Clinical Microbiology Laboratory Research Articles

Update on Accepted Novel Bacterial Isolates Derived from Human Clinical Specimens and Taxonomic Revisions Published in 2020 and 2021.

A number of factors, including microbiome analyses and the increased utilization of whole-genome sequencing in the clinical microbiology laboratory, has contributed to the explosion of novel prokaryotic species discovery, as well as bacterial taxonomy revision. This review attempts to summarize such changes relative to human clinical specimens that occurred in 2020 and 2021, per primary publication in the International Journal of Systematic and Evolutionary Microbiology or acceptance on Validation Lists published by the International Journal of Systematic and Evolutionary Microbiology. Of particular significance among valid and effectively published taxa within the past 2 years were novel Corynebacterium spp., coagulase-positive staphylococci, Pandoraea spp., and members of family Yersiniaceae. Noteworthy taxonomic revisions include those within the Bacillus and Lactobacillus genera, family Staphylococcaceae (including unifications of subspecies designations to species level taxa), Elizabethkingia spp., and former members of Clostridium spp. and Bacteroides spp. Revisions within the Brucella genus have the potential to cause deleterious effects unless the relevance of such changes is properly communicated by microbiologists to stakeholders in clinical practice, infection prevention, and public health.

An Update on Novel Taxa and Revised Taxonomic Status of Bacteria Isolated from Domestic Animals Described in 2018 to 2021.

Novel bacterial taxonomy and nomenclature revisions can have significant impacts on clinical practice, disease epidemiology, and veterinary microbiology laboratory operations. Expansion of research on the microbiota of humans, animals, and insects has significant potential impacts on the taxonomy of organisms of clinical interest. Implications of taxonomic changes may be especially important when considering zoonotic diseases. Here, we address novel taxonomy and nomenclature revisions of veterinary significance. Noteworthy discussion centers around descriptions of novel mastitis pathogens in Streptococcaceae, Staphylococcaceae, and Actinomycetaceae; bovine reproductive tract pathogens in Corynebacteriaceae; novel members of Mannheimia spp., Leptospira spp., and Mycobacterium spp.; the transfer of Ochrobactrum spp. to Brucella spp.; and revisions to the genus Mycoplasma.

Diagnostic yield of broad-range 16s rRNA gene PCR varies by sample type and is improved by the addition of qPCR panels targeting the most common causative organisms.

Introduction. Molecular techniques are used in the clinical microbiology laboratory to support culture-based diagnosis of infection and are particularly useful for detecting difficult to culture bacteria or following empirical antimicrobial treatment.Hypothesis/Gap Statement. Broad-range 16S rRNA PCR is a valuable tool that detects a wide range of bacterial species. Diagnostic yield is low for some sample types but can be improved with the addition of qPCR panels targeting common bacterial pathogens.Aim. To evaluate the performance of a broad-range 16S rRNA gene PCR and the additional diagnostic yield of targeted qPCR applied to specimens according to a local testing algorithm.Methodology. In total, 6130 primary clinical samples were collected as part of standard clinical practice from patients with suspected infection during a 17 month period. Overall, 5497 samples were tested by broad-range 16S rRNA gene PCR and a panel of targeted real-time qPCR assays were performed on selected samples according to a local testing algorithm. An additional 633 samples were tested by real-time qPCR only. The 16S rRNA gene PCR was performed using two assays targeting different regions of the 16S rRNA gene. Laboratory developed qPCR assays for seven common bacterial pathogens were also performed. Data was extracted retrospectively from Epic Beaker Laboratory Information Management System (LIMS).Results. Broad-range 16S rRNA gene PCR improves diagnostic yield in culture-negative samples and detects a large range of bacterial species. Streptococcus spp., Staphylococcus spp. and the Enterobacteriaceae family are detected the most frequently in samples with a single causative organism, but mixed samples frequently contained anaerobic species. The highest diagnostic yield was obtained from abscess, pus and empyema samples; 44.9 % were positive by 16S and 61 % were positive by the combined 16S and targeted qPCR testing algorithm. Samples with a particularly low diagnostic yield were blood, with 3.3 % of samples positive by 16S and CSF with 4.8 % of samples positive by 16S. The increased diagnostic yield of adding targeted qPCR is largest (~threefold) in these two sample types.Conclusion. Broad-range PCR is a powerful technique that can detect a very large range of bacterial pathogens but has limited diagnostic sensitivity. The data in this report supports a testing strategy that combines broad-range and targeted bacterial PCR assays for maximizing diagnosis of infection in culture-negative specimens. This is particularly justified for blood and CSF samples. Alternative approaches, such as metagenomic sequencing, are needed to provide the breadth of broad-range PCR and the sensitivity of targeted qPCR panels.

1837. Genotypic and Phenotypic Diversity of Contemporaneous Carbapenem Resistant Klebsiella pneumoniae from Blood Cultures of Individual Patients

Abstract Background The longstanding paradigm is that almost all bloodstream infections (BSIs) stem from a single, clonal organism. We hypothesized that carbapenem resistant K. pneumoniae (CRKP) from individual patients (pts) with BSIs are genetically diverse and manifest phenotypic differences that are not typically recognized by the clinical microbiology laboratory at time of diagnosis. Methods We streaked blood culture (BC) broth from 6 pts with CRKP BSI onto blood agar plates, and randomly picked 9 colonies (strains) for HiSeq (Illumina) whole genome sequence. Single BSI isolates recovered by the clinical micro lab from individual pts underwent short- and long-read MinION (ONT) sequencing. Results 2 pts were infected with clade 1 (B, G), and 4 with clade 2 (A, D, F, J) ST258 KPC-producing KP. Strains from individual pts clustered by cgSNP phylogeny (Fig. 1A-B). BC bottles from each pt harbored genetically heterogenous KP populations, with strains differing from each other by cgSNPs (Fig. 1B), presence/absence of specific antibiotic resistance genes (Fig. 1A), mutations of capsular genes (Fig 2) and at other loci involved in host interactions, and/or loss of plasmids or plasmid-borne genes (Fig. 1A). Differences in capsular gene composition were observed in KL107 capsule type strains from pts A, D and J (Fig. 2). Pangenome analyses showed accessory gene composition diversity among strains from all pts (Fig. 3). Intra-pt genetically diverse strains exhibited differences in antibiotic resistance (Fig. 4), viscosity and mucosity, capsular content, and resistance to serum and macrophage killing. Various strains from pts A and J differed in ability to cause target organ infections or mortality in a mouse model of intravenous disseminated infection (Fig. 5). Conclusion We identified genotypic and phenotypic variant strains of ST258 KP from BSIs of individual patients that were not recognized at time of diagnosis. Our data suggest a new, population-based paradigm for BSIs by CRKP. The findings potentially have profound implications for medical, microbiology laboratory and infection prevention practices, and for understanding emergence of antibiotic resistance and pathogenesis. Disclosures Cornelius J. Clancy, MD, receives research funding paid to his institution from Astellas and Merck: Grant/Research Support|serves as an advisory Board member for Astellas, Cidara, and Scynexis, served on the advisory board for Merck, Qpex Biopharma, and Shionogi: Advisor/Consultant|Venatorx and Needham & Associates: Advisor/Consultant.

