CARD 2020: antibiotic resistome surveillance with the comprehensive antibiotic resistance database.

The Comprehensive Antibiotic Resistance Database (CARD; https://card.mcmaster.ca) is a curated resource providing reference DNA and protein sequences, detection models and bioinformatics tools on the molecular basis of bacterial antimicrobial resistance (AMR). CARD focuses on providing high-quality reference data and molecular sequences within a controlled vocabulary, the Antibiotic Resistance Ontology (ARO), designed by the CARD biocuration team to integrate with software development efforts for resistome analysis and prediction, such as CARD's Resistance Gene Identifier (RGI) software. Since 2017, CARD has expanded through extensive curation of reference sequences, revision of the ontological structure, curation of over 500 new AMR detection models, development of a new classification paradigm and expansion of analytical tools. Most notably, a new Resistomes & Variants module provides analysis and statistical summary of in silico predicted resistance variants from 82 pathogens and over 100 000 genomes. By adding these resistance variants to CARD, we are able to summarize predicted resistance using the information included in CARD, identify trends in AMR mobility and determine previously undescribed and novel resistance variants. Here, we describe updates and recent expansions to CARD and its biocuration process, including new resources for community biocuration of AMR molecular reference data.

Workflow and Throughput of Commercial Assays to Detect Mycoplasma genitalium and Macrolide Resistance-Mediating Mutations.

ABSTRACT: Aptima Mycoplasma genitalium (MG) required the shortest and STD6 the longest time to detect MG in clinical samples. ResistancePlus MG detected MG and macrolide resistance-mediating mutations simultaneously. Times were influenced by specimen numbers. M. genitalium positives from the other 2 assays required increased time for macrolide resistance-mediating mutation sequencing.

Comparison of Assays for the Diagnosis of Mycoplasma genitalium and Macrolide Resistance Mutations in Self-Collected Vaginal Swabs and Urine.

BACKGROUND: The objective was to compare commercial assays on clinical specimens for Mycoplasma genitalium (MG) detection and macrolide resistance mutation (MRM) frequency. METHODS: Three self-collected vaginal swabs (VS) and a first-void urine (FVU) from 300 consented women were tested by Aptima MG (AMG), ResistancePlus MG (RPMG) and Seeplex STD6 ACE (STD6) for detection of MG. Aptima MG and STD6 MG positives were tested for MRM using MG 23S rRNA polymerase chain reaction with Sanger sequencing (23SMGSS) compared with MRM determination in the RPMG assay. Unique AMG positives were tested with confirmatory Aptima assays. RESULTS: M. genitalium prevalence ranged from 7.1% to 19.7%, influenced by the assay used and the specimen tested. Overall agreements for MG detection were 96.3% (κ = 0.91) for VS and 93.3% (κ = 0.72) for FVU between AMG and RPMG with lower agreements with STD6. Using a rotating reference standard, sensitivities on VS and FVU were 100% and 100% for AMG, 100% and 83.3% for RPMG, and 54.2% and 48.4% for STD6. Specificities were high for RPMG and STD6 and AMG detected extra positives, most of which were confirmed. Macrolide resistance mutation frequency rates testing VS and FVU were 50% (24/48) and 58.1% (18/31) by RPMG compared with 52.5% (31/59) and 23.5% (12/51) by 23SMGSS. MRM overall agreements between RPMG and 23SMGSS were 73.2% (κ = 0.41) for VS and 76.0% (κ = 0.52) for FVU. CONCLUSIONS: Aptima MG detected more cases of MG infections. ResistancePlus MG detection was more effective on VS than on FVU. Seeplex STD6 ACE performance was inferior. The MRM detection component of RPMG agreed with results from 23SMGSS most of the time.

Diagnostic challenges of oral and cutaneous Kaposi's sarcoma in resource-constrained settings.

