Clinical Microbiology Laboratory Research Articles

Evaluation of a new rapid immunochromatographic assay for the detection of GES-producing Gram-negative bacteria.

As carbapenemase-producing Enterobacterales are increasingly reported worldwide, their rapid detection is crucial to reduce their spread and prevent infections and outbreaks. Lateral flow immunoassays (LFIAs) have become major tools for the detection of carbapenemases. However, as for most commercially available assays, only the five main carbapenemases are targeted. Here, we have developed and evaluated an LFIA prototype for the rapid and reliable detection of the increasingly identified GES-type β-lactamases. The GES LFIA was validated on 103 well-characterized Gram-negative isolates expressing various β-lactamases grown on Mueller-Hinton (MH) agar, chromogenic, and chromogenic/selective media. The limit of detection of the assay was 106 cfu per test with bacteria grown on MH agar plates. GES LFIA accurately detected GES-type β-lactamases irrespective of the culture media and the bacterial host. The GES LFIA was not able to distinguish between GES-ESBLs and GES-carbapenemases. Because GES enzymes are still rare, their detection as an ESBL or a carbapenemase remains important, especially because extensive use of carbapenems to treat ESBL infections may select for GES variants capable of hydrolysing carbapenems. The GES LFIA is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of GES-type β-lactamases. Combining it with immunochromatographic assays targeting the five main carbapenemases (KPC, NDM, VIM, IMP and OXA-48) would improve the overall sensitivity for the most frequently encountered carbapenemases and ESBLs, especially in non-fermenters.

Artificial intelligence-aided rapid and accurate identification of clinical fungal infections by single-cell Raman spectroscopy.

Integrating artificial intelligence and new diagnostic platforms into routine clinical microbiology laboratory procedures has grown increasingly intriguing, holding promises of reducing turnaround time and cost and maximizing efficiency. At least one billion people are suffering from fungal infections, leading to over 1.6 million mortality every year. Despite the increasing demand for fungal diagnosis, current approaches suffer from manual bias, long cultivation time (from days to months), and low sensitivity (only 50% produce positive fungal cultures). Delayed and inaccurate treatments consequently lead to higher hospital costs, mobility and mortality rates. Here, we developed single-cell Raman spectroscopy and artificial intelligence to achieve rapid identification of infectious fungi. The classification between fungi and bacteria infections was initially achieved with 100% sensitivity and specificity using single-cell Raman spectra (SCRS). Then, we constructed a Raman dataset from clinical fungal isolates obtained from 94 patients, consisting of 115,129 SCRS. By training a classification model with an optimized clinical feedback loop, just 5 cells per patient (acquisition time 2 s per cell) made the most accurate classification. This protocol has achieved 100% accuracies for fungal identification at the species level. This protocol was transformed to assessing clinical samples of urinary tract infection, obtaining the correct diagnosis from raw sample-to-result within 1 h.

Prevalence and molecular characterization of methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Staphylococcus aureus (VRSA) in a tertiary care hospital

Resistance shown by Staphylococcus aureus to methicillin; mediated by mecA, and vancomycin; mediated by vanA, has led to difficulty in treatment of related infections. Despite reports showing methicillin resistant S. aureus (MRSA) and vancomycin resistant S. aureus (VRSA) in Nepal, and need for their regular surveillance, no study has been conducted on it in our hospital. So, this study is aimed to determine prevalence of MRSA, VRSA and their molecular characterization along with antibiogram. A descriptive cross-sectional study was conducted from August to December, 2022 in Clinical Microbiology Laboratory of NMCTH among S. aureus (n=160) isolated from various clinical specimens after receiving ethical approval from NMC-IRC. AST was done by modified Kirby-Bauer’s disc diffusion method. MRSA and VRSA were detected by cefoxitin disc method and agar dilution method respectively. Inducible clindamycin resistance was detected by D-test. Resistant genes (mecA, PVL, and vanA) were detected using conventional PCR. Prevalence of MRSA was found to be 31.2% (50/160) but none of the isolates were resistant to vancomycin. Total 7 (46.6%) mecA and 7 (46.6%) PVL genes were detected among 15 selected MRSA isolates but vanA was not found. All the MRSA isolates were susceptibile to co-trimoxazole, tigecycline, chloramphenicol, vancomycin, teicoplanin and linezolid. The resistance rate against ciprofloxacin, ofloxacin, and clindamycin was 52.0%, 44.0%, and 68.0% (20.0% iMLSB, 28.0% cMLSB and 16.0% MS-phenotypes) respectively. Prompt implementation of hospital antibiotic policy and AMR Act by government along with regular surveillance of MRSA and VRSA seems essential to contain MRSA infections. Co-trimoxazole could be treatment option against MRSA in our setting, keeping vancomycin in reserve. However, large scale studies are required to establish this conclusion.

Antimicrobial susceptibility testing: An updated primer for clinicians in the era of antimicrobial resistance: Insights from the Society of Infectious Diseases Pharmacists.

Antimicrobial susceptibility testing (AST) is a critical function of the clinical microbiology laboratory and is essential for optimizing care of patients with infectious diseases, monitoring antimicrobial resistance (AMR) trends, and informing public health initiatives. Several methods are available for performing AST including broth microdilution, agar dilution, and disk diffusion. Technological advances such as the development of commercial automated susceptibility testing platforms and the advent of rapid diagnostic tests have improved the rapidity, robustness, and clinical application of AST. Numerous accrediting and regulatory agencies are involved in the process of AST and setting and revising breakpoints, including the U.S. Food and Drug Administration and the Clinical and Laboratory Standards Institute. Challenges to optimizing AST include the emergence of new resistance mechanisms, the development of new antimicrobial agents, and generation of new data requiring updates and revisions to established methods and breakpoints. Together, the challenges in AST methods and their interpretation create important opportunities for well-informed clinicians to improve patient outcomes and provide value to antimicrobial stewardship programs, especially in the setting of rapidly changing and increasing AMR. Addressing AST challenges will involve continued development of new technologies along with collaboration between clinicians and the laboratory to facilitate optimal antimicrobial use, combat the increasing burden of AMR, and inform the development of novel antimicrobials. This updated primer serves to reinforce important principles of AST, and to provide guidance on their implementation and optimization.

Characterization of SARS-CoV-2 Distribution and Microbial Succession in a Clinical Microbiology Testing Facility during the SARS-CoV-2 Pandemic.

The exchange of microbes between humans and the built environment is a dynamic process that has significant impact on health. Most studies exploring the microbiome of the built environment have been predicated on improving our understanding of pathogen emergence, persistence, and transmission. Previous studies have demonstrated that SARS-CoV-2 presence significantly correlates with the proportional abundance of specific bacteria on surfaces in the built environment. However, in these studies, SARS-CoV-2 originated from infected patients. Here, we perform a similar assessment for a clinical microbiology lab while staff were handling SARS-CoV-2 infected samples. The goal of this study was to understand the distribution and dynamics of microbial population on various surfaces within different sections of a clinical microbiology lab during a short period of 2020 Coronavirus disease (COVID-19) pandemic. We sampled floors, benches, and sinks in 3 sections (bacteriology, molecular microbiology, and COVID) of an active clinical microbiology lab over a 3-month period. Although floor samples harbored SARS-CoV-2, it was rarely identified on other surfaces, and bacterial diversity was significantly greater on floors than sinks and benches. The floors were primarily colonized by bacteria common to natural environments (e.g., soils), and benchtops harbored a greater proportion of human-associated microbes, including Staphylococcus and Streptococcus. Finally, we show that the microbial composition of these surfaces did not change over time and remained stable. Despite finding viruses on the floors, no lab-acquired infections were reported during the study period, which suggests that lab safety protocols and sanitation practices were sufficient to prevent pathogen exposures. IMPORTANCE For decades, diagnostic clinical laboratories have been an integral part of the health care systems that perform diagnostic tests on patient's specimens in bulk on a regular basis. Understanding their microbiota should assist in designing and implementing disinfection, and cleaning regime in more effective way. To our knowledge, there is a lack of information on the composition and dynamics of microbiota in the clinical laboratory environments, and, through this study, we have tried to fill that gap. This study has wider implications as understanding the makeup of microbes on various surfaces within clinical laboratories could help identify any pathogenic bacterial taxa that could have colonized these surfaces, and might act as a potential source of laboratory-acquired infections. Mapping the microbial community within these built environments may also be critical in assessing the reliability of laboratory safety and sanitation practices to lower any potential risk of exposures to health care workers.

Combination of UC-3500 and UF-5000 as a quick and effective method to exclude bacterial urinary tract infection

BackgroundOur study aims to evaluate the performance of the combination of Sysmex urine dry chemistry analyzer UC-3500 and urine particle analyzer UF-5000 in screening bacterial urinary tract infection (UTI). MethodsWe analyzed 2000 urine specimens from patients with suspected UTI by using a urine dry chemistry analyzer (UC-3500) and a fully automated sediment analyzer (UF-5000). After being tested by the instrument, all specimens were sent to our clinical microbiology laboratory for culture. In addition, 600 urine specimens were selected to evaluate the accuracy of the six screening strategies established in this study. ResultsThe consistency of UF-5000 bacterial classification and bacterial culture was fair (Kappa = 0.339). The counts of WBC and BACT elevated with sequential group designs (P < 0.001). The cut-off value of WBC was 32.20/μL for males (AUC, 0.942, 95%CI, 0.930–0.955) and 39.15/μL for females (AUC, 0.931, 95%CI, 0.914–0.948). The sensitivity and specificity of WBC were relatively higher than those of BACT. Strategy④ and Strategy⑥ in all six strategies had a good negative predictive value (NPV) which was 98.73%. ConclusionUF-5000 bacterial classification cannot be used as a practical reference. 32.20/μL (male) and 39.15/μL (female) for WBC as well as 22.35/μL (male) and 127.25/μL (female) for BACT were used as cut-off values to effectively determine whether UTI occurs. WBC, BACT and LEU joint screening programs were suitable to rapidly and effectively exclude bacterial UTI.

Genomic and clinical case characterisation of Staphylococcus haemolyticus isolated from dogs and cats in the United States, including strains with high-level mupirocin tolerance.

Staphylococcus haemolyticus is a coagulase-negative commensal organism of both people and companion animals. It has pathogenic potential and when cultured is often meticillin- and multidrug-resistant. To characterise the clinical features of dogs and cats with clinical skin disease that had positive S.haemolyticus skin cultures, and to employ whole-genome sequencing (WGS) to identify resistance genes and characterise the genetic relatedness of strains. Isolates were identified by the institutional clinical microbiology laboratory by routine aerobic culture and susceptibility from seven veterinary hospitals across the United States. Then, WGS and analysis of each isolate were performed and clinical data collected via a retrospective clinician questionnaire. S. haemolyticus was identified from superficial (seven of 12) and deep (five of 12) cutaneous infections in our study. Most animals had received antimicrobials (10 of 12) and/or immunomodulatory drugs (nine of 12) within the six months before culture. WGS analysis revealed a variety of genetic lineages and a wide array of antimicrobial resistance genes. Meticillin resistance was identified in nine of 12 isolates and four of 12 isolates demonstrated mupirocin tolerance. Staphylococcus haemolyticus may be an under-recognised pathogen in companion animals, and its demonstrated potential for multidrug-resistance, meticillin-resistance, and high-level mupirocin tolerance may create a therapeutic challenge. Further studies should evaluate the prior antimicrobial use and immunocompromised status as risk factors for infection with S.haemolyticus.

Culturing clinical Methanobrevibacter smithii using GG medium in a minimal anaerobe atmosphere.

Methanobrevibacter smithii (M. smithii), the most prevalent and abundant gut methanogen, detoxifies hydrogen into methane and is, therefore, of paramount importance for the equilibrium of the gut microbiota. The isolation by culture of M. smithii has routinely relied upon hydrogen‑carbon dioxide-enriched, oxygen-deprived atmospheres. In this study, we developed a medium referred to as “GG”, which allowed for M. smithii growth and isolation by culture in an oxygen-deprived atmosphere, with no supply of either hydrogen or carbon dioxide, making it easier to detect M. smithii by culture in clinical microbiology laboratories.

