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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 14 months 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 6–9 months, 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 14 months, 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. [ABSTRACT FROM AUTHOR]

Abstract: Background: Kaposi's sarcoma (KS) continues to be the most common human immunodeficiency virus (HIV)-associated neoplasm with considerable morbidity and mortality. While lesions normally resolve upon initiation of antiretroviral therapy (ART), recrudescence or unmasking of KS lesions may occur as part of immune reconstitution inflammatory syndrome (IRIS). Treatment of unmasking KS-IRIS is not yet standardised. Objectives: To report the successful treatment of a patient with fulminating mucocutaneous unmasking KS-IRIS by maintaining ART and using pegylated liposomal doxorubicin (PLD). Study design: The patient, a 39-year-old HIV-positive male with no previous history of KS presented with a 2-week history of cutaneous and oral KS lesions that had disseminated rapidly over the preceding 4 days. The KS lesions appeared 8 weeks after recommencing ART. At the time of this presentation, his CD4+ count was 742cells/mm3 with a HIV viral load <400copies/ml. ART was maintained and treatment with PLD commenced. Results: Despite the rapid dissemination of KS lesions, virus was undetectable in plasma. In a late-stage vasoformative lesion, immunohistochemistry (IHC) for human herpesvirus 8 (HHV-8) antigen was light and diffuse, with stippled deposits within endothelial cell nuclei. Virus extracted from the lesion was HHV-8 subtype A. The patient responded well to PLD, relapsed a year later, but after further PLD, has remained well for the following 5 years. Conclusion: Despite the absence of HHV-8 viraemia, this is clearly a case of unmasking KS-IRIS. It demonstrates that this entity can be successfully treated by maintaining ART and administering PLD. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

• Oral swabs are an easy, self-collection method for measuring EBV DNA and antibodies. • Oral swabs can be dried within 5 days without affecting positivity. • EBV-DNA positivity was associated with all symptom scores in the previous 3 to 6 months. • Oral swabs are potentially useful for surveillance of past and recent EBV infection, and in clinical microbiology as a tool for noninvasive surveillance testing. 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. [ABSTRACT FROM AUTHOR]

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. • Oral flocked swabs are an easy, self-collection method for measuring viral antibodies. • Viral IgG is stable on dried oral flocked swabs for at least 2 years. • Oral swabs are highly sensitive for CMV, VZV, and EBV IgG. • Oral swabs are potentially useful for surveillance and clinical microbiology. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Acute respiratory illnesses (ARIs) with unconfirmed infectious aetiologies peak at different times of the year. Molecular diagnostic assays reduce the number of unconfirmed ARIs compared to serology- or culture-based techniques. Screening of 888 inpatient and outpatient respiratory specimens spanning late autumn through to early spring, 2004, identified the presence of a human coronavirus (HCoV) on 74 occasions (8.3% of all specimens and 26.3% of all respiratory virus detections). Prevalence peaked in August (late winter in the southern hemisphere) when they were detected in 21.9% of specimens tested. HCoV-HKU1 and HCoV-OC43 comprised 82.4% of all HCoVs detected. Positive specimens were used to develop novel reverse transcriptase real-time PCRs (RT-rtPCRs) for HCoV detection. An objective clinical severity score was assigned to each positive HCoV patient. Severity scores were similar to those from a random selection of young children who were positive for respiratory syncytial virus at a different time but from the same specimen population. During the cooler months of 2004, sensitive and specific RT-rtPCRs identified the concurrent circulation of all four HCoVs, a quarter of which co-occurred with another virus and most of which were from children under the age of two years. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The first positive case of COVID-19 in Canada was reported on January 25, 2020, in the city of Toronto, Ontario. Over the following four months, the number of individuals diagnosed with COVID-19 in Ontario grew to 28,263 cases. A state of emergency was announced by the Premier of Ontario on March 17, 2020, and the provincial health care system prepared for a predicted surge of COVID-19 patients requiring hospitalization. The Chief Medical Officer of Health and the Minister of Health guided the changes in the system in response to the evolving needs and science related to COVID-19. The pandemic required a rapid, concerted, and coordinated effort from all sectors of the system to optimize and maximize the capacity of the health system. The response to the pandemic in Ontario was complex with some sectors experiencing multiple outbreaks of COVID-19 (i.e. long-term care homes and hospitals). Notably, numerous sectors shifted to virtual delivery of care. By the end of May 2020, it was announced that hospitals would gradually resume postponed or cancelled services. This paper explores the impact of the COVID-19 pandemic on multiple health system sectors (i.e., public health, primary care, long-term care, emergency medical services, and hospitals) in Ontario from January to May 2020. Given the scope of the sectors contributing to the health system in Ontario, this analysis of a regional response to COVID-19 provides insight on how to improve responses and better prepare for future health emergencies. [ABSTRACT FROM AUTHOR]

In late January 2020, the first COVID-19 case was reported in Canada. By March 5, 2020, community spread of the virus was identified and by May 26, 2020, close to 86,000 patients had COVID-19 and 6,566 had died. As COVID-19 cases increased, provincial and territorial governments announced states of public health emergency between March 13 and 20, 2020. This paper examines Canada's public health response to the COVID-19 pandemic during the first four months (January to May 2020) by overviewing the actions undertaken by the federal (national) and regional (provincial/territorial) governments. Canada's jurisdictional public health structures, public health responses, technological and research endeavours, and public opinion on the pandemic measures are described. As the pandemic unravelled, the federal and provincial/territorial governments unrolled a series of stringent public health interventions and restrictions, including physical distancing and gathering size restrictions; closures of borders, schools, and non-essential businesses and services; cancellations of non-essential medical services; and limitations on visitors in hospital and long-term care facilities. In late May 2020, there was a gradual decrease in the daily numbers of new COVID-19 cases seen across most jurisdictions, which has led the provinces and territories to prepare phased re-opening. Overall, the COVID-19 pandemic in Canada and the substantial amount of formative health and policy-related data being created provide an insight on how to improve responses and better prepare for future health emergencies. [ABSTRACT FROM AUTHOR]

• Testing agreement between AmpFire HPV and cobas HPV was strong on FNA and fair on OPS. • AmpFire HPV and cobas HPV testing of FNA showed similar ability to predict p16 positive tumors. • AmpFire HPV provided results more rapidly than cobas HPV. 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. [ABSTRACT FROM AUTHOR]

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, andAspergillus tamariiwere 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. