Public Health Microbiology Laboratories Research Articles

Seek and you shall find: Yersinia enterocolitica in Ireland’s drinking water

IntroductionThree Yersinia species were identified from samples of drinking water from diverse geographic regions of Ireland. Conventional commercial biochemical identification systems classified them as Yersinia enterocolitica. Since this organism is the most common cause of bacterial gastroenteritis in some countries, further investigation was warranted. The aim of the study was to provide a microbial characterisation of three Yersinia species, to determine their pathogenicity, and to review the incidence rate of Yersinia enterocolitica detection in our region.MethodsOrganism identification was performed using conventional commercial diagnostic systems MALDI-TOF, API 20E, API 50CHE, TREK Sensititre GNID and Vitek 2 GN, and whole genome sequencing (WGS) was performed. Historical data for detections was extracted from the lab system for 2008 to 2023.ResultsAll three isolates gave “good” identifications of Yersinia enterocolitica on conventional systems. Further analysis by WGS matched two of the isolates with recently described Yersinia proxima, and the third was a member of the non-pathogenic Yersinia enterocolitica clade 1Aa.DiscussionOur analysis of these three isolates deemed them to be Yersinia species not known currently to be pathogenic, but determining this necessitated the use of next-generation sequencing and advanced bioinformatics. Our work highlights the importance of having this technology available to public laboratories, either locally or in a national reference laboratory. The introduction of molecular technologies for the detection of Yersinia species may increase the rate of detections. Accurate identification of significant pathogens in environmental, public health and clinical microbiology laboratories is critically important for the protection of society.

FDA's proposed rule for the regulation of laboratory-developed tests.

In October 2023, the Food and Drug Administration (FDA) released a proposed rule that ends enforcement discretion for laboratory-developed tests (LDTs). The FDA's proposal outlines a five-stage implementation to begin regulating LDTs as they do for commercial in vitro diagnostics (IVDs), including modified FDA-approved/cleared tests. We outline here concerns from the clinical and public health microbiology laboratory perspective. It is our opinion that LDTs performed by individual Clinical Laboratory Improvement Amendments-certified diagnostic laboratories should not be regulated in the same way as commercial IVDs. This rule, if finalized, will negatively impact the diagnostic services currently offered by clinical and public health laboratories and, therefore, patients and the providers who care for them. Ending enforcement discretion will likely stifle diagnostic innovation and decrease access to diagnostic testing and health equity. Furthermore, the lack of infrastructure, including personnel and funding, at the FDA and diagnostic laboratories to support the required submissions for review is an obstacle. Like the FDA, diagnostic laboratories prioritize patient safety, accurate clinical diagnostics, and health equity. Since the scope of the LDT landscape is currently unknown, we are supportive of a registration process, along with non-burdensome adverse event reporting, to first understand the scope of clinical use of LDTs and any associated safety concerns. Any regulatory rule should be based on data that have been gathered systematically, not anecdotes or case reports. A rule must also balance the potential negative impact to patient care with realistic safety risks for infectious disease diagnostics.

Implementation of routine genomic surveillance provided insights into a locally acquired outbreak caused by a rare clade of Salmonella enterica serovar Enteritidis in Queensland, Australia.

Salmonellosis is a significant public health problem globally. In Australia, Salmonella enterica serovar Enteritidis is one of the main causes of salmonellosis. This study reports how the implementation of routine genetic surveillance of isolates from human S. Enteritidis cases enabled identification of the likely source of an outbreak that occurred in a remote town in Far North Queensland, Australia. This study included patient, food and water samples collected during an outbreak investigation. S. Enteritidis of the novel sequence type 5438 was isolated from all seven patient samples and one bore water sample but not any of the food samples. Both whole-genome single nucleotide polymorphism (SNP) and core-genome multilocus sequence typing analysis revealed that S. Enteritidis isolated from outbreak-related patient samples and the bore water isolates clustered together with fewer than five SNP differences and ten allelic differences. This genetic relatedness informed the outbreak response team around public health interventions and no further cases were identified post-treatment of the bore water. This disease cluster was identified through the routine sequencing of S. Enteritidis performed by the state public health laboratory in an actionable time frame. Additionally, genomic surveillance captured a case with unknown epidemiological links to the affected community, ruled out a simultaneous outbreak in an adjacent state as the source and provided evidence for the likely source preventing further transmission. Therefore, this report provides compelling support for the implementation of whole-genome sequencing based genotyping methods in public health microbiology laboratories for better outbreak detection and management.

