis differed from the sequences of the similar genes in the most closely related species B. garinii. The dendrogram of the concatenated nucleotide sequences of recA and ospA genes demonstrated that it was totally consistent with the results of identification of the isolates based on the MLSA protocol.
 Conclusion. The optimized approach to MLSA of the B. burgdorferi sensu lato group suggests that species identification should be based on the concatenated analysis of loci of only two genes (recA and ospA) out of 7 loci recommended by the MLSA protocol.

Factors impacting the detection of weed seed contaminants in seed lots.

The setting and following of phytosanitary standards for weed seeds can lessen the impacts of weeds on agriculture. Standards adopted by seed companies, laboratories and regulators ensure the contamination rates do not exceed some thresholds. Globally sample size standards are set based on the amount needed to obtain a contaminant in a random sample of the seed lot, not detectability. New Zealand requires a 95% confidence that the maximum pest limit of 0.01% of quarantine weed seed contamination is not exceeded in an imported seed lot. We examined 24 samples each containing approximately 150 000 seeds of either perennial ryegrass (12 samples) or white clover seeds (12 samples) that were then spiked with seeds (contaminants) from 12 non-crop species (3-8 seeds of each). We considered factors that may impact detection rates: shape, color, size, and texture relative to the crop, and technician (including a commercial seed laboratory). A linear mixed model fitted to the data indicated significant observer, crop, and seed color, shape, and size effects on detection. Detectability increased by 20% ± 7.7 (± standard error)when seeds had a distinct shape or color (28% ± 8.1), or were larger (23% ± 8.7) rather than smaller, relative to the crop. Commercial laboratory identifications were usually correct at the level of genus, and species for common weeds, but some misidentifications occurred. Sample sizes for border inspections should be based on detectability of regulated weed seeds in the crop in combination with weed risk for the crop and location. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Feasibility of Cornstarch Masks for Dry Facial Skin Care

This study is based on the problem of dry facial skin that causes a person's lack of self-confidence due to complaints of dark spots, dull, scaly skin, and fine wrinkles on the face. Corn is one of the vegetables that can be used as an ingredient for traditional skin beauty treatments and contains vitamin E which is beneficial for dry skin. The purpose of this study is to analyze the feasibility of cornmeal masks in terms of laboratory tests, organoleptic tests, hedonic tests This research method is an experiment and a type of quantitative descriptive research of cornmeal masks. Data collection methods in the form of observation, documentation, and questionnaires Research instruments are laboratory tests, organoleptic tests, hedonic tests and identification tests. The collected data is processed using a percentage descriptive analysis formula. The results of laboratory test studies are Vitamin A contained in cornmeal masks, namely 82 mg / 100gr, Vitamin B1 contained in cornmeal masks 5.5 mg / 100gr and Vitamin C contained in cornmeal masks 7.92 gr / 100gr. The results of the texture organoleptic test found that 86% of the panelists said the texture of cornmeal was very smooth, 100% of the panelists said the aroma of the cornmeal mask had a strong aroma typical of corn, 71% of the panelists said the stickiness of the cornmeal mask was quite close to the results of the hedonic test the panelists' likeness could be shown that 100% of the panelists liked the cornstarch mask. Flour Cornstarch masks have the feasibility for this to be used as a natural mask for treatment.

POST COVID-19 CONDITION: NEEN FOR CONSENSUS DEFINITION

During literature review we come across to different definitions of prolonged illness associated with SARS-CoV2 infection, which made it difficult to characterize and compare COVID-19 cases by epidemiological and clinical patterns. Our study is an attempt to assess post-COVID-19 frequency in the context of the case definitions. Prevalence of post-COVID-19 condition ranges from 10% up to 77.8% over the reviewed studies and tends to increase over time along with mutation of the SARS-CoV-2 virus. Consensus definition of Post-COVID Conditions is essential for identification and characterization of clinical, pathophysiological, laboratory, imaging and epidemiologic features of all forms of COVID-19 manifestation. It will help us better understand the disease entity, set up best-practice standards of care and elaborate effective control and preventive measures. 

Performance Evaluation of Bruker Biotyper, ASTA MicroIDSys, and VITEK-MS and Three Extraction Methods for Filamentous Fungal Identification in Clinical Laboratories.

Filamentous fungi are a major cause of life-threatening infections in immunocompromised patients; thus, rapid and accurate identification is critical. Filamentous fungal identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been demonstrated with high sensitivity and reproducibility; however, its wider application has been limited in clinical laboratories because of practical challenges such as database availability or lack of standardization. In this study, we compared the performance of the Bruker Biotyper, ASTA MicroIDSys, and Vitek MS for 84 clinical filamentous fungal isolates. Moreover, the sensitivity of three independent sample preparation methods (direct, on plate, in tube) was compared. Bruker Biotyper identified 71.43% (60/84) of isolates correctly (species, genus, or complex/group level). ASTA MicroIDSys and Vitek MS showed accuracy rates of 70.24% (59/84) and 55.95% (47/84), respectively. We found that any difference in sensitivity may be attributed to the database of the systems. In addition, the "in tube" method showed the highest sensitivity among the three methods; however, there was no statistical difference among them. For the broader application of MALDI-TOF MS for filamentous fungal identification, further studies from multiple perspectives are required.

Veratrum parviflorum poisoning: identification of steroidal alkaloids in patient blood and breast milk

Introduction The Veratrum genus is composed of plants containing a diverse set of steroidal alkaloids. Veratrum plant material has been utilized for centuries as herbal medicines, however the alkaloids have such a low therapeutic index that they are not used in modern medicine. Here we report an incident of inadvertent ingestion of V. parviflorum by hikers in Georgia that allowed detection, and in several instances identification of alkaloids from the plant, and correlated their presence within patient blood and breast milk specimens. Case history Eight patients, three male and five female, presented in the spring of 2020 and 2021 with symptoms requiring emergent medical attention after ingestion of Veratrum parviflorum. All patients believed the plants to be a local native species of wild leek, Allium tricoccum, locally known as ramps. Plants were identified using photographs as well as fresh and cooked plant material provided by patients, in consultation with botanists at the University of Georgia Herbarium. Written consent was obtained from all patients for collection of blood and breast milk specimens for laboratory identification of Veratrum alkaloids. Methods V. parviflorum plant material, and patient serum and breast milk were analyzed by high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF) to identify steroidal alkaloids. Results The V. parviflorum extract was confirmed to contain cyclopamine, veratramine, jervine, and muldamine. Two out of the eight patients had detectable concentrations of Veratrum alkaloids. Of the alkaloids identified in the plant, cyclopamine and jervine were detected within patient serum, and cyclopamine and veratramine were observed to be present in breast milk. Discussion Toxicity resulting from Veratrum steroidal alkaloids has primarily been reported from V. album and V. viride. This is the second report of V. parviflorum poisoning. The present work reports for the first time the presence of muldamine and jervine within V. parviflorum. This work provides the first instance of identification of Veratrum alkaloids in breast milk. Thus, the findings presented herein add to literature record causative agents contributing to the toxicity of V. parviflorum when ingested and potential for secondary poisoning through breastfeeding. Conclusion V. parviflorum toxicity was observed to cause nausea, vomiting, hypotension, bradycardia, abdominal pain, light-headedness, blurred vision, and tingling in the arms. Patients experiencing mild symptoms improved with supportive care, IV fluids, and antiemetics, but hemodynamically unstable patients required atropine and vasopressors. This study demonstrated that more lipophilic Veratrum alkaloids can be passed along in breast milk, which suggests additional precautions may be critical to limit further poisonings.

Necrotizing hepatitis caused by Clostridium novyi type B in a dog with no predisposing liver lesions: a case report

BackgroundInfectious necrotic hepatitis (INH) is typically a disease of ruminants caused by Clostridium novyi type B. Growth of the causative agent is supported by development of an anaerobic environment within the liver. In dogs, C. novyi is rare and has only been previously reported as a post-mortem diagnosis. In one case, infection was secondary to metastatic pancreatic adenocarcinoma and the other was presumptively diagnosed on histopathology of a hepatic lesion in a dog initially presented for acute collapse.Case presentationAn 8-year-old spayed, female mixed breed dog was presented for acute onset of hyporexia and vomiting. Serum biochemistry revealed elevated hepatocellular injury and cholestatic liver enzymes. Ultrasound revealed peritoneal fluid accumulation and multiple hepatic masses. Cytologic examination of liver aspirates and peritoneal fluid revealed frequent 4 × 1 μm bacilli with a terminal endospore. Anaerobic bacterial growth isolated from the fluid sample could not be identified using typical laboratory identification techniques. Long-read, whole genome sequencing was performed, and the organism was identified as Clostridium novyi type B. Antimicrobial and hepatic support treatment were initiated. The patient re-presented 27 days later, and the follow up liver aspirate with cytology revealed no appreciable bacteria and anaerobic culture was negative. The patient was presented four months later and a large hepatic mass and peritoneal fluid were again identified on abdominal ultrasound. Cytologic examination of the peritoneal fluid revealed bacilli similar to those identified on initial presentation. The patient was euthanized. The most significant finding on necropsy was necrotizing hepatitis with intralesional endospore-forming bacilli compatible with recurrence of Clostridium novyi type B. There was no identifiable cause of an anaerobic insult to the liver.ConclusionsThis case demonstrates the diagnostic utility of using cytology as part of the initial diagnostic work up for infectious hepatitis. The cytologic findings coupled with whole genome sequencing and anaerobic culture were crucial for the identification and classification of the organism identified on fine needle aspirate. Clostridium novyi type B should be considered when bacilli organisms containing a terminal endospore are identified on liver aspirates collected from canine patients.

A glimpse into the genotype and clinical importance of non tuberculous mycobacteria among pulmonary tuberculosis patients: The case of Ethiopia.

Laboratory identification of nontuberculous mycobacteria (NTM) species is not regularly performed while, they have a public health importance with a prevalence of more than 5% among pulmonary tuberculosis (PTB) patients in Ethiopia. Hence, this study aimed to identify the NTM species and their clinical significance among PTB patients. A retrospective study was conducted at the Ethiopian Public Health Institution’s (EPHI’s) national TB referral laboratory. Stored NTM isolates were genotyped using GenoType Mycobacterium CM/AS kit (Hain Life science, Germany). Data pertinent to the study was extracted from the EPHI’s database and patients’ medical records. Between January 2 & December 28 of 2017, a total of 3,834 samples were processed from 698 TB patients of whom 50% were female. Among 3,317 samples with mycobacterial culture results 7.3% were NTM and majority of them were identified from smear negative TB patients. M. simiae was the /predominant NTM among the genotyped isolates. All the studied NTM species were not clinically important however, considering the similarity of clinical and radiologic findings between NTM and MTBC infected patients, integrating NTM species identification in the routine TB laboratory diagnosis may augment clinicians’ decision particularly in DR-TB patients. Additional similar prospective study with a larger sample size is recommended. Moreover, urgent improvements on patients’ record keeping practice are required in the studied hospitals.

Early Diagnosis of Interstitial Lung Disease

Interstitial Lung Disease (ILD) includes more than 200 diseases that involve the interstitial lung. The diagnosis of ILD depends on the onset of symptoms, causes, and clinical manifestations. An anamnesis comprehensive and physical examination are essential in diagnosing ILD. In addition, laboratory tests are carried out in certain clinical conditions. The analysis of biomarkers in ILD is helpful for diagnosis, disease monitoring, and prediction of prognosis. Pulmonary function studies support the diagnosis of ILD and as a predictor of prognosis. High-resolution Computed Tomography (HRCT) is the main diagnostic procedure in ILD patients. In certain conditions, a lung biopsy may be considered. Multidisciplinary discussion (MDD) enhances accurate diagnosis. An accurate early diagnosis of ILD is necessary to ensure that patients receive optimal management, reduce the risk of developing pulmonary fibrosis and reduce the risk of death. Early diagnosis of ILD define as early identification of symptoms, laboratory and radiological findings at the early stage of the disease

MALDI-TOF MS for pathogenic bacteria analysis

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) has been widely used for bacterial identification in clinical laboratories. However, the current technique needs bacterial culture to obtain purified single colonies for MS analysis. It is also limited in direct bacterial antimicrobial resistance (AMR) analysis. The two limitations restrict fast clinical diagnosis of bacterial infectious diseases and the choice of suitable antibiotic drugs in a timely manner. In the past years, we focus on bacterial identification and bacterial AMR analysis by MALDI-TOF MS, and developed a number of new methods with respect to sample pretreatment, mass spectrometry data acquisition and mass spectrometry data mining, with the aim of more accurate and more rapid bacterial identification as well as direct bacterial AMR analysis. Microfluidic chips and functional nanomaterials can be used to extract bacterial cells directly from the raw materials to shorten or even avoid the bacterial culture step. In some sample, the total bacterial cell amount is limited and hence it is necessary to further enhance the MS analysis sensitivity, and the single cell mass spectrometry techniques may play an important role in bacterial identification in the future. In view of bacterial AMR analysis, it is necessary to collect more molecular information of bacterial cells by mass spectrometry and to develop advanced data mining strategies based on machine learning and deep learning techniques. It is expected that the MALDI-TOF MS technique can further advance clinical treatment of bacterial infection diseases in combination with the development of sample pretreatment methods, MS techniques and data analysis algorithms.

Laporan Pertama Kasus Infestasi Spinturnix spp. pada Kelelawar Pemakan Buah (Rousettus spp.)

Rousettus spp. is one of the wild animals that may transmit various diseases including zoonotic. Rousettus spp. or known as fruit bat is found in Java island and belongs to the Pteropodidae family. Generally, Rousettus spp. consumes fruit and floral products. This case report aims to detect ectoparasites in fruit bats (Rousettus spp.) found at Djawatan Banyuwangi, East Java. The samples in this case report are seven fruit bats (Rousettus spp.). Based on the results of laboratory identification using the whole mount method, from 7 samples of fruit bats (Rousettus spp.), there are five fruit bats (Rousettus spp.) were infected with Spinturnix spp.. Spinturnix spp. that obtained from fruit bats (Rousettus spp.) were examined using a binocular microscope (Olympus CX-23, Tokyo Japan) with a magnification of 100 times. Images of ectoparasites and bats were taken using a Nikon d5300 camera. Total amount of Spinturnix spp. that was successfully obtained from 5 fruit bats (Rousettus spp.) are 25 individuals.

Identification of Mycoplasma pneumoniae-associated pneumonia cases among hospitalized patients using CLART® microarray technology.

ObjectivesCommunity-acquired pneumonia (CAP) is a global health condition that affects populations from all age groups. The laboratory identification of Mycoplasma pneumoniae as a causative agent of CAP is challenging because of its atypical and fastidious nature. Therefore, this study assessed the diagnostic potential of PneumoCLART bacteria® in identifying M. pneumoniae as a causative agent of pneumonia in hospitalized adults.MethodsThis prospective study used a cross-sectional approach to assess the diagnostic potential of PneumoCLART bacteria® for detecting M. pneumoniae in sputum samples procured from 27 patients with pneumonia who required hospitalization.ResultsThe PneumoCLART bacteria® results illustrated that 7 of 27 patients with pneumonia were positive for M. pneumoniae (26%). However, the quality of sputum varied among the M. pneumoniae-positive and M. pneumoniae-negative samples. Fifty percent of the specimens obtained from patients positive for M. pneumoniae were saliva-contaminated and unsuitable for analysis.ConclusionsBecause the leukocyte count was low and sputum specimens were saliva-contaminated, these findings require further validation to prove the utility of CLART® microarray technology for the identification of M. pneumoniae in pneumonia-positive patients. Conclusively, this prospective study included a small number of clinical samples, which likely affected its outcomes.

Improved semiautomated detection of TRBC-restricted Sézary cells unveils a spectrum of clonal cluster immunophenotypes

Letter to Blood| December 29, 2022 Improved semiautomated detection of TRBC-restricted Sézary cells unveils a spectrum of clonal cluster immunophenotypes Pedro Horna, Pedro Horna Division of Hematopathology, Mayo Clinic, Rochester, MN Search for other works by this author on: This Site PubMed Google Scholar Gregory Otteson, Gregory Otteson Division of Hematopathology, Mayo Clinic, Rochester, MN Search for other works by this author on: This Site PubMed Google Scholar Min Shi, Min Shi Division of Hematopathology, Mayo Clinic, Rochester, MN Search for other works by this author on: This Site PubMed Google Scholar Jansen N. Seheult, Jansen N. Seheult Division of Hematopathology, Mayo Clinic, Rochester, MN Search for other works by this author on: This Site PubMed Google Scholar Dragan Jevremovic, Dragan Jevremovic Division of Hematopathology, Mayo Clinic, Rochester, MN Search for other works by this author on: This Site PubMed Google Scholar Horatiu Olteanu Horatiu Olteanu Division of Hematopathology, Mayo Clinic, Rochester, MN https://orcid.org/0000-0001-5783-4485 Search for other works by this author on: This Site PubMed Google Scholar Blood (2022) 140 (26): 2852–2856. https://doi.org/10.1182/blood.2022017548 Article history Submitted: June 24, 2022 Accepted: August 21, 2022 Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Request Permissions Cite Icon Cite Search Site Citation Pedro Horna, Gregory Otteson, Min Shi, Jansen N. Seheult, Dragan Jevremovic, Horatiu Olteanu; Improved semiautomated detection of TRBC-restricted Sézary cells unveils a spectrum of clonal cluster immunophenotypes. Blood 2022; 140 (26): 2852–2856. doi: https://doi.org/10.1182/blood.2022017548 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBlood Search Subjects: Lymphoid Neoplasia TO THE EDITOR: Peripheral blood flow cytometry is the test of choice to detect circulating neoplastic T-cells (Sézary cells) in patients with mycosis fungoides (MF) or Sézary syndrome (SS).1-4 These 2 clinically distinct subtypes of cutaneous T-cell lymphoma (CTCL) harbor neoplastic CD4+ T cells with largely identical immunophenotypic properties, and share a staging scheme that includes blood ratings based on absolute numbers of Sézary cells (B0: <250, B1: ≥250 and <1000, and B2: ≥1000 cells/μL).5 Flow cytometric identification of Sézary cells also plays a critical role in distinguishing SS from reactive erythroderma, and in the assessment of therapy response. Unfortunately, the laboratory identification of Sézary cells has remained challenging, given that their most frequent immunophenotypic characteristics (lack of CD7 and CD26 expression) are shared with common benign CD4+ T-cell subsets, whereas immunophenotypic properties specific for neoplasia (eg,...

In silico prediction, human hepatocyte metabolism, LC-HRMS/MS analysis, and targeted/nontargeted data-mining to characterize 4-acetoxy-N,N-diisopropyl-tryptamine metabolism

Tryptamine intoxications and fatalities are increasing, although these novel psychoactive substances (NPS) are not controlled in most countries. There are few data on the metabolic pathways and enzymes involved in tryptamines biotransformation. 4-acetoxy- N,N -diisopropyltryptamine (4-AcO-DiPT) is a synthetic tryptamine related to 4-hydroxy- N,N -diisopropyltryptamine (4-OH-DiPT), 4-acetyloxy- N,N -dipropyltryptamine (4-AcO-DPT) and 4-acetoxy- N,N -dimethyltryptamine (4-AcO-DMT). The aim was to determine the best 4-AcO-DiPT metabolites to identify 4-AcO-DiPT consumption through human hepatocyte metabolism and high-resolution mass spectrometry. 4-AcO-DiPT metabolites were predicted in silico with GLORYx freeware to assist in metabolite identification. 4-AcO-DiPT was incubated with 10-donor-pooled human hepatocytes, and sample analysis performed with reversed-phase liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) in positive- and negative-ion modes. LC-HRMS/MS raw data were separately processed with targeted and nontargeted data-mining. A total of 47 phase I and II metabolites were predicted, and six metabolites were identified after 3 h incubation following ester hydrolysis, O-glucuronidation, O-sulfation, N-oxidation and N-dealkylation. All second-generation metabolites derived from the only first-generation metabolite detected after ester hydrolysis (4-OH-DiPT). Interestingly, the most intense metabolite (4-OH-DiPT) was also detected in the control samples without hepatocytes at 0 and 3 h and at 0 h incubation with hepatocytes but at much lower intensity than after 3 h incubation with hepatocytes, meaning that, although 4-OH-DiPT is formed through enzymatic reaction, it is also spontaneously formed during incubation to a lesser extent. This indicates that 4-OH-DiPT formation was overestimated in our experiments. When analyzing authentic samples, digestion (with β-glucuronidase/sulfatase) is preferred to increase the 4-OH-DiPT signal. Since the parent drug is most likely degraded and the acetyl group is eliminated quickly, toxicologists could report 4-OH-DiPT positive cases that actually were from 4-AcO-DiPT intake. The second most intense metabolite was 4-OH-iPT-sulfate followed by 4-OH-DiPT-glucuronide, indicating that glucuronidation and sulfation are common in this tryptamine's metabolic pathway. Others propose that tryptamines do not have a common metabolic pathway and that metabolism changes depending on the nature and position of their substituents, with demethylation, hydroxylation and dealkylation the most common phase I reactions, followed by glucuronidation or sulfation. 4-OH-DiPT, 4-OH-iPT and 4-OH-DiPT-N-oxide are proposed as biomarkers of 4-AcO-DiPT consumption, but the rapid enzymatic hydrolysis and lower spontaneous hydrolysis of 4-AcO-DiPT to 4-OH-DiPT might create a problem in discerning 4-AcO-DiPT from 4-OH-DiPT consumption. If the parent drug is present even in low concentrations, the ingested drug would be clear, but there is no available information regarding detection of this drug in authentic samples. These results require in vivo confirmation, which is challenging due to the small number of 4-AcO-DiPT seizures in recent years. Psychedelic tryptamine use is low but increasing, raising the importance of laboratory identification of specific metabolites to verify intake and identify potential public health NPS outbreaks. Since the parent drug is rapidly hydrolyzed and it is currently unknown whether 4-AcO-DiPT is present in authentic biological samples, identifying its metabolites is highly useful throughout, but especially late in its time course. 4-OH-DiPT, 4-OH-iPT and 4-OH-DiPT- N -oxide are optimal biomarkers to identify 4-AcO-DiPT consumption.

High-resolution quadrupole time-of-flight mass spectrometry, a powerful technique to include in analytical scheme for the identification of drugs and chemicals in seizures: A case study

A seizure containing 37 items was analyzed with laboratory identification scheme for drugs and chemicals in seized products applying accredited methods based on gas chromatography mass spectrometry (GC-MS) and liquid chromatography with photodiode-array detection (UPLC-PDA), a category A technique combined with category B and C techniques. No identification was obtained for 1 item [analogue of lysergic acid diethylamide (LSD)] and none detected or identified by GC-MS and/or UPLC-PDA for 8 items. All items were subsequently analyzed using ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-Tof) to investigate whether it is appropriate to include this technique in laboratory identification scheme for seized materials. The seizure consisted of individual packages containing blotter papers, capsules and powders, respectively. The powders were present in sealed alu-bags labeled ‘not for human consumption’ and Falcon® tubes, presumably purchased as ‘research chemical’. Methanolic solutions were analyzed. The GC-MS method was described elsewere (Coopman V. Toxicol Anal Clin 2018;30:75–79). Automated library search was performed against NIST, SWGDRUG and CAYMAN mass spectral library and mass spectra published in scientific literature were consulted. The UPLC-PDA analysis was performed with a Waters Acquity UPLC H-Class system. The compounds were separeted on an Acquity UPLC C18 column using gradient elution with a mobile phase consisting of 0.1% (v/v) formic acid aqueous solution and 0.1% (v/v) formic acid in acetonitrile, and photodiode-arra y detection. Automated library search was performed with Empower™ 3 software against in-house and Waters library. A Waters Acquity UPLC I-Class with Xevo G2-XS QTof was used, equipped with UNIFI® scientific information system and Forensic Toxicology Screening Application from Waters with data acquisition parameters for accurate mass analysis in positive and negative mode. Analogues of LSD (1P-LSD, ETH-LAD, AL-LAD) and a designer benzodiazepine (clonazolam) respectively, were identified in blotter paper by UPLC-PDA, GC-MS and confirmed by UPLC-Q-Tof. The unknown analogue of LSD was identified as ALD-52 by UPLC-Q-Tof. 4-fluoromethylphenidate (4F-MPH), N-ethyldeschloroketamine (O-PCE) and mirtagynine were identified in powder by UPLC-PDA, GC-MS and UPLC-Q-Tof. O-PCE was not present in the Forensic Toxicology Screening Application library and was therefore not automatically identified. Salicylic acid, caffeine, trazodone, pseudoephedrine, melatonin and ETH-LAD respectively, were identified in tablet or capsule with routine laboratory identification scheme and confirmed by UPLC-Q-Tof. In a capsule found to contain pseudoephedrine and unknown, a library hit was found for the designer drug N-methyl-2-aminoindane (NM-2AI) by UPLC-Q-Tof. Laboratory identification scheme for the identification of drugs and chemicals in seizures is based on GC-MS and UPLC-PDA, a category A technique and category B, C techniques (SWGDRUG recommendations). Not all drugs and chemicals were detected with both techniques. When identification is obtained with one or any of the applied techniques, a more extensive analysis is recommended. UPLC-Q-Tof is a sensitive and powerful technique, appropriate to include in laboratory identification scheme for seized materials when identification is obtained with one or any of the applied techniques or if the presence of low concentration, a NPS or designer drugs is suspected, allowing confirmation or identification.

β-LACTAMASE PRODUCING GRAM NEGATIVE BACTERIA: MAIN ISSUES, DIAGNOSTIC METHODS AND MEANS OF REDUCING THEIR BURDEN IN ROMANIA

Bacterial resistance to antibiotics is a globally recognized healthcare problem. Β-lactam resistance în Gram negative bacteria, mediated through mecanisms like ESBLs (extended spectrum beta-lactamases), cephalosporinases and carbapenemases, is one of the most important. This study aims to identify the most important tests which can be applied in the laboratory in order to correctly identify Gram-negative bacteria (Enterobacterales, Pseudomonas spp., Acidentobacter spp.) which are resistant to this class of antibiotics and their resistance mechanisms, apply them on the microorganisms isolated in a clinical hospital from Bucharest, Romania, describe and report the findings, followed by the proposal of measures that can reduce the burden of these bacteria and their resistance mechanisms. In order to support the national effort in the fight against the antimicrobial resistance phenomenon, a new phenotipic laboratory identification test is proposed, using resources available in Romania. Keywords: antimicrobial resistance, β-lactamase, carbapenemase