2224. Aminopenicillins versus Non-aminopenicillins for Treatment of Enterococcal Cystitis

Abstract Background Aminopenicillins achieve urinary concentrations that overcome aminopenicillin resistance in Enterococcus spp. lower urinary tract infections (UTI). With this rationale, our clinical microbiology laboratory discontinued routine identification on Enterococcus urine isolates in 2012 and report “aminopenicillins are predictably reliable for uncomplicated enterococcal UTI”. The study objective was to compare outcomes of patients treated with aminopenicillins (AP) versus non-aminopenicillins (NAP) for enterococcal cystitis. Methods IRB approved, retrospective cohort of adults hospitalized with symptomatic enterococcal cystitis from 2013-2021. Exclusion: definitive E. faecalis, enterococcal UTI in past year, review of systems unavailable/altered mental status, urinary instrumentation except for stent/catheter, systemic infection, fever, genitourinary trauma, renal transplant. Primary endpoint: clinical and microbiologic success at 14 days, defined as resolution of symptoms without new symptoms and no repeat culture growth of index organism. Logistic regression evaluated characteristics associated with 14-day failure. Sample size of 178 calculated for non-inferiority using α=0.025, β=0.8, 15% non-inferiority margin. Results 178 subjects included, 89 AP, 89 NAP . VRE identified: 73 (82%) AP and 76 (85%) NAP (P=0.50). Amoxicillin (36/89, 40%) and ampicillin (36/89, 40%) used most in AP. Linezolid (41, 46%) and fosfomycin (30, 34%) most common agents in NAP. 14-day clinical composite endpoint was no different between AP and NAP groups, respectively (74 [83%] vs. 73 [82%], P=0.84). No variables independently predicted failure (Table 1). Non-inferiority analysis for AP vs. NAP for 14-day composite: mean difference 1.1% (-0.14 to 0.127, 97.5% CI; P=0.42). Definite E. faecium subgroup: 14-day clinical composite success for was no different between AP and NAP groups, respectively (27/34 [79%] vs. 53/66 [80%], P=0.916). Conclusion AP were non-inferior to NAP, supporting their use as first-line therapy for cystitis due to enterococci, including E. faecium. This stewardship strategy has implications to preserve VRE active therapies (e.g. linezolid) for serious infections. Disclosures All Authors: No reported disclosures.

The clinical significance of in-house metagenomic next-generation sequencing for bronchoalveolar lavage fluid diagnostics in patients with lower respiratory tract infections.

Metagenomic next-generation sequencing (mNGS) technology has the potential to detect a wide range of pathogenic microorganisms. However, reports on the diagnostic value and clinical significance of different platforms of mNGS for patients with lower respiratory tract infections (LRTIs) remain scarce. A total of 306 patients with suspected LRTIs were enrolled from January 2019 to December 2021. The diagnostic performance of conventional methods and mNGS on bronchoalveolar lavage fluid (BALF) were compared. BALF mNGS was performed using a commercial and an in-house laboratory. The diagnostic value and the clinical implications of mNGS for LRTIs were analyzed for the different platforms. The positive rate of mNGS in the in-house group was higher than that in the commercial group (85.26% vs. 70.67%, p < 0.001). mNGS significantly increased the pathogen detection rate compared with conventional methods [from 70.67% vs. 22.67% (p < 0.001) to 85.26% vs. 30.77% (p < 0.001)]. The pathogens detected using mNGS included bacteria, fungi, viruses, and atypical pathogens. The in-house platform performed well on a wider spectrum of microbial distribution. Furthermore, it showed an advantage in detecting mixed pathogens in immunocompromised patients. Among the mNGS positive cases, 34 (32.0%) cases had their antibiotics adjusted in the commercial group, while 51 (38.3%) cases had a change of treatment in the in-house group. Moreover, the turnaround time of mNGS and the time from mNGS to discharge in the in-house group were significantly shorter than those in the commercial group. In-house mNGS had a higher detection rate and can show a wider spectrum of pathogens, with potential benefits for the clinic by shortening the turnaround time and hospitalization, and it may be more suitable for clinical microbiology laboratories.

Epidemiology of Escherichia coli as a Critical Pathogen of Bloodstream Infection Patients in Tertiary Referral Hospital

Bloodstream infections (BSI), caused primarily by multidrug-resistant Escherichia coli, are a significant cause of morbidity and mortality worldwide. This study aims to evaluate the epidemiology of E. coli as a critical pathogen in patients with bloodstream infections in a tertiary referral hospital. This is a retrospective study using a descriptive observational research design. This study used a medical record instrument for bloodstream patients in Dr. Soetomo Hospital's inpatient ward with Gram-negative bacteria results of blood cultures in the Clinical Microbiology Laboratory from April 2021 to September 2021. The observed variables include; antimicrobial sensitivity, patient clinical characteristics, demographic data, clinical diagnosis, and clinical outcome. In 6 months, 276 Gram-negative bloodstream infection patients were treated at Dr. Soetomo Hospital. The proportion of E. coli was 17 %. The main characteristics of patients were over 60 years old (28%), and 54% were female. 63% of E. coli were ESBL, and 9% were carbapenem-resistant microorganisms. High antimicrobial resistance was found in quinolones (100%), ampicillin (93%), piperacillin (74%), tetracycline (72%), ceftriaxone (66%), cefotaxime (65%), ceftazidime (60%), cefazolin (65%), and trimethoprim-sulfamethoxazole (65%). The most common potential determinant profile discovered was linked to immunocompromised status due to malignancy. The high number of antimicrobial-resistant bacteria showed the importance of strict infection control and updated epidemiology data as a guide for empirical antimicrobial therapy.

Evaluation of chromogenic agar medium, can it be a suitable alternative to conventional culture system for identification of uropathogens?

Urinary tract infections (UTI) account for major proportion of outpatient load and hospital admission globally. In most of the clinical microbiology laboratories MacConkey agar (MAC) and Cystine lactose electrolyte-deficient (CLED) agar are being used for identification of uropathogens. The main objective of the present study was to evaluate the usefulness of HiCromeTM UTI by comparing isolation rate and presumptive identification of uropathogens against CLED and MAC agar. This study was conducted over a period of three months on 672 non-duplicate midstream and/or catheter-catch urine samples. All samples were inoculated on to HiCromeTM UTI, CLED agar and MacConkey agar. Among the 672 samples received for culture, 113 (16.8%) showed significant growth. Among the 672 samples, 95 (14.1%) showed growth of a single organism while 18 (2.7%) showed polymicrobial growth. The rate of isolation and presumptive identification of the isolates and polymicrobial growth was found significantly higher on HiCromeTM UTI Agar. HiCromeTM UTI Agar has the potential to streamline processing of samples for urine culture in a way that will reduce the workload for technicians, reduce turnaround time which in turn will benefit the laboratory ultimately leading to better patient care.

Phenotypic and Genotypic Detection of Extended-Spectrum β-Lactamases (ESBL) among Escherichia coli Isolated from Symptomatic Female's Genital Tract Infection

Clinical E. coli isolates (36) from women (aged 18-45 years) with symptomatic genital tract infection were detected phenotypically and genotypically for ESBL production. Phenotypically, most (30/36: 83.3%) isolates were resistant to cefotaxime (CTX) and more than half of them (26/36: 72.2%) were resistant to ceftazidime (CAZ). All (100%) and 25 (69.4%) of them were ESBLs producers by screening and confirmatory tests, respectively. Genotypically, ESBL genotypes were detected in 31/36 (86.1%) of isolates. All four ESBL genotypes were found among these isolates with predominance of CTX-M-type (26/36: 72.2%) followed by SHV-type (22/36: 61.1%), OXA-type (7/36: 19.4%), and TEM-type (1/36: 2.7%). Of These isolates, 25/36 (69.4%) had two types of ESBL genes. Seventeen (17/22: 77.2%) of SHV-type positive isolates were CTX-M positive. Six (6/7: 85.7%) of OXA-type and the TEM-type positives were also CTX-M- positive whereas the other OXA-type positive isolate was SHV-type positive. Most (22/25: 88%) isolates with two types of ESBLs were resistant to both CTX and CAZ. It can be concluded that, in female's genital tract infection, ESBL production, especially CTX-M-type, can be added as another factor, in addition to virulence factors, that select for certain strains to survive and cause disease and as vaginal E. coli is a reservoir along the fecal-vaginal-urinary/neonatal course of transmission in extraintestinal E. coli infections, our clinicalmicrobiology labs and clinicians need to be aware of the presence of these ESBL-producing organisms and should conduct surveillance studies to ascertain this.

Applications of MALDI-TOF Mass Spectrometry to the Identification of Parasites and Arthropod Vectors of Human Diseases.

Arthropod vectors and parasites are identified morphologically or, more recently, by molecular methods. Both methods are time consuming and require expertise and, in the case of molecular methods, specific devices. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification of bacteria has meant a major change in clinical microbiology laboratories because of its simplicity, speed and specificity, and its capacity to identify microorganisms, in some cases, directly from the sample (urine cultures, blood cultures). Recently, MALDI-TOF MS has been shown as useful for the identification of some parasites. On the other hand, the identification of vector arthropods and the control of their populations is essential for the control of diseases transmitted by arthropods, and in this aspect, it is crucial to have fast, simple and reliable methods for their identification. Ticks are blood-sucking arthropods with a worldwide distribution, that behave as efficient vectors of a wide group of human and animal pathogens, including bacteria, protozoa, viruses, and even helminths. They are capable of parasitizing numerous species of mammals, birds and reptiles. They constitute the second group of vectors of human diseases, after mosquitoes. MALDI-TOF MS has been shown as useful for the identification of different tick species, such as Ixodes, Rhipicephalus and Amblyomma. Some studies even suggest the possibility of being able to determine, through MALDI-TOF MS, if the arthropod is a carrier of certain microorganisms. Regarding mosquitoes, the main group of vector arthropods, the possibility of using MALDI-TOF MS for the identification of different species of Aedes and Anopheles has also been demonstrated. In this review, we address the possibilities of this technology for the identification of parasites and arthropod vectors, its characteristics, advantages and possible limitations.