OBJECTIVES: Kaposi's sarcoma (KS), caused by HHV-8, is the most frequent HIV-associated malignancy worldwide and remains a major scourge in Sub-Saharan Africa. KS is also endemic in much of Africa. There is a risk of misdiagnosis based solely on clinical appearance and haematoxylin and eosin (H&E) staining, especially with other reactive and neoplastic vascular proliferations which occur in the mouth. This study examined oral and cutaneous biopsies from clinically diagnosed lesions of KS in Kenya, using histopathology supplemented with immunohistochemistry (IHC) and polymerase chain reaction (PCR) for HHV-8 as confirmation of diagnosis. METHODS: Biopsies of 49 lesions (28 oral, 21 cutaneous) previously diagnosed as 'KS' were re-examined by H&E staining and IHC targeting HHV-8 LANA-1. Positive controls were sections from embedded BCBL-1 cell lines. Negative controls were from three different HHV-8-negative biopsies. Confirmation of HHV-8 immunohistochemistry was sought by PCR and by determining the HHV-8 ORFK1 subtype. RESULTS: Whilst most cases were confirmed, 12 oral and 4 cutaneous lesions displayed clinical and histological features of KS but were negative to HHV-8 IHC. These oral lesions were re-diagnosed as pyogenic granulomata (n = 6), deep mycosis (n = 1), inflamed mucosa (n = 2) or 'uncertain but not KS' (n = 3). Whilst PCR is usually helpful in differentiating HHV-8 disease, all samples were HHV-8 PCR positive, with identical sequences, suggesting cross-contamination of samples in the original pathology laboratories. CONCLUSION: HHV-8 IHC is essential for the correct diagnosis of KS, but due to the high level of contamination in resource-poor settings, PCR is inadvisable.

Protocol for assessing maternal, environmental and epigenetic risk factors for dental caries in children.

BACKGROUND: Expenditure on dental and oral health services in Australia is $3.4 billion AUD annually. This is the sixth highest health cost and accounts for 7 % of total national health expenditure. Approximately 49 % of Australian children aged 6 years have caries experience in their deciduous teeth and this is rising. The aetiology of dental caries involves a complex interplay of individual, behavioural, social, economic, political and environmental conditions, and there is increasing interest in genetic predisposition and epigenetic modification. METHODS: The Oral Health Sub-study; a cross sectional study of a birth cohort began in November 2012 by examining mothers and their children who were six years old by the time of initiation of the study, which is ongoing. Data from detailed questionnaires of families from birth onwards and data on mothers' knowledge, attitudes and practices towards oral health collected at the time of clinical examination are used. Subjects' height, weight and mid-waist circumference are taken and Body Mass Index (BMI) computed, using an electronic Bio-Impedance balance. Dental caries experience is scored using the International Caries Detection and Assessment System (ICDAS). Saliva is collected for physiological measures. Salivary Deoxyribose Nucleic Acid (DNA) is extracted for genetic studies including epigenetics using the SeqCap Epi Enrichment Kit. Targets of interest are being confirmed by pyrosequencing to identify potential epigenetic markers of caries risk. DISCUSSION: This study will examine a wide range of potential determinants for childhood dental caries and evaluate inter-relationships amongst them. The findings will provide an evidence base to plan and implement improved preventive strategies.

HPV genotype-specific distribution and attributable risk in cervical intraepithelial neoplasia in a referral population with a history of LSIL.

BACKGROUND AND OBJECTIVE: CINtec PLUS and cobas HPV tests (Roche) were previously ascertained for triaging an LSIL referral population [1]. As part of this study, genotype-specific distribution and attributable risk of high-risk (HR)-HPV in cervical intraepithelial neoplasia (CIN) were determined. METHODS: Archived cervical specimens in ThinPrep PreservCyt (Hologic Inc) from the LSIL referral population (n= 533) were genotyped using the Anyplex II HPV HR test (Anyplex, Seegene Inc). Since the study specimens had been in storage in ambient temperature for 31-47 months since collection, Anyplex results were compared with that of the initial cobas testing of fresh specimens to validate the suitability and stability of specimens for the present study. RESULTS: Overall, Anyplex test was positive in 63% (336/533) vs. 55.7% (297/533) for cobas test. Anyplex test performed identical to cobas test identifying 93.2% (82/88) of ⩾CIN2/adenocarcinoma in situ (AIS). Anyplex test detected genotypes 16/18 in 15.7% (36/230) ⩽CIN1 vs. 45.5% (40/88) ⩾CIN2/AIS; the corresponding figures were 13.5% (31/230) and 45.5% (40/48) for the cobas test. Genotype 16 showed increasing attribution, 13.2% in CIN1, 27.1% in CIN2 and 40% in CIN3/AIS. Of the 12 other high-risk (OHR) types collectively identified by cobas, Anyplex test specifically detected, in decreasing order, genotypes 51, 31, 35, 56, 39, and 45 as the most frequent types, often in multiple-type infections, in 64.8% ⩾CIN2. Regardless, estimated attribution was evident for each of the 12 OHR types in ⩾CIN2. Multiple-type infections were more frequent than single-type infections in all CIN grades. CONCLUSIONS: Attributable risk of all HR-HPV genotypes targeted by both Anyplex and cobas tests was evident in ⩾CIN2/AIS Testing for these genotypes in HPV primary cervical screening and cytology triage could identify those at increased risk of cervical cancer and also be beneficial in the management of LSIL referral populations.

Detection of human herpesvirus 8 by quantitative polymerase chain reaction: development and standardisation of methods.

BACKGROUND: Human herpesvirus 8 (HHV-8), the aetiological agent of Kaposi's sarcoma (KS), multicentric Castleman's disease (MCD), and primary effusion lymphoma (PEL) is rare in Australia, but endemic in Sub-Saharan Africa, parts of South-east Asia and Oceania. While the treatment of external KS lesions can be monitored by clinical observation, the internal lesions of KS, MCD and PEL require extensive and expensive internal imaging, or autopsy. In patients with MCD and PEL, if HHV-8 viraemia is not reduced quickly, ~50% die within 24 months. HHV-8 qPCR is a valuable tool for monitoring HHV-8 viraemia, but is not available in many parts of the world, including those with high prevalence of KS and HHV-8. METHODS: A new molecular facility with stringent three-phase workflow was established, adhering to NPAAC and CLSI guidelines. Three fully validated quantitative assays were developed: two for detection and quantification of HHV-8; one for GAPDH, necessary for normalisation of viral loads in tissue and peripheral blood. RESULTS: The HHV-8 ORF73 and ORF26 qPCR assays were 100% specific. All qPCR assays, displayed a broad dynamic range (102 to 1010 copies/µL TE Buffer) with a limit of detection of 4.85x103, 5.61x102, and 2.59x102 copies/µL TE Buffer and a limit of quantification of 4.85x103, 3.01x102, and 1.38x102 copies/µL TE Buffer for HHV-8 ORF73, HHV-8 ORF26, and GAPDH respectively.The assays were tested on a panel of 35 KS biopsies from Queensland. All were HHV-8 qPCR positive with average viral load of 2.96x105 HHV-8 copies/µL DNA extract (range: 4.37x103 to 1.47x106 copies/µL DNA extract): When normalised these equate to an average viral load of 2.44x104 HHV-8 copies/103 cells (range: 2.20x102 to 7.38x105 HHV-8 copies/103 cells). CONCLUSIONS: These are the first fully optimised, validated and MIQE compliant HHV-8 qPCR assays established in Australia. They worked well for qualitative detection of HHV-8 in archival tissue, and are well-suited for quantitative detection in whole blood. They are now available for research, for clinical diagnosis of HHV-8 infection, and for monitoring treatment efficacy.

Tumor Temperature: Friend or Foe of Virus-Based Cancer Immunotherapy.

The temperature of a solid tumor is often dissimilar to baseline body temperature and, compared to healthy tissues, may be elevated, reduced, or a mix of both. The temperature of a tumor is dependent on metabolic activity and vascularization and can change due to tumor progression, treatment, or cancer type. Despite the need to function optimally within temperature-variable tumors, oncolytic viruses (OVs) are primarily tested at 37 °C in vitro. Furthermore, animal species utilized to test oncolytic viruses, such as mice, dogs, cats, and non-human primates, poorly recapitulate the temperature profile of humans. In this review, we discuss the importance of temperature as a variable for OV immunotherapy of solid tumors. Accumulating evidence supports that the temperature sensitivity of OVs lies on a spectrum, with some OVs likely hindered but others enhanced by elevated temperatures. We suggest that in vitro temperature sensitivity screening be performed for all OVs destined for the clinic to identify potential hinderances or benefits with regard to elevated temperature. Furthermore, we provide recommendations for the clinical use of temperature and OVs.

Human Herpesvirus 8 in Australia: DNAemia and Cumulative Exposure in Blood Donors.

Human herpesvirus 8 (HHV-8), the causative agent of Kaposi's sarcoma, multicentric Castleman's disease and primary effusion lymphoma, predominantly manifests in immunocompromised individuals. However, infection in immunocompetent individuals does occur. The prevalence of HHV-8 exposure in blood donors from non-endemic countries ranges between 1.2% and 7.3%. Nothing was known about the prevalence in Australian blood donors. Therefore, this study investigated the active and cumulative exposure of HHV-8 in this cohort. Plasma samples (n = 480) were collected from eastern Australian blood donors and were tested for HHV-8 DNA by qPCR, and for HHV-8 antibodies by two different ELISAs. Samples initially positive on either ELISA were retested in duplicate on both, and on a mock-coated ELISA. Any samples positive two or three out of the three times tested on at least one ELISA, and repeat negative on the mock-coated ELISA, were assigned as repeat positive. None of the 480 samples tested contained HHV-8 DNA. Serological testing revealed 28 samples (5.83%; 95% CI: 3.74−7.93%) had antibodies to HHV-8. There was no difference (p > 0.05) in seropositivity between sex or with increasing age. This is the first study to show serological evidence of cumulative HHV-8 exposure and no HHV-8 DNAemia within a select blood donor population in Australia. Our molecular and serological data is consistent with published results for blood donors residing in HHV-8 non-endemic countries, which shows the prevalence to be very low.

Profiling the Human Oral Mycobiome in Tissue and Saliva Using ITS2 DNA Metabarcoding Compared to a Fungal-Specific Database.

The advent of high-throughput sequencing has caused a paradigm shift from the one-pathogen one-disease model to the significance of dysbiosis of the oral microbiome, including the oral mycobiome. The oral mycobiome can be profiled by a method modified from that used to profile the bacteriome with 16S rRNA gene primers. The first modification is to include an initial fungus lysis step that ensures representative yields of fungal DNA. The second step is to use a reliable target, the ITS1 and/or ITS2 regions of the 23S rRNA, to define the oral fungal population, and modifications of library preparation required to deal with the variable sized amplicons generated. In this chapter, a proven microbiomic approach to identify fungal populations in oral tissue samples associated with cancer is described. This approach is also applicable to the study of the salivary mycobiome in both healthy and diseased individuals.

Whole-Genome Sequencing of Pathogens in Saliva : A Target-Enrichment Approach for SARS-CoV-2.

Outbreak analysis and transmission surveillance of viruses can be performed via whole-genome sequencing after viral isolation. Such techniques have recently been applied to characterize and monitor SARS-CoV-2 , the etiological agent of the COVID-19 pandemic. However, the isolation and culture of SARS-CoV-2 is time consuming and requires biosafety level 3 containment, which is not ideal for many resource-constrained settings. An alternate method, bait capture allows target enrichment and sequencing of the entire SARS-CoV-2 genome eliminating the need for viral culture. This method uses a set of hybridization probes known as "baits" that span the genome and provide sensitive, accurate, and minimal off-target hybridization. Baits can be designed to detect any known virus or bacteria in a wide variety of specimen types, including oral secretions. The bait capture method presented herein allows the whole genome of SARS-CoV-2 in saliva to be sequenced without the need to culture and provides an outline of bait design and bioinformatic analysis to guide a bioinformatician.

Self-collected oral flocked swabs to measure prevalence of Epstein-Barr Virus antibodies and DNA amongst university students.

Epstein-Barr Virus (EBV) exposure and illness is common in undergraduate university students and may affect academic achievement, social life, and quality of life. We designed a study to measure EBV exposure (EBV-IgG, either Epstein-Barr nuclear antigen 1 (EBNA-1)-IgG or viral capsid antigen (VCA)-IgG) and current viral shedding (EBV-DNA) using self-collected oral swabs among university undergraduate students. Of 184 students enrolled, 129 (70.1%) tested positive for EBV-IgG. Salivopositivity was associated with being in a current relationship, but not with enrollment year. Forty (21.7%) of the participants tested positive for EBV-DNA, which was associated with all symptom scores, including history of sore throat, fever, swollen glands, muscle weakness, and fatigue in the previous 6 months. Our findings suggest that noninvasive, self-collected oral flocked swabs are feasible and potentially valuable for measuring EBV IgG antibodies and DNA.

Comparison of Alinity m HPV and cobas HPV assays on cervical specimens in diverse storage media.

OBJECTIVE: To assess the concordance of high-risk HPV (HR-HPV) testing with the Alinity assay on cervical samples collected with diverse collection/storage protocols (ThinPrep, SurePath, Cervicollect) and to assess inter-assay concordance of HR-HPV testing of cervical cell specimens with Alinity m HR HPV assay (Alinity) vs cobas® 4800 HPV assay (cobas). METHODS: Specimens were obtained from 560 women attending a Women's Health clinic. Two specimens were obtained from each woman with combinations of two of the three collection devices and aliquots were tested by the two assays. RESULTS: Alinity showed an agreement of 93.9%, Kappa = 0.89 (263/280) between ThinPrep and SurePath specimens; 97.5%, Kappa = 0.95 (347/356) and 92.9%, Kappa = 0.85 (104/112) between ThinPrep and SurePath aliquots taken before or after cytology processing, respectively. Cervi-Collect specimens showed an agreement of 94.6%, Kappa = 0.89 (265/280) with ThinPrep specimens. Compared to cobas, Alinity showed agreements of 94.3%, Kappa = 0.88 (395/419) and 91.8%, Kappa = 0.82 (257/280) between ThinPrep and SurePath specimens, respectively. Alinity and cobas detected genotypes 16/18 and other high-risk HPV types at similar rates and showed similar correlations with cytology grades. CONCLUSIONS: Compared to cobas, Alinity performed equally well for detecting HPV in cervical specimens obtained with ThinPrep and SurePath. The Cervi-Collect device compared well to the other collection methods. Alinity is a reliable assay for simultaneous detection of HPV-16/18 and other high-risk genotypes in cervical specimens.

Non-invasive detection of IgG antibodies from common pathogenic viruses using oral flocked swabs.

Salivary antibodies are useful in surveillance and vaccination studies. However, low antibody levels and degradation by endonucleases are problematic. Oral flocked swabs are a potential non-invasive alternative for detecting viral antibodies. Seroprevalence for Cytomegalovirus (CMV), Varicella-Zoster virus (VZV), Epstein-Barr virus (EBV), Measles and Mumps IgG antibodies were determined from 50 matched serum, saliva and swabs samples from healthy volunteers using commercial ELISAs. CMV IgG, VZV IgG, and EBV EBNA-1 IgG, VCA IgG, and Measles IgG swab versus serum sensitivities were 95.8%, 96.0%, 92.1%, 95.5%, 84.5%, respectively, and swabs correlated well with saliva. Sensitivity of Mumps IgG in swabs and saliva was poor at 60.5%, and 68.2%, respectively. Specificities for IgG antibodies were 100% for CMV, EBV and Mumps, but could not be determined for VZV and Measles due to exclusively seropositive volunteers. Except for Mumps IgG, swabs correlate well with serum, are easy to self-collect and are stable at room temperature.

Performance of AmpFire HPV assay on neck cervical lymph node aspirate and oropharyngeal samples.

Early determination of high-risk human papillomaviruses causing oropharyngeal squamous cell carcinomas (OPSCC) may influence treatment. The objectives were to evaluate the performance of a new rapid isothermal nucleic acid amplification point of care HPV test (AmpFire HPV) on fine needle neck aspirates (FNA) of cervical lymph nodes and oropharyngeal swabs and saliva (OPS) which had been previously tested by the cobas HPV assay. The comparison was performed on 56 FNA and 81 OPS. The two assays showed strong agreement (94.6 %, K = 0.88) on FNA and fair agreement (65.4 %, K = 0.34) on OPS. AmpFire HPV performed on FNA demonstrated a sensitivity of 76.7 % and specificity of 81.8 % for the prediction of p16 antigens in OPSCC with results available in 1.5 h.

A dysbiotic mycobiome dominated by Candida albicans is identified within oral squamous-cell carcinomas.

The aim of this study was to characterize the mycobiome associated with oral squamous-cell carcinoma (OSCC). DNA was extracted from 52 tissue biopsies (cases: 25 OSCC; controls: 27 intra-oral fibro-epithelial polyps [FEP]) and sequenced for the fungal internal transcribed spacer 2 region using Illumina™ 2 x300bp chemistry. Merged reads were classified to species level using a BLASTN-algorithm with UNITE's named species sequences as reference. Downstream analyses were performed using QIIME™ and linear discriminant analysis effect size. A total of 364 species representing 160 genera and two phyla (Ascomycota and Basidiomycota) were identified, with Candida and Malassezia making up 48% and 11% of the average mycobiome, respectively. However, only five species and four genera were detected in ≥50% of the samples. The species richness and diversity were significantly lower in OSCC. Genera Candida, Hannaella, and Gibberella were overrepresented in OSCC; Alternaria and  Trametes  were more abundant in FEP. Species-wise, Candida albicans, Candida etchellsii, and a Hannaella luteola-like species were enriched in OSCC, while aHanseniaspora uvarum-like species, Malassezia restricta, and Aspergillus tamarii were the most significantly abundant in FEP. In conclusion, a dysbiotic mycobiome dominated by C. albicans was found in association with OSCC, a finding worth further investigation.

Evidence of human coronavirus HKU1 and human bocavirus in Australian children.

Undiagnosed cases of respiratory tract disease suspected of an infectious aetiology peak during the winter months. Since studies applying molecular diagnostic assays usually report reductions in the number of undiagnosed cases of infectious disease compared to traditional techniques, we applied PCR assays to investigate the role of two recently described viruses, namely human coronavirus (HCoV) HKU1 and human bocavirus (HBoV), in a hospital-based paediatric population. Both viruses were found among Australia children with upper or lower respiratory tract disease during the autumn and winter of 2004, contributing to 21.1% of all microbial diagnoses, with individual incidences of 3.1% (HCoV-HKU1) and 5.6% (HBoV) among 324 specimens. HBoV was found to coincide with another virus in more than half of all instances and displayed a single genetic lineage, whilst HCoV-HKU1 was more likely to occur in the absence of another microbe and strains could be divided into two genetic lineages which we propose be termed HCoV-HKU1 type A and type B. Children under the age of 2 years were most at risk of infection by these viruses which contribute significantly to the microbial burden among patients with respiratory tract disease during the colder months.

Emerging role of bacteria in oral carcinogenesis: a review with special reference to perio-pathogenic bacteria.

Oral cancer, primarily oral squamous cell carcinoma (OSCC), continues to be a major global health problem with high incidence and low survival rates. While the major risk factors for this malignancy, mostly lifestyle related, have been identified, around 15% of oral cancer cases remain unexplained. In light of evidence implicating bacteria in the aetiology of some cancer types, several epidemiological studies have been conducted in the last decade, employing methodologies ranging from traditional culture techniques to 16S rRNA metagenomics, to assess the possible role of bacteria in OSCC. While these studies have demonstrated differences in microbial composition between cancerous and healthy tissues, they have failed to agree on specific bacteria or patterns of oral microbial dysbiosis to implicate in OSCC. On the contrary, some oral taxa, particularly Porphyromonas gingivalis and Fusobacterium nucleatum, show strong oral carcinogenic potential in vitro and in animal studies. Bacteria are thought to contribute to oral carcinogenesis via inhibition of apoptosis, activation of cell proliferation, promotion of cellular invasion, induction of chronic inflammation, and production of carcinogens. This narrative review provides a critical analysis of and an update on the association between bacteria and oral carcinogenesis and the possible mechanisms underlying it.

Detecting DNA viruses in oral fluids: evaluation of collection and storage methods.

Storing saliva for nucleic acid diagnostics is problematic in resource-constrained settings. DNA Genotek's OMNIgene™·DISCOVER kit aims to stabilise microbial DNA at room temperature. We evaluate this for long-term storage, determining DNA quantity/purity and human herpesvirus 8 (HHV-8) load as indicator. Viral loads and DNA degradation were assayed over 14months in HHV-8-negative saliva spiked with cell-associated and cell-free virus and saliva collected fresh frozen and into kits from 10 HIV-positive patients. Viral loads remained constant for 6-9months, yielding high quantities of DNA: subsequent losses were ≤48%. Patient samples, frozen or kit stored, produced pure DNA of comparable concentration. Higher HHV-8 detection in frozen saliva resulted from losses during ethanol precipitation using kits. After 14months, DNA degradation was significant in frozen saliva, but that in kits had integrity similar to fresh samples. Storing frozen saliva is detrimental. This kit is well suited for collection, long-term storage, and assay of viral DNA in resource-constrained settings.