Candidiasis profile at the outpatient department of the university of cape coast hospital in the central region of Ghana: a retrospective study

IntroductionVulvovaginal candidiasis (VVC) is a public health problem with an estimated 138 million women globally experiencing recurrent VVC annually. The microscopic diagnosis of VVC has low sensitivity, but it remains an essential tool for diagnosis as the microbiological culture methods are limited to advanced clinical microbiology laboratories in developing countries. The study retrospectively analyzed the presence of red blood cells (RBCs), epithelial cells (ECs), pus cells (PCs) and Candida albicans positive in wet mount preparation of urine or high vaginal swabs (HVS) samples to test for their sensitivity and specificity for the diagnosis of candidiasis.MethodsThe study is a retrospective analysis at the Outpatient Department of the University of Cape Coast between 2013 and 2020. All urine and high vagina swabs (HVS) cultures samples using Sabourauds dextrose agar with wet mount data were analyzed. 2 × 2 contingency diagnostic test was used to ascertain the diagnostic accuracy of red blood cells (RBCs), epithelial cells (ECs), pus cells (PCs), and Candida albicans positive in wet mount preparation of urine or high vaginal swabs (HVS) samples for the diagnosis of candidiasis. The association of candidiasis among patients' demographics was analyzed using relative risk (RR) analysis.ResultsThe high prevalence of candida infection was among female subjects 97.1% (831/856) compared to males 2.9% (25/856). The microscopic profiles which characterized candida infection were pus cells 96.4% (825/856), epithelial cells 98.7% (845/856), red blood cells (RBCs) 7.6% (65/856) and Candida albicans positive 63.2% (541/856). There was a lower risk of Candida infections among male patients compared to female patients RR (95% CI) = 0.061 (0.041–0.088). The sensitivity (95%) for detecting Candida albicans positive and red blood cells (0.62 (0.59–0.65)), Candida albicans positive and pus cells (0.75 (0.72–0.78)) and Candida albicans positive and epithelial cells (0.95 (0.92–0.96)) with corresponding specificity (95% CI) of 0.63 (0.60–0.67), 0.69 (0.66–0.72) and 0.74 (0.71–0.76) were detected among the high vaginal swab samples.ConclusionIn conclusion, the study has shown that the presence of PCs, ECs, RBCs or ratio of RBCs/ECs and RBCs/PCs in the wet mount preparation from urine or HVS can enhance microscopic diagnosis of VVC cases.

Clinical distribution and drug resistance characteristics of carbapenem-resistant Klebsiella pneumoniae

To explore the clinical distribution and drug resistance characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP), in order to provide reference for the prevention and treatment of CRKP infection. Retrospective analysis was performed on 510 clinical isolates of CRKP from January 2017 to December 2021, and strain identification and drug sensitivity tests were conducted by MALDI-TOF mass spectrometer and VITEK-2 Compact microbial drug sensitivity analyzer. The carbapenemase phenotype of CRKP strain was detected by carbapenemase inhibitor enhancement test. The CRKP strain was further categorized by immunochromogenic method and polymerase chain reaction (PCR) was used for gene detection. The results showed that 302 strains (59.2%) were derived from sputum, 127 strains (24.9%) from urine and 47 strains (9.2%) from blood. 231 (45.3%) were mainly distributed in intensive care, followed by 108 (21.2%) in respiratory medicine and 79 (15.5%) in neurosurgery. Drug susceptibility test result shows that the resistant rate of tigecycline increased from 1.0% in 2017 to 10.1% in 2021, the difference was statistically significant (χ2=14.444,P<0.05). The results of carbapenemase inhibitor enhancement test showed that 461 carbapenemase strains (90.4%) of 510 CRKP strains, including 450 serinase strains (88.2%), 9 metalloenzyme strains (1.8%), and 2 strains (0.4%) produced both serine and metalloenzyme. 49 strains (9.6%) did not produce enzymes. Further typing by immunochromogenic assay showed that 461 CRKP strains were KPC 450 (97.6%) and IMP 2 (0.4%). 7 NDM (1.5%); 2 strains of KPC+NDM (0.4%); PCR results were as follows: 450 strains of blaKPC (97.6%), 2 strains of blaIMP (0.4%), 7 strains of blaNDM (1.5%), and 2 strains of blaKPC+NDM (0.4%). In conclusion, CRKP strains mainly originated from sputum specimens and distributed in intensive care department, and the drug resistance characteristics were mainly KPC type in carbapenemase production. Clinical microbiology laboratory should strengthen the monitoring of CRKP strains, so as to provide reference for preventing CRKP infection and reducing the production of bacterial drug resistance.