Evaluation of Nested broad-range PCR for Pathogen Detection in Negative Blood Cultures

Background: Bloodstream infections are a major cause of morbidity and mortality among all age groups. In the laboratory, it is routinely diagnosed by performing a blood culture which is considered an imperfect gold standard. The diagnostic limitations and uncertainties of blood cultures are related to low sensitivity, particularly in antibiotic-exposed cases, and extended time for pathogen detection that increases turn-around-time of the results. An alternative and recently introduced nucleic acid amplification method for 16S rDNA, a conserved DNA sequence common to all bacteria, has gained significant. It has high sensitivity and an ability to detect organisms that are non-cultivable or nonviable owing to prior antibiotic treatment and where the organism fails to grow on a culture medium. Objectives: To determine the utility of broad-range PCR in the detection of pathogens from negative blood culture was the aim of our study. Materials and Methods: A total of 50 negative blood cultures after 5 days of incubation on BacT/Alert continuously monitored blood culture system were included in this study. From each blood broth sample, DNA was isolated which was subjected to a broad range 16S rDNA nested PCR. The PCR products obtained were visualized on agarose gel electrophoresis and the results were interpreted. Results: Broad range bacterial 16S rDNA nested PCR detected the desired amplicon in 4/50 (8%) of the blood culture-negative samples. Conclusion: The broad range bacterial nested PCR method if used in combination with routine culture techniques in clinical microbiology laboratories will increase the detection of Bloodstream pathogens.

First-person video experiences as a vicarious, virtual alternative to in-person basic science labs.

The global COVID-19 pandemic required the teaching of basic science instructional laboratories be done in a remote, online format termed emergency remote teaching (ERT). The aims of this study were to: (1) share strategies for ERT of basic science instructional laboratories and (2) assess student perceptions of the experience of virtual demonstrations that were recorded from the first-person perspective of the professor. Laboratories for courses in gross anatomy, neuroanatomy, and clinical microbiology were adapted to ERT by creating videos that allowed the students to view the laboratory activities through the eyes and hands of the faculty. A unique 5-question survey instrument was created to collect students' perceptions of gross anatomy, neuroanatomy, and clinical microbiology virtual lab experiences. Percentage of responses were calculated for 4 close-ended questions. Qualitative content analysis was conducted on the single open-ended question. Two additional close-ended questions were used for assessing perception of gross anatomy labs only. Videos of gross anatomy, neuroanatomy, and clinical microbiology laboratory activities mimicked the student experience through the camera lens as labs were performed by faculty members engaged in either dissection, viewing structures or doing experiments, respectively. In all 3 basic science courses, over 70% of students strongly agreed or agreed that the videos created a sense of being in the laboratory. Use of video technology allowed faculty to mimic the student experience of being in basic science laboratories, and, importantly, allowed the student to virtually participate in the learning experience.

Spa Typing of Methicillin-Resistant Staphylococcus aureus Based on Whole-Genome Sequencing: the Impact of the Assembler.

Sequencing of the spa gene of methicillin-resistant Staphylococcus aureus (MRSA) is used for assigning spa types to e.g., detect transmission and control outbreaks. Traditionally, spa typing is performed by Sanger sequencing but has in recent years been replaced by whole-genome sequencing (WGS) in some laboratories. Spa typing by WGS involves de novo assembly of millions of short sequencing reads into larger contiguous sequences, from which the spa type is then determined. The choice of assembly program therefore potentially impacts the spa typing result. In this study, WGS of 1,754 MRSA isolates was followed by de novo assembly using the assembly programs SPAdes (with two different sets of parameters) and SKESA. The spa types were assigned and compared to the spa types obtained by Sanger sequencing, regarding the latter as the correct spa types. SPAdes with the two different settings resulted in assembly of the correct spa type for 84.8% and 97.6% of the isolates, respectively, while SKESA assembled the correct spa type in 98.6% of cases. The misassembled spa types were generally two spa repeats shorter than the correct spa type and mainly included spa types with repetition of the same repeats. WGS-based spa typing is thus very accurate compared to Sanger sequencing, when the best assembly program for this purpose is used. IMPORTANCE spa typing of methicillin-resistant Staphylococcus aureus (MRSA) is widely used by clinicians, infection control workers, and researchers both in local outbreak investigations and as an easy way to communicate and compare MRSA types between laboratories and countries. Traditionally, spa types are determined by Sanger sequencing, but in recent years a whole-genome sequencing (WGS)-based approach has become increasingly used. In this study, we compared spa typing by WGS using different methods for assembling the genome from short sequencing reads and compared to Sanger sequencing as the gold standard. We find substantial differences in correct assembly of spa types between the assembly methods. Our findings are therefore important for the quality of WGS based spa typing data being exchanged by clinical microbiology laboratories.

How MALDI-TOF Mass Spectrometry Technology Contributes to Microbial Infection Control in Healthcare Settings.

Healthcare settings have been utilizing matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) since 2010. MALDI-TOF MS has various benefits over the conventional method of biochemical identification, including ease of use, speed, accuracy, and low cost. This approach can solve many of the obstacles to identifying bacteria, fungi and viruses. As technology advanced, more and more databases kept track of spectra, allowing species with similar morphological, genotypic, and biochemical traits to be identified. Using MALDI-TOF MS for identification has become more accurate and quicker due to advances in sample preparation and database enrichment. Rapid sample detection and colony identification using MALDI-TOF MS have produced promising results. A key application of MALDI-TOF MS is quickly identifying highly virulent and drug-resistant diseases. Here, we present a review of the scientific literature assessing the effectiveness of MALDI-TOF MS for locating clinically relevant pathogenic bacteria, fungi, and viruses. MALDI-TOF MS is a useful strategy for locating clinical pathogens, however, it also has some drawbacks. A small number of spectra in the database and inherent similarities among organisms can make it difficult to distinguish between different species, which can result in misidentifications. The majority of the time additional testing may correct these problems, which happen very seldom. In conclusion, infectious illness diagnosis and clinical care are being revolutionized by the use of MALDI-TOF MS in the clinical microbiology laboratory.

24/7 workflow for bloodstream infection diagnostics in microbiology laboratories: the first step to improve clinical management.

We aimed to evaluate the impact of an uninterrupted workflow regarding blood cultures on turnaround time and antibiotic prescription. Monomicrobial episodes of bacteremia were retrospectively evaluated before and after a continuous 24/7 workflow was implemented in our clinical microbiology laboratory (pre- and post-intervention periods; PREIP and POSTIP). Primary outcome was the time from specimen collection to the first change in antibiotic therapy. Secondary outcomes included the time from specimen collection to effective antibiotic therapy and to antibiotic susceptibility testing results (or turnaround time), as well as hospital length of stay and all-cause mortality at 30days. A total of 548 episodes of bacteremia were included in the final analysis. There was no difference in PREIP and POSTIP regarding patient characteristics and causative bacteria. In POSTIP, the mean time to the first change in antibiotic therapy was reduced by 10.4h (p<0.001). The time to effective antibiotic therapy and theturnaround time were respectively reduced by 4.8h (p<0.001) and 5.1h (p=0.006) in POSTIP. There was no difference in mean hospital length of stay or mortality between the two groups. Around the clock processing of blood cultures allows for a reduction in turnaround time, which in turn reduces the delay until effective antibiotic therapy prescription.