Next Generation and Other Sequencing Technologies in Diagnostic Microbiology and Infectious Diseases.

Next-generation sequencing (NGS) technologies have become increasingly available for use in the clinical microbiology diagnostic environment. There are three main applications of these technologies in the clinical microbiology laboratory: whole genome sequencing (WGS), targeted metagenomics sequencing and shotgun metagenomics sequencing. These applications are being utilized for initial identification of pathogenic organisms, the detection of antimicrobial resistance mechanisms and for epidemiologic tracking of organisms within and outside hospital systems. In this review, we analyze these three applications and provide a comprehensive summary of how these applications are currently being used in public health, basic research, and clinical microbiology laboratory environments. In the public health arena, WGS is being used to identify and epidemiologically track food borne outbreaks and disease surveillance. In clinical hospital systems, WGS is used to identify multi-drug-resistant nosocomial infections and track the transmission of these organisms. In addition, we examine how metagenomics sequencing approaches (targeted and shotgun) are being used to circumvent the traditional and biased microbiology culture methods to identify potential pathogens directly from specimens. We also expand on the important factors to consider when implementing these technologies, and what is possible for these technologies in infectious disease diagnosis in the next 5 years.

Epidemiological and microbiological investigation of a large increase in vibriosis, northern Europe, 2018.

BackgroundVibriosis cases in Northern European countries and countries bordering the Baltic Sea increased during heatwaves in 2014 and 2018.AimWe describe the epidemiology of vibriosis and the genetic diversity of Vibrio spp. isolates from Norway, Sweden, Denmark, Finland, Poland and Estonia in 2018, a year with an exceptionally warm summer.MethodsIn a retrospective study, we analysed demographics, geographical distribution, seasonality, causative species and severity of non-travel-related vibriosis cases in 2018. Data sources included surveillance systems, national laboratory notification databases and/or nationwide surveys to public health microbiology laboratories. Moreover, we performed whole genome sequencing and multilocus sequence typing of available isolates from 2014 to 2018 to map their genetic diversity.ResultsIn 2018, we identified 445 non-travel-related vibriosis cases in the study countries, considerably more than the median of 126 cases between 2014 and 2017 (range: 87–272). The main reported mode of transmission was exposure to seawater. We observed a species-specific geographical disparity of vibriosis cases across the Nordic-Baltic region. Severe vibriosis was associated with infections caused by Vibrio vulnificus (adjOR: 17.2; 95% CI: 3.3–90.5) or Vibrio parahaemolyticus (adjOR: 2.1; 95% CI: 1.0–4.5), age ≥ 65 years (65–79 years: adjOR: 3.9; 95% CI: 1.7–8.7; ≥ 80 years: adjOR: 15.5; 95% CI: 4.4–54.3) or acquiring infections during summer (adjOR: 5.1; 95% CI: 2.4–10.9). Although phylogenetic analysis revealed diversity between Vibrio spp. isolates, two V. vulnificus clusters were identified.ConclusionShared sentinel surveillance for vibriosis during summer may be valuable to monitor this emerging public health issue.

Complete microbial genomes for public health in Australia and the Southwest Pacific.

Complete genomes of microbial pathogens are essential for the phylogenomic analyses that increasingly underpin core public health laboratory activities. Here, we announce a BioProject (PRJNA556438) dedicated to sharing complete genomes chosen to represent a range of pathogenic bacteria with regional importance to Australia and the Southwest Pacific; enriching the catalogue of globally available complete genomes for public health while providing valuable strains to regional public health microbiology laboratories. In this first step, we present 26 complete high-quality bacterial genomes. Additionally, we describe here a framework for reconstructing complete microbial genomes and highlight some of the challenges and considerations for accurate and reproducible genome reconstruction.

Novel biotechnological approaches for monitoring and immunization against resistant to antibiotics Escherichia coli and other pathogenic bacteria

The application of next-generation molecular, biochemical and immunological methods for developing new vaccines, antimicrobial compounds, probiotics and prebiotics for zoonotic infection control has been fundamental to the understanding and preservation of the symbiotic relationship between animals and humans. With increasing rates of antibiotic use, resistant bacterial infections have become more difficult to diagnose, treat, and eradicate, thereby elevating the importance of surveillance and prevention programs. Effective surveillance relies on the availability of rapid, cost-effective methods to monitor pathogenic bacterial isolates. In this opinion article, we summarize the results of some research program initiatives for the improvement of live vaccines against avian enterotoxigenic Escherichia coli using virulence factor gene deletion and engineered vaccine vectors based on probiotics. We also describe methods for the detection of pathogenic bacterial strains in eco-environmental headspace and aerosols, as well as samples of animal and human breath, based on the composition of volatile organic compounds and fatty acid methyl esters. We explain how the introduction of these low-cost biotechnologies and protocols will provide the opportunity to enhance co-operation between networks of resistance surveillance programs and integrated routine workflows of veterinary and clinical public health microbiology laboratories.

Hepatitis A seroprevalence in Erzurum, Turkey.

Hepatitis A Virus (HAV), reportedly the most common cause of acute viral hepatitis in developing countries, infects millions of people worldwide each year. The aim of the study is to investigate the seropositivity of anti-hepatitis A virus (HAV) IgG and IgM in all age groups in Erzurum, and to determine the effect of various factors such as age, gender, climatic conditions and HAV vaccination (included in 2012 in the National Immunization Schedule on seroprevalence) on the seropositivity. The serological results of 25,007 individuals referred to Erzurum Public Health Microbiology Laboratory between January 2015 - December 2018 were retrospectively reviewed to test for the presence of anti-HAV IgG and IgM. The patient ages were 0-93 years. Serum samples were analyzed by ELISA. S/CO values of ≥1.00 and >1.21 were considered positive for anti-HAV IgG and IgM, respectively; results below this value were considered negative. Anti-HAV IgG and IgM seropositivities were 87.3% and 0.2%, respectively. Anti-HAV IgG prevalence - 88.5% and 86.4%, anti-HAV IgM positivity - 0.1% and 0.3% in men and women. Anti-HAV IgG seroprevalence - 87%, 73.2%, 58.7%, 75.2%, 86.1%, 89.8%, 96.1%, 99.1%, 99.1% and 99.3%, respectively, at 0-4, 5-9 10-14, 15-19, 20-24, 25-29, 30-39, 40-49, 50-59 and >60 age groups. Anti-HAV IgM seropositivity - 0, 0.1%, 0.7%, 0.7%, 0.3%, 0, 0.1%, 0.2%, 0.1%, and 0.2%, respectively, in the same age groups. Anti-HAV IgM positivity was the highest in November - 36(0.97%. In Erzurum, anti-HAV IgG prevalence is tremendously high, whereas prevalence of anti-HAV IgM is exceptionally low, especially in the paediatric age group. Therefore, HAV vaccine is provided free of charge in Turkey, including Erzurum, since 2012.

Evaluation of the NG-Test MCR-1 Lateral Flow Assay and EDTA-Colistin Broth Disk Elution Methods To Detect Plasmid-Mediated Colistin Resistance among Gram-Negative Bacterial Isolates.

Plasmid-mediated colistin resistance (PMCR) is a global public health concern, given its ease of transmissibility. The purpose of this study was to evaluate two methods for the detection of PMCR from bacterial colonies: (i) the NG-Test MCR-1 lateral flow immunoassay (LFA; NG Biotech, Guipry, France) and (ii) the EDTA-colistin broth disk elution (EDTA-CBDE) screening test method. These methods were evaluated using a cohort of contemporary, clinical Gram-negative bacillus isolates from 3 U.S. academic medical centers (126 isolates of the Enterobacterales, 50 Pseudomonas aeruginosa isolates, and 50 Acinetobacter species isolates; 1 isolate was mcr positive) and 12 mcr-positive CDC-FDA Antibiotic Resistance (AR) Isolate Bank isolates for which reference broth microdilution colistin susceptibility results were available. Eleven (4.6%) isolates were strongly positive by the MCR-1 LFA, with an additional 8 (3.4%) isolates yielding faintly positive results. The positive percent agreement (PPA) and negative percent agreement (NPA) for MCR-1 detection were 100% and 96.1%, respectively. Upon repeat testing, only a single false-positive MCR-2 producer remained, as the isolates with initially faintly positive results were negative. The EDTA-CBDE screening method had an overall PPA and NPA of 100% and 94.3%, respectively. The NPA for the EDTA-CBDE method was slightly lower at 94.2% with Enterobacterales, whereas it was 96.0% with P. aeruginosa The MCR-1 LFA and EDTA-CBDE methods are both accurate and user-friendly methods for the detection of PMCR. Despite the rarity of PMCR among clinical isolates in the United States, these methods are valuable tools that may be implemented in public health and clinical microbiology laboratories to further discern the mechanism of resistance among colistin-resistant Gram-negative isolates and to detect PMCR for infection prevention and control purposes.

Influenza Viruses

Objective: The influenza viruses classified in the Orthomyxo viridae family have three types, A, B, C according to the irantigenic properties. Influenza A virus can lead to epidemics as well as pandemics with different subtypes depending on hemagglutinin and neuraminidase glycoproteins. Influenza viruses have been monitored by the World Health Organization (WHO) since 1952, due to their public health threat. In this study, it was aimed to determine the influenza data causing epidemics and loss of life in our country and in the world. Method: During the 4 (four) year period from January 2016 to December 2019, samples were taken from patients with suspected SARI in the form of nasal swabs who applied to hospitals in our city's centers and districts with influenza-like symptoms. Turkey Public Health Microbiology Laboratory samples sent to the Virology Department were examined by molecular methods. Findings: In 2016, 276 samples with SARI were examined; 128 (46.3%) specimens were positive for influenza A and 117 (42.3%) were negative. 17 (6.4%) samples were identified as influenza B, while 14 (5%) samples were identified as Rhinovirus. 65 (50.7%) samples of influenza A patients were identified as influenza A (H1N1) pdm09 and 63 (49.3%) samples as influenza A H3N2. Influenza A and Influenza B decreased significantly in 2017, 2018 and 2019. Discussion and conclusion: It is a disease that changes every year in terms of influenza factors and effects. In line with this information, while the number of patients was quite high in 2016, Influenza A and Influenza B decreased significantly in 2017, 2018 and 2019. Molecular typing will be facilitated in that the influenza vaccine to be administered in the following year will include identification genictypes. As a result, individuals who are at risk should be vaccinated; necessary measures should be taken to prevent outbreaks; A sample should be taken from every patient suspected of SARI and examined in terms of influenza and molecular typing should be done. Key words: Severe acute respiratory infection(SARI), influenza, epidemic Special Issue of Health Sciences DOI: 10.7176/JSTR/6-03-43

Research of hepatitis a virus antibodies in children 0-18 aged in the province of Erzurum, Turkey