Comparison between the time to detection of pathogenic and nonpathogenic microorganisms from blood culture specimens using the BACTECTM FX machine to determine the optimal incubation time

Introduction: Bloodstream infections can lead to a high rate of life-threatening sepsis, morbidity and mortality, requiring early and accurate detection. The gold standard for diagnosing bacteremia is blood culture. The recommended incubation time of blood cultures is five days minimum, but studies have shown that five-day incubation is unnecessary. This study aims to determine the optimal blood incubation time on the BACTECTM FX machine. Methods: This is an analytic observational study with a cross-sectional retrospective approach. The samples resulted from tests in the Clinical Microbiology Laboratory and medical record data of patients at Dr. Soetomo Regional Public Hospital, Surabaya, with suspected bacteremia and fungemia-positive blood cultures based on tests with the BACTECTM FX machine who met the inclusion criteria. Samples were analyzed for Time to Detection (TTD) and identification results. TTD of 96–120 hours was analyzed to assess the occurrence of Laboratory-Confirmed Bloodstream Infection (LCBI). Results: From a total of 2,221 samples, 875 (39.4%) pathogenic microorganisms and 1,346 (60.6%) nonpathogenic microorganisms were identified. At TTD &lt; 24 hours, the number of pathogenic microorganisms was higher, reaching 86.6%, than that of nonpathogenic microorganisms, reaching 68.8%. Whereas at TTD of 24–&lt; 48 hours, nonpathogenic microorganisms had a higher count, reaching 23.1%, than pathogenic microorganisms, reaching 10.1%. A total of 2,203 (99.2%) microorganisms were identified at TTD &lt; 96 hours, and as many as 18 (0.8%) microorganisms with TTD &gt; 96 hours. Conclusion: There is no difference in the distribution of pathogenic and nonpathogenic microorganisms at TTD &lt; 96 hours and TTD 96–120 hours. Based on tests using the BACTECTM FX machine, five LCBI-group microorganisms based on NHSN criteria were found with TTD of 96–120 hours, namely Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Trichosporon asahi, and coagulase-negative Staphylococcus, and thirteen non-LCBI-group microorganisms were identified.

The Molecular Technology Revolution and Bacterial Identification: Unexpected Consequences for Clinical Microbiologists

The introduction of sophisticated molecular technologies (16S rRNA gene sequencing, matrix-assisted laser desorption ionization–time of flight [MALDI-TOF], and whole-genome sequencing) into many clinical and diagnostic microbiology laboratories has brought with it enhanced capabilities for the accurate identification of many prokaryotic species not resolvable by common phenotypic or commercially automated methods. Along with this heightened capacity to provide highly accurate bacterial identifications have come some indirect consequences that may not be entirely appreciated by the scientific community. Some examples of these consequences are a transitional approach to training and a different skill set for current laboratorians, a quickly changing bacterial taxonomy, and peer-reviewed literature requiring much closer scrutiny. This article provides an overview of the present situation and challenges to microbiologists as the field moves forward.

Comparison of in vitro fosfomycin susceptibility testing methods with agar dilution for carbapenem resistant Klebsiella pneumoniae and Escherichiacoli.

The emergence of Extensively drug resistant (XDR) pathogens like Carbapenem Resistant Klebsiella pneumoniae (CR Kpn) and Carbapenem Resistant Escherichia coli (CR Eco) has limited therapeutic options for treating them. Fosfomycin a broad-spectrum antibiotic, has emerged as a potential treatment option in combination with other agents. It is therefore important that accurate drug susceptibility testing (DST) results of fosfomycin should be available to all clinical microbiology laboratories. Agar dilution which is the recommended method for fosfomycin DST is not convenient to adopt in a routine set-up. This study aimed to determine the susceptibility pattern of CR Kpn and CR Eco to fosfomycin and to evaluate the discrepancies of the available manual MIC based alternative methods. Agar dilution (AD), broth microdilution (BMD), E-test and Ezy MIC test were performed on 235 CR-Kpn and Eco isolates respectively. Of 177 CR Kpn, 31.63% (n​=​56/177) of the isolates were susceptible by AD. Categorical Agreement (CA) by BMD, E-test and Ezy MIC were lower than the acceptable limit while Very Major Errors (VMEs) and Major Errors (MEs) were beyond the acceptable limits. In the case of CR Eco, 96.55% (n​=​56/58) were susceptible by AD. CA of 100% (n​=​58/58) was shown by both BMD and Ezy MIC while 86.20% (n​=​50/58) was shown by E-test, with no VME observed for CR Eco. ME was only observed for E-test method. The alternative methods were in poor agreement with AD method for CR Kpn and for CR Eco, BMD and Ezy MIC have shown reliable results.