P14 The antibiotic resistance profile of bacteria causing acute respiratory tract infections in Khartoum, Sudan

BackgroundAcute respiratory tract infections (ARTIs) are the most common indication for antibiotic prescriptions. This is associated with irrational prescription and correlated with antimicrobial resistance especially in low-income countries including Sudan. This is a cross-sectional study aimed at identifying bacterial pathogens causing ARTIs and their susceptibility to the commonly prescribed antibiotics.MethodsBacterial pathogens (51 isolates) were obtained from 29 ARTI patients from two hospitals in Khartoum in the time period March 2021 to March 2022. Samples were collected from each patient using both nasopharyngeal and oropharyngeal swabs. They were then cultured using blood and chocolate agar. The isolated colonies were identified using Gram staining technique, biochemical test, and selective media. Antibiotic susceptibility tests (AST) were done using the Kirby–Bauer disc diffusion susceptibility test protocol. Susceptible strains were determined following CLSI guidelines. The susceptibility of isolates was determined for different antibiotics representing the important classes including co-amoxiclav, cefixime, penicillin G, oxacillin, ceftriaxone, erythromycin, levofloxacin, ciprofloxacin and meropenem.ResultsIsolates were found to be mostly (82.3%) Gram positive (Staphylococcus aureus and Streptococcus pyogenes). Pseudomonas aeruginosa was the most abundant (17.9%) Gram-negative bacteria. S. aureus isolates were found to be 100% susceptible to levofloxacin, ciprofloxacin and meropenem. S. aureus exhibited less susceptibility to ceftriaxone (75%), and the least susceptibility to co-amoxiclav (37.5%), cefixime (31.3%), oxacillin (18.8) and erythromycin (25%). S. pyogenes isolates were found to be completely susceptible (100%) to co-amoxiclav, levofloxacin, ciprofloxacin and ceftriaxone. S. pyogenes had less susceptibility to vancomycin (92%), and the least susceptibility to cefixime (38.5%) and erythromycin (26.9%). P. aeruginosa isolates were found to be 100% susceptible to levofloxacin, ciprofloxacin and ceftriaxone, with less susceptibility to ceftriaxone (66%) and the least susceptibility to co-amoxiclav (22.2%), cefixime (33.3%), erythromycin (11.1%), vancomycin (11.1%) and oxacillin.ConclusionsCo-amoxiclav is extensively used in Sudan empirically. The results show that S. aureus and P. aeruginosa are increasingly becoming resistant to this drug. However, S. pyogenes is still susceptible to it. Conversely resistance to erythromycin and cephalosporins is shown in all the different isolated species, however these drugs are still widely used without even culture and ASTs. Luckily, non-susceptibility to ciprofloxacin and levofloxacin was not detected here. Obviously, laboratory identification and ASTs are much needed along with surveillance programmes in hospitals. Together with rational use of antibiotics these measures will help to slow down the progress of the deadly AMR pandemic.

P16 Strengthening surveillance systems for antimicrobial resistance in urinary tract infections in Kenya

BackgroundUrinary tract infections (UTIs) are amongst the most common conditions leading to outpatient visits and are also one of the most common causes of inappropriate antibiotic usage. Addressing antimicrobial resistance (AMR) requires strengthening surveillance system in Kenya where UTI is a problem with health systems having limited capacity for accurate laboratory identification of UTI pathogens. Empirical treatment to manage UTIs is the most common problem due to limited microbiology capacity. These include shortages of suitable training, equipment, quality control procedures and professional expertise to make an accurate clinical interpretation of urine cultures. This leads to reporting normal flora as pathogens.ObjectivesTo deliver training on the identification and antibiotic susceptibility testing of bacterial isolates from urine infections at participating hospitals in the Kenyan AMR surveillance network and measure competency levels in staff participating in the training with the aim of strengthening the Kenyan surveillance system.MethodsA 1 day baseline visit to the five sites was made between October and November 2021 using a standardized checklist to assess laboratory capacity. This was followed by urine processing training conducted between November 2021 and January 2022 in Malindi, Jaramogi, Kitale, Nakuru and Nyeri County Hospitals. Training materials were developed with the input of Aga Khan University Hospital. A 5 day in-person training course was delivered at each of the five hospitals, which targeted an average of 2–3 laboratory staff per hospital. The competencies of the laboratory staff were assessed by written pre-assessment and post-assessment tests. A standardized practical assessment was achieved by use of five different control organisms. The scores were done by doing an average performance score at each hospital laboratory.ResultsOut of 13 trainees, 8 were male and 5 were female. The median number of years since qualification for these staff was 6 (IQR 2–9). The median number of total months of microbiology training time was 2.3 (IQR 1–7). The median pre-training and post-training scores were 46% and 90%, respectively; non-parametric Wilcoxon rank test showed statistical difference (P < 0.0001) between pre-training and post-training. All participants improved from pre-to post- training assessment from the 5 day training with marked improvement amongst from staff with lowest previous level of laboratory training.ConclusionsTraining microbiology staff in the accurate processing of urine sample will be an important activity for a Kenyan AMR surveillance system. These training materials, if delivered by an experienced trainer, can achieve a clear improvement in knowledge levels and practical competence for real-life microbiology identifications.