Aim: The aim of the study was to compare the anti-HAV IgG and Anti-HAV IgM seropositivity in children aged 0-18 in Erzurum with studies in our country and other countries, and also to evaluate the effect of seasons on hepatitis A infection.Material and Methods: The study was conducted on the serum samples of 5086 patients aged 0-18 years, who were considered to have Hepatitis A or who applied with different complaints as a result of the examination of family physicians in Erzurum between 2015 and 2018. The results of these patients, obtained from Anti-HAV IgM and Anti-HAV IgG analysis by ELISA method in the Public Health Microbiology Laboratory, were evaluated retrospectively. Pearson chi-square test was used to analyze the categorical data such as gender, age and seasonal differences in anti-HAV IgM and Anti-HAV IgG positivity rates and p 0.05 was considered significant.Results: Anti-HAV IgG seropositivity was 69.0% (3510/5086) and Anti-HAV IgM positivity was 0.7% (33/5063). Anti-HAV IgM was positive in 1.1% of girls and 0.1% of boys. Anti-HAV IgG positivity was 89.9%, 83.3%, 62.5% 61.9% and 74.5% in the 0-3 age group, 4-7 age group, 8-11 age group, 12-15 age group and 16-18 age groups, respectively. Anti-HAV IgM positivity was 0, 0.2%, 0.2%, 1.0%, 0.9% in the 0-3 age group, 4-7 age group, 8-11 age group, 12-15 age group and 16-18 age groups, respectively. The highest anti-HAV IgM positivity was found in November (1.7%).Conclusion: Total anti-HAV IgG seropositivity rate in our province showed many similarities with studies in Turkey and developing countries. The prevalence of anti-HAV IgM was the highest in November.

Virological Quality of Urban Rivers and Hospitals Wastewaters in Addis Ababa, Ethiopia

Background:Polluted rivers and hospital wastewater become a greater concern because of their public health and environmental hazards with high tendency to result in epidemics.Methods and Materials:The current study investigated 84 samples of Urban rivers and 30 samples of hospitals wastewaters in Addis Ababa, Ethiopia between February and April, 2017. The simultaneous detection of male-specific and somatic Coliphages from the samples was carried out usingEscherichia coliCB390 as the host according to the single agar layer plaque assay at public health microbiology laboratory of Ethiopian Public Health Institute.Results:Of the total 114 samples tested, coliphages were detected in 44 (52.4%) and 3 (10%) samples of urban rivers and hospital waste waters, respect ively. Total coliphages enumerations ranged from <1pfu/100ml to 5.2×103pfu/100ml for urban rivers and <1pfu/100ml to 4.92×103pfu/100ml for hospitals wastewaters.Conclusion:The detection of total coliphages in our study settings warrants the possibility that the pollution of urban rivers and hospital wastewaters may be a source for pathogenic viral infections. Unless coliphages, viral and fecal indicators are also examined in the waters by public health agencies, waterborne infections cause a major risk to public health.

Investing in Public Health Microbiology Laboratories in Western Balkan Countries Enhances Health Security From Communicable Disease Threats in Europe

The European Centre for Disease Prevention and Control (ECDC), under the EU enlargement policy, has supported national efforts of Western Balkan countries to strengthen their communicable disease prevention and control systems. The new EU strategy “A credible enlargement perspective for and enhanced EU engagement with the Western Balkans” advocates transformation processes that will build the foundation of EU-oriented national reforms. Well-functioning public health microbiology laboratories are key for early detection and control of infectious diseases, and thus maintaining and enhancing health security in Europe. In order to help Western Balkan countries to improve their national capacities, ECDC facilitated needs assessments and identified key areas for advancement toward effective public health microbiology systems. Countries identified gaps in their laboratory data reporting and exchange systems. Harmonized and effective procedures for handling of highly contagious agents and cross-border transportation of biological samples were often lacking, as well as the systematic use of diagnostic testing at the primary care level or referral of patients, in particular for detection of antimicrobial resistance. There is a clear need to address the financial investment required for sustaining sufficient numbers of skilled laboratory workforce, laboratory supplies, and the development of new methods and techniques, including investment in emerging laboratory technologies, such as molecular typing by whole genome sequencing. This article highlights the key areas for investing in public health microbiology laboratories in Western Balkan countries needed to strengthen health security in Europe.

Brucella abortus RB51 Strain Sent in a Proficiency Testing Panel to Clinical and Public Health Laboratories

In early 2017, a laboratory proficiency testing panel, designed to assess laboratory competency for culture and identification of bacterial organisms, was distributed to clinical and public health microbiology laboratories. The panel included a Brucella abortus RB51 isolate. Although B. abortus RB51 is an attenuated Brucella species strain, it still poses some risk to laboratory personnel, particularly if safety precautions and personal protective equipment are not adequately deployed. The PT event highlighted the challenges that clinical and public health microbiology laboratories face when handling samples that can contain highly transmissible organisms that can lead to laboratory-acquired infections. This review describes the B. abortus RB51 PT event, its associated exposures, and subsequent medical interventions. General considerations for handling Brucella species, including RB51, in the clinical microbiology laboratory are also discussed. Finally, we describe lessons learned from this event, including improving a “culture of safety” in the clinical laboratory.

Networking of Public Health Microbiology Laboratories Bolsters Europe's Defenses against Infectious Diseases.

In an era of global health threats caused by epidemics of infectious diseases and rising multidrug resistance, microbiology laboratories provide essential scientific evidence for risk assessment, prevention, and control. Microbiology has been at the core of European infectious disease surveillance networks for decades. Since 2010, these networks have been coordinated by the European Centre for Disease Prevention and Control (ECDC). Activities delivered in these networks include harmonization of laboratory diagnostic, antimicrobial susceptibility and molecular typing methods, multicentre method validation, technical capacity mapping, training of laboratory staff, and continuing quality assessment of laboratory testing. Cooperation among the European laboratory networks in the past 7 years has proved successful in strengthening epidemic preparedness by enabling adaptive capabilities for rapid detection of emerging pathogens across Europe. In partnership with food safety authorities, international public health agencies and learned societies, ECDC-supported laboratory networks have also progressed harmonization of routinely used antimicrobial susceptibility and molecular typing methods, thereby significantly advancing the quality, comparability and precision of microbiological information gathered by ECDC for surveillance for zoonotic diseases and multidrug-resistant pathogens in Europe. ECDC continues to act as a catalyst for sustaining continuous practice improvements and strengthening wider access to laboratory capacity across the European Union. Key priorities include optimization and broader use of rapid diagnostics, further integration of whole-genome sequencing in surveillance and electronic linkage of laboratory and public health systems. This article highlights some of the network contributions to public health in Europe and the role that ECDC plays managing these networks.

Re-evaluation of chronic hepatitis B and hepatitis C patients lost to follow-up: results of the Northern Holland hepatitis retrieval project

BackgroundMany persons infected with Hepatitis B virus (HBV) and Hepatitis C virus (HCV) in the past are now lost to follow-up. The aim of the Northern Holland Hepatitis Retrieval Project (NHHRP) is to retrieve and re-evaluate persons previously diagnosed with HBV or HCV and bring them back into care. Chronic HBV infection was defined as two positive Hepatitis B surface antigen (HBsAg) tests within 6 months and chronic HCV infection with 2 positive HCV RNA tests by polymerase chain reaction (PCR).MethodsData files of the local public health services and microbiology laboratory were explored to identify all registered HBV and HCV cases in the Alkmaar region, the Netherlands, for the past 15 years. Identified cases were compared with patients currently known in our hospital. Patients without follow-up in primary or hospital care were approached via their primary health care physician and invited for evaluation at our hospitalResultsIn total, 552 cases of HBV were identified. 356 (64.5%) had no follow-up. Only 113/356 (31.7%) were eligible for retrieval and 44.2% were evaluated in our hospital resulting in a change of management in 22/50 (44%) of patients. Four hundred ninety nine cases of HCV were identified, 150/499 (30.1%) were lost to follow-up. Only 20/150 (13.3%) were eligible for retrieval and 4/20 (20%) were evaluated at our clinic. Resulting in a change of management in 3/4 (75%).ConclusionOnly a limited part of HBV and HCV persons lost to follow-up is eligible for retrieval, nonetheless re-evaluation of these persons will lead to a change of management in the majority of persons.

Towards a genomics-informed, real-time, global pathogen surveillance system.

The recent Ebola and Zika epidemics demonstrate the need for the continuous surveillance, rapid diagnosis and real-time tracking of emerging infectious diseases. Fast, affordable sequencing of pathogen genomes - now a staple of the public health microbiology laboratory in well-resourced settings - can affect each of these areas. Coupling genomic diagnostics and epidemiology to innovative digital disease detection platforms raises the possibility of an open, global, digital pathogen surveillance system. When informed by a One Health approach, in which human, animal and environmental health are considered together, such a genomics-based system has profound potential to improve public health in settings lacking robust laboratory capacity.

Right-Sizing Technology in the Era of Consumer-Driven Health Care

Technology for modern clinical and public health microbiology laboratories has evolved at an impressive rate over the last two decades. Contemporary diagnostics can rapidly provide powerful data that can impact patient lives and support infectious disease outbreak investigations. At the same time, dramatic changes to health care delivery are putting new pressures on a system that is now focusing on patient-centric, value-driven, convenient care. For laboratories, balancing all these demands in a cost-contained environment remains a challenge. This article explores the current and future directions of diagnostics in our dynamic health care environment.

Validation and Implementation of Clinical Laboratory Improvements Act-Compliant Whole-Genome Sequencing in the Public Health Microbiology Laboratory.

ABSTRACTPublic health microbiology laboratories (PHLs) are on the cusp of unprecedented improvements in pathogen identification, antibiotic resistance detection, and outbreak investigation by using whole-genome sequencing (WGS). However, considerable challenges remain due to the lack of common standards. Here, we describe the validation of WGS on the Illumina platform for routine use in PHLs according to Clinical Laboratory Improvements Act (CLIA) guidelines for laboratory-developed tests (LDTs). We developed a validation panel comprising 10 Enterobacteriaceae isolates, 5 Gram-positive cocci, 5 Gram-negative nonfermenting species, 9 Mycobacterium tuberculosis isolates, and 5 miscellaneous bacteria. The genome coverage range was 15.71× to 216.4× (average, 79.72×; median, 71.55×); the limit of detection (LOD) for single nucleotide polymorphisms (SNPs) was 60×. The accuracy, reproducibility, and repeatability of base calling were >99.9%. The accuracy of phylogenetic analysis was 100%. The specificity and sensitivity inferred from multilocus sequence typing (MLST) and genome-wide SNP-based phylogenetic assays were 100%. The following objectives were accomplished: (i) the establishment of the performance specifications for WGS applications in PHLs according to CLIA guidelines, (ii) the development of quality assurance and quality control measures, (iii) the development of a reporting format for end users with or without WGS expertise, (iv) the availability of a validation set of microorganisms, and (v) the creation of a modular template for the validation of WGS processes in PHLs. The validation panel, sequencing analytics, and raw sequences could facilitate multilaboratory comparisons of WGS data. Additionally, the WGS performance specifications and modular template are adaptable for the validation of other platforms and reagent kits.

Characterisation of invasive clinical Haemophilus influenzae isolates in Queensland, Australia using whole-genome sequencing.

Haemophilus influenzae is an important aetiological organism of both adult and child respiratory disease. The number of non-typeable (NTHi) invasive H. influenzae isolates referred to the Queensland (QLD) Public Health Microbiology laboratory has increased notably year-by-year. In this study we used whole-genome sequencing to molecularly characterise 100 referred invasive H. influenzae, including 74 NTHi isolates over a 15-year period, observing the carriage of capsular and putative virulence genes, including the major adhesins, antimicrobial resistance genes and population diversity. Encapsulated isolates were largely clonal, however NTHi isolates displayed high genetic variability by MLST and single nucleotide polymorphism typing with no dominant clone observed. The only mechanism for β-lactam resistance identified in the QLD isolates was β-lactamase production. No single set of virulence determinants was conclusively associated with invasive QLD NTHi isolates.