Increasing Parechovirus Central Nervous System Infections and the Enduring Value of Clinical Microbiology Laboratories.

In early spring 2022, clinical microbiologists in Tennessee noticed an increasing trend of parechovirus (PeV) central nervous system (CNS) infections in infants <3 months through routine monitoring of test results for quality assurance. This initiated a discussion on ClinMicroNet, an online forum for clinical microbiologists supported by the American Society of Microbiology (ASM), about the unseasonably early circulation of PeV and increased CNS infections. Additional microbiologists in the United States noted similar trends, though reports differed by geographic region. The Tennessee trend was reported to the Tennessee Department of Health and then to the Centers for Disease Control and Prevention (CDC). These and other observations prompted release of a CDC Health Alert Network Health Advisory on July 12, 2022, which warned of increased PeV-A3 circulation in the United States. The CDC Health Advisory recommended inclusion of PeV in the differential for patients, particularly infants, with meningoencephalitis during seasons of high transmission (summer and fall). Notably, the presentation of meningoencephalitis in the Tennessee cases differed from conventional presentations in that cerebrospinal fluid pleocytosis was absent in most of the children. A case series of the Tennessee patients was thereafter reported in the CDC Morbidity and Mortality Weekly Report (MMWR), detailing 23 PeV CNS infections detected with a US Food and Drug Administration-approved multiplex molecular panel in infants <3 months over 6 weeks (1).

Molecular Characterization and Clinical Relevance of Taxonomic Reassignment of Staphylococcus schleiferi Subspecies into Two Separate Species, Staphylococcus schleiferi and Staphylococcus coagulans.

Staphylococcus schleiferi is an opportunistic pathogen in humans and dogs. Recent taxonomic reassignment of its subspecies (S. schleiferi subsp. schleiferi and S. schleiferi subsp. coagulans) into two separate species (S. schleiferi and S. coagulans) lacks supporting data for diagnostic implications and clinical relevance. We aimed to confirm the reclassification of S. schleiferi by using genomic and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) data for a large set of isolates from humans and animals to investigate their molecular epidemiology and clinical relevance. Routine MALDI-TOF analysis and Illumina sequencing were performed on 165 S. schleiferi isolates from the Netherlands. With 33 publicly available genomes, the study included 198 genomes from 149 dogs, 34 humans, and 15 other sources. The Type Strain Genome Server was used to identify species in the genomes, and the MALDI-TOF MS database was extended to improve species differentiation. MALDI-TOF did not discriminate between S. schleiferi and S. coagulans. Genome phylogeny distinguished the two species in two monophyletic clusters. S. schleiferi isolates originated from humans, while S. coagulans isolates were found in animals and three human isolates clustering with the animal isolates. The sialidase B gene (nanB) was a unique marker gene for S. schleiferi, whereas the chrA gene was exclusive for S. coagulans. The mecA gene was exclusively detected in S. coagulans, as were the lnu(A), blaZ, erm(B/C), tet(O/M), and aac(6')-aph(2'') genes. The MALDI-TOF database extension did not improve differentiation between the two species. Even though our whole-genome sequencing-based approach showed clear differentiation between these two species, it remains critical to identify S. schleiferi and S. coagulans correctly in routine diagnostics. IMPORTANCE This study clearly shows that S. schleiferi is a concern in human hospital settings, whereas S. coagulans predominantly causes infections in animals. S. coagulans is more resistant to antibiotics and can sometimes transmit to humans via exposure to infected dogs. Even though genome-based methods can clearly differentiate the two species, current diagnostic methods used routinely in clinical microbiology laboratories cannot distinguish the two bacterial species.

Wound swab quality grading is dependent on Gram smear screening approach

Superficial skin swab collections are inherently low-quality and may be of little clinical value due to their poor sensitivity and specificity. Clinical microbiology laboratories can use Gram smears to screen and differentiate higher and lower quality specimens to direct the extent of potential pathogen work up, including antimicrobial susceptibility testing (AST). We compared the impact of two different smear grading approaches to our current reporting practices for superficial wound swab cultures. Two variations of the Q score methodology (low power under 10X (QS10) and high power under 100X (QS100) were compared to our existing oil immersion method (OM100) (100X). We further evaluated the QS100 method by scoring superficial swab smears previously screened by OM100 from cultures submitted between November 2018 and December 2019. No significant difference in the number of low-quality specimens (N = 50) was identified by QS10 or QS100 grading (N = 9; 18%; N = 8; 16% respectively). Among 968 additional QS100 screened smears, 67 (6.9%) low quality swabs were identified and 7.4% fewer organisms (76/1020 organisms) would require reporting with AST. Implementing the Q score for superficial wound swab cultures would provide minimal improvements in their clinical relevance, laboratory quality and efficiency in our laboratory due to the low number of poor-quality swabs received.