Insights from biosurveillance: non-fatal opioid overdoses in Rhode Island 2019-21.

Opioids biosurveillance is a new approach to public health surveillance of non-fatal overdoses that relies upon laboratory analysis of residual biospecimens from hospitals treating opioids overdoses. In Rhode Island (RI), USA, hospitals report suspected opioid overdoses to the Department of Health. Residual specimens associated with these overdoses are submitted to the State Health Laboratories for further characterization. This surveillance project aimed to characterize non-fatal overdoses through toxicological testing of urine specimens associated with non-fatal overdoses during the initial 2-year period of biosurveillance implementation in RI to assess the feasibility and public health utility of this approach. This study included individuals who presented for treatment for a suspected opioid overdose in 10 RI hospitals between July 2019 and June 2021. Urine samples were received for 1354 unique overdose encounters corresponding to reported overdoses. Some individuals experienced multiple overdoses during this time. Urine samples were extracted and then analyzed by liquid chromatography tandem mass spectrometry with a panel consisting of 1033 opiates, synthetic opioids, fentanyl analogs and select metabolites. Temporal and spatial trends were evaluated for the studied population. A total of 1354 samples were tested for the presence of opioids in urine collected from individuals who experienced a suspected overdose. Fentanyl (and/or norfentanyl) was present in 79% of all samples in which opioids were found (n = 1033). Fentanyl analogs varied in their contribution to these totals, with observations ranging as high as 35% of all opioid-containing samples in August 2019 and May 2021. Laboratory identification of opioids involved in suspected overdoses shows that fentanyl and its analogs are the main drivers of the opioids overdose epidemic in Rhode Island, USA. The biosurveillance approach is unique in its ability to quantify contributions of novel fentanyl analogs to the burden of overdoses.

Identifying Pneumonia Subtypes from Electronic Health Records Using Rule-Based Algorithms

The International Classification of Disease (ICD) coding for pneumonia classification is based on causal organism or use of general pneumonia codes, creating challenges for epidemiological evaluations where pneumonia is standardly subtyped by settings, exposures, and time of emergence. Pneumonia subtype classification requires data available in electronic health records (EHRs), frequently in nonstructured formats including radiological interpretation or clinical notes that complicate electronic classification. The current study undertook development of a rule-based pneumonia subtyping algorithm for stratifying pneumonia by the setting in which it emerged using information documented in the EHR. Pneumonia subtype classification was developed by interrogating patient information within the EHR of a large private Health System. ICD coding was mined in the EHR applying requirements for "rule of two" pneumonia-related codes or one ICD code and radiologically confirmed pneumonia validated by natural language processing and/or documented antibiotic prescriptions. A rule-based algorithm flow chart was created to support subclassification based on features including symptomatic patient point of entry into the health care system timing of pneumonia emergence and identification of clinical, laboratory, or medication orders that informed definition of the pneumonia subclassification algorithm. Data from 65,904 study-eligible patients with 91,998 episodes of pneumonia diagnoses documented by 380,509 encounters were analyzed, while 8,611 episodes were excluded following Natural Language Processing classification of pneumonia status as "negative" or "unknown." Subtyping of 83,387 episodes identified: community-acquired (54.5%), hospital-acquired (20%), aspiration-related (10.7%), health care-acquired (5%), and ventilator-associated (0.4%) cases, and 9.4% cases were not classifiable by the algorithm. Study outcome indicated capacity to achieve electronic pneumonia subtype classification based on interrogation of big data available in the EHR. Examination of portability of the algorithm to achieve rule-based pneumonia classification in other health systems remains to be explored.