Nasopharyngeal aspirates in children with severe community-acquired pneumonia collected within 3 days before bronchoscopy can partially reflect the pathogens in bronchoalveolar lavage fluids

BackgroundThere is little evidence about consistency between nasopharyngeal and pulmonary pathogens in children with severe pneumonia. This study aims to compare the difference of pathogens between nasopharyngeal aspirates (NPAs) collected before bronchoscopy and bronchoalveolar lavage fluids (BALFs) in children with severe community-acquired pneumonia (SCAP).MethodsNPAs and BALFs were collected form pediatric SCAP cases hospitalized from January 2018 to March 2019. NPAs were colleced within 3 days before bronchoscopy. Samples were detected by direct immunofluorescence assay (DFA) for seven respiratory viruses and by routine bacterial culture in the clinical microbiology laboratory. Respiratory syncytial virus (RSV), Adenovirus (ADV), Influenza virus types A, B (IV-A and IV-B), Parainfluenza virus 1–3 (PIV1-3) were detected with a commercial assay. The virological and bacteriological detention results of NPAs were compared with the results of BALFs.ResultsIn total 204 cases with mean age of 3.4 ± 2.8 years (IQR, 1 month-14 years) were included in the study. Both NPA and BALF were collected from those cases. The positive rates of pathogen in NPAs and BALFs were 25.0% (51/204) and 36.7% (75/204), respectively (x2 = 6.614, P = 0.010). Respiratory viruses were found in 16.1% (33/204) from NPAs and 32.3% (66/204) from BALFs (x2 = 14.524, P < 0.001). RSV and ADV were the two most frequent detected viruses in NPAs and BALFs. High consistentcy of pathogens between NPAs and BALFs was observed, and 96.9% (32/33) viruses detected in NPAs were also found in BALFs. While bacteria were isolated from 12.7% (26/204) and 10.7% (22/204) of the two kinds of samples, respectively (x2 = 0.378, P = 0.539). In addition, Haemophilus influenzae (HI) was the dominant germ in both samples.ConclusionThe DFA method used to detect seven respiratory viruses from NPAs collected within 3 days before bronchoscopy can partially reflect the pathogens in the lungs in children with SCAP.

Identification and genomic analysis of a Vibrio cholerae strain isolated from a patient with bloodstream infection

Vibrio cholerae is a bacterium ubiquitous in aquatic environments which can cause widespread infection worldwide. V. cholerae gradually became a rare species of bacteria in clinical microbiology laboratories with the control of the cholera epidemic. In this study, we isolated a V. cholerae strain, named VCHL017, from the blood of an elderly patient without gastrointestinal symptoms. The patient had a history of hookworm infection and multiple myeloma. Furthermore, she was immunocompromised, and received long-term chemotherapy and antimicrobial agents. VCHL017 was inoculated on blood agar and thiosulfate citrate bile salt sucrose plates (TCBS) to observe morphological characteristics. Then this isolate was identified by matrix-assisted laser desorption/ionization time-of-flight spectrometry (MALDI-TOF MS). The minimum inhibitory concentrations (MICs) for cefazolin, ceftazidime, cefepime, meropenem, tetracycline, ciprofloxacin, chloramphenicol, and gentamicin of VCHL017 were determined by the microbroth dilution method. PCR and serum agglutination tests were used to determine whether the serogroups of the isolate belonged to the O1/O139 and cholera toxin encoding genes. Finally, the genomic features and phylogeny of VCHL017 were analyzed by whole genome sequencing (WGS). VCHL017 was a non-O1/O139 V cholerae strain that did not carry the ctxA gene. Antimicrobial susceptibility tests revealed that VCHL017 was susceptive to chloramphenicol and tetracycline. Although it did not carry the genes encoding the cholera toxin, WGS indicated that VCHL017 carried a variety of other virulence factors. By calculating the average nucleotide identity (ANI), we precisely identified the species of VCHL017 as V. cholerae. There are also A171S and A202S missense mutations in gyrA of VCHL017. The phylogenetic analysis indicated that VCHL017 was closely related to V. cholerae strains isolated from aquatic environments. Our results suggest that continuous monitoring is necessary for non-O1/O139 V cholerae strains isolated from outside the digestive tract, which could be pathogenic through multiple virulence factors.

Un percorso di integrazione tra strutture ospedaliere e servizi territoriali

The forty years of work in the Ser.D. have taught us to grasp changes, often beyond laws and resources, proposing the correct organizational evolutions. The evolution of consumption and states of dependence, the new consumption of young people, the challenges to the limit of risks, the addictive behaviors such as chemic sex recall the need for new reading paradigms, a new clinic and new operational tools. A collaboration between the SMEL Clinical Chemical Analysis and Microbiology Laboratory of the Lodi Hospital and the Ser.D. has begun a few months ago, aimed at introducing a new protocol for the research of substances of abuse on the keratin matrix in drug addiction monitoring. Furthermore, with this type of analysis, the laboratory can provide its Ser.D. with a more effective analysis tool in monitoring patients. The high specificity of the test is able to determine greater diagnostic reliability with a consequent improvement in the specificity of treatment.

Direct, Rapid Detection of Pathogens from Urine Samples.

The problem of rapidly detecting pathogens directly from clinical samples poses significant analytical challenges. Addressing this issue in relation to urinary tract infections, we propose an effective protocol and related immunomagnetic test kits enabling versatile screening for the presence of pathogenic bacteria in unprocessed urine samples. To achieve this, the components of a typical immunomagnetic separation protocol were optimized towards the sensitive assessment of the aggregates formed out of immunomagnetically tagged target pathogens collected from clinical samples. Specifically, a dedicated immunomagnetic material was developed via the functionalization of standardized, micron-sized magnetic beads with generic antibodies against gram-specific bacterial constituents with mannan binding lectin. As such, we demonstrate efficient procedures for achieving the enhanced, specific, and pathogen-mediated cluster formation of these tailored affinity-coated magnetic beads in complex samples. We further show how cluster analysis, in conjunction with the use of nonspecific, inexpensive fluorescent dye, allows for a straightforward optical assessment of the bacterial load directly from urine samples. The optimized sensing protocol and related kits provide, in less than 60 min, qualitative (positive/negative) information on the bacterial load with 85% specificity and 96% sensitivity, which is appropriate to empower clinical microscopy with a new analytic dimension. The procedure is prone to automation, can be conveniently used in clinical microbiology laboratories and, since it preserves the viability of the captured bacteria, can be interfaced with downstream analyses and antimicrobial susceptibility testing. Moreover, the study emphasizes a suite of practical validation assays that are useful for bringing the tool-box of immunomagnetic materials outside the academic laboratory and into real-life applications.

The Antibiotic Resistance Rates in Multidrug Resistant Acinetobacter Isolates from Microbiological Culture Samples of Adult Patients in Turkey: A Retrospective Study

Objective: Acinetobacter species lead to extremely serious infections, particularly in hospitalized patients, and in patients with impaired host defense. The high rates of resistance against several antibiotics detected in recent years have created serious issues in treatments of different diseases. We aimed to examine antibiotic resistance profiles of Acinetobacter species isolated from patients who are treated as outpatients in polyclinics or hospitalized in services or intensive care units (ICU), against various antimicrobial therapies. Materials and Methods: Antibiotic resistance of Acinetobacter strains isolated from 533 clinical samples collected between 2017-2021 years in Bandırma State Hospital Clinical Microbiology Laboratory were evaluated retrospectively. The identification of isolates and antibiotic susceptibility tests were performed by BD Phoenix (Becton Dickinson, USA) automated system. Results: Most of Acinetobacter strains were isolated from respiratory secretions (32.5%) and from urine (24.4%). Of species, 63.8% were Acinetobacter baumannii, 34.9% Acinetobacter baumannii complex, 1.1% other Acinetobacter spp., 0.2% Acinetobacter lwoffii. Resistance rates to antibiotics were found as following: ciprofloxacin 91.1%, meropenem 91.3%, imipenem 89.2%, gentamicin 82.5%, trimethoprim-sulfamethaxasol 78.6%, amikacin 66.3% (highest in 2020), aztreonam 99.0% (significantly decreased in 2020), ceftriaxone 100%, ampicillin 100%, amoxicillin-clavulanate 100%, ertapenemicin 100%, cefuroxime 100%, netilmicin 62.5%, nitrofurantion 100%, colistin 4.7% and levofloxacin 87.1%. The samples collected from patients hospitalized in service and ICU were found more resistant against Ciprofloxacin, Levofloxacin, Meropenem, Imipenem, Trimethoprim/Sulfamethoxazole, Gentamicin and Amikacin (P&lt;0.0001). Conclusion: Colistin resistance against Acinetobacter infections was observed to be low, hence colistin could be utilized in treatments. Infection control measures have to be taken in services and ICU, and rational antibiotic use policies should be applied so as to prevent the spread of infection