Microbiology diagnostic approach in identifying Streptococcus pneumoniae: Case report of Streptococcal meningitis

Introduction: Bacterial meningitis remains a serious global health problem. Streptococcus pneumoniae as the causative organism, is fastidious. Microbiology diagnostic approach is needed to identify S.pneumoniae and antimicrobial susceptibility tests (AST). This study aims to report S. pneumoniae meningitis has been successfully cured strongly related to diagnostic stewardship. Case: A 1 year old child who fell, suffering from vomiting, fever and seizures was brought to the Emergency Department. Multi-slice Computerized Tomography shows cerebral edema and intracerebral hemorrhage. Laboratory blood tests and cerebrospinal fluid analysis strongly indicate bacterial meningitis. Presumptive Gram microscopy of Cerebrospinal Fluid (CSF) and initial identification of CSF cultures on lysed blood agar under microaerophilic conditions, consistently showed lancet-shaped and encapsulated Gram-positive diplococci. Our Clinical Microbiology Laboratory finally identified the growth of S.pneumoniae from blood and CSF cultures. Patients were treated with cefepime 50 mg/kg body weight/8 hours in the first 10 days as empirical and switched later definitively to ciprofloxacin 10 mg/kg body weight/12 hours according to the AST result on day 11 to day 26. On the 31ˢᵗ day, the patient recovered and was discharged. Conclusion: The case of a 1 year-old child with S. pneumoniae meningitis has been successfully cured strongly related to diagnostic stewardship by the Clinical Microbiology Laboratory. Supplementation media with sodium bicarbonate can improve S. pneumoniae recovery as a basis for identification and AST. Proper inoculation and AST need to be accelerated because S.pneumoniae easily undergoes autolysis.

Evaluasi Kejadian Infeksi pada Pasien Luka Bakar yang Dirawat Inap di RSUD dr. Zainoel Abidin

This study examines the prevalence of infection in burn patients as represented by the clinical spesimen culture results. The purpose of this study was to obtain data on the prevalence, distribution and susceptibility pattern of microbes isolated from clinical spesimens of burn patients hospitalized at RSUD dr. dr. Zainoel Abidin. This study is descriptive observational. The retrospective data obtained from the Plastic Surgery Department and the Clinical Microbiology Laboratory. The results showed the prevalence of infection in burn patients who were hospitalized at RSUD dr. Zainoel Abidin based on clinical spesimen culture of 4.7%. Microbial patterns isolated from burn patients were Gram positive bacteria (37.5%) consisting of Staphylococcus aureus and Staphylococcus hominis. Gram negatif bacteria (62.5%) isolated consisted of Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae. The clinical spesimens that showed the most bacterial growth were pus followed by blood and joint fluid. Patients with less than 40% wound area, showed the growth of Staphylococcus aureus, Acinetobacter baumannii and Klebsiella pneumoniae. Patients with wound area above 40%, showed the growth of Staphylococcus aureus, Staphylococcus hominis, Acinetobacter baumannii and Pseudomonas aeruginosa was found. The group of Gram positive bacteria had good sensitivity to the antibiotics vancomycin (100%), linezolid (100%), tigecycline (100%), clindamycin (66.7%), tetracycline (66.7%) and trimethoprim-sulfamethoxazole (66, 7%). The group of Gram negatif bacteria showed high sensitivity to Amikacin (100%).

Development and Optimization of an Unbiased, Metagenomics-Based Pathogen Detection Workflow for Infectious Disease and Biosurveillance Applications

Rapid, specific, and sensitive identification of microbial pathogens is critical to infectious disease diagnosis and surveillance. Classical culture-based methods can be applied to a broad range of pathogens but have long turnaround times. Molecular methods, such as PCR, are time-effective but are not comprehensive and may not detect novel strains. Metagenomic shotgun next-generation sequencing (NGS) promises specific identification and characterization of any pathogen (viruses, bacteria, fungi, and protozoa) in a less biased way. Despite its great potential, NGS has yet to be widely adopted by clinical microbiology laboratories due in part to the absence of standardized workflows. Here, we describe a sample-to-answer workflow called PanGIA (Pan-Genomics for Infectious Agents) that includes simplified, standardized wet-lab procedures and data analysis with an easy-to-use bioinformatics tool. PanGIA is an end-to-end, multi-use workflow that can be used for pathogen detection and related applications, such as biosurveillance and biothreat detection. We performed a comprehensive survey and assessment of current, commercially available wet-lab technologies and open-source bioinformatics tools for each workflow component. The workflow includes total nucleic acid extraction from clinical human whole blood and environmental microbial forensic swabs as sample inputs, host nucleic acid depletion, dual DNA and RNA library preparation, shotgun sequencing on an Illumina MiSeq, and sequencing data analysis. The PanGIA workflow can be completed within 24 h and is currently compatible with bacteria and viruses. Here, we present data from the development and application of the clinical and environmental workflows, enabling the specific detection of pathogens associated with bloodstream infections and environmental biosurveillance, without the need for targeted assay development.

Hypervirulent Klebsiella pneumoniae Causing Neonatal Bloodstream Infections: Emergence of NDM-1-Producing Hypervirulent ST11-K2 and ST15-K54 Strains Possessing pLVPK-Associated Markers.

Klebsiella pneumoniae is a major cause of neonatal sepsis. Hypervirulent Klebsiella pneumoniae (hvKP) that cause invasive infections and/or carbapenem-resistant hvKP (CR-hvKP) limit therapeutic options. Such strains causing neonatal sepsis have rarely been studied. Characterization of neonatal septicemic hvKP/CR-hvKP strains in terms of resistance and virulence was carried out. Antibiotic susceptibility, molecular characterization, evaluation of clonality, in vitro virulence, and transmissibility of carbapenemase genes were evaluated. Whole-genome sequencing (WGS) and mouse lethality assays were performed on strains harboring pLVPK-associated markers. About one-fourth (26%, 28/107) of the studied strains, leading to mortality in 39% (11/28) of the infected neonates, were categorized as hvKP. hvKP-K2 was the prevalent pathotype (64.2%, 18/28), but K54 and K57 were also identified. Most strains were clonally diverse belonging to 12 sequence types, of which ST14 was most common. Majority of hvKPs possessed virulence determinants, strong biofilm-forming, and high serum resistance ability. Nine hvKPs were carbapenem-resistant, harboring blaNDM-1/blaNDM-5 on conjugative plasmids of different replicon types. Two NDM-1-producing high-risk clones, ST11 and ST15, had pLVPK-associated markers (rmpA, rmpA2, iroBCDEN, iucABCDiutA, and peg-344), of which one co-transferred the markers along with blaNDM-1. The 2 strains revealed high inter-genomic resemblance with the other hvKP reference genomes, and were lethal in mouse model. To the best of our knowledge, this study is the first to report on the NDM-1-producing hvKP ST11-K2 and ST15-K54 strains causing fatal neonatal sepsis. The presence of pLVPK-associated markers and blaNDM-1 in high-risk clones, and the co-transmission of these genes via conjugation calls for surveillance of these strains. IMPORTANCE Klebsiella pneumoniae is a leading cause of sepsis in newborns and adults. Among the 2 major pathotypes of K. pneumoniae, classical (cKP) and hypervirulent (hvKP), hvKP causes community-acquired severe fatal invasive infections in even healthy individuals, as it possesses several virulence factors. The lack of comprehensive studies on neonatal septicemic hvKPs prompted this work. Nearly 26% diverse hvKP strains were recovered possessing several resistance and virulence determinants. The majority of them exhibited strong biofilm-forming and high serum resistance ability. Nine of these strains were also carbapenem (last-resort antibiotic)-resistant, of which 2 high-risk clones (ST11-K2 and ST15-K54) harbored markers (pLVPK) noted for their virulence, and were lethal in the mouse model. Genome-level characterization of the high-risk clones showed resemblance with the other hvKP reference genomes. The presence of transmissible carbapenem-resistant gene, blaNDM, along with pLVPK-markers calls for vigilance, as most clinical microbiology laboratories do not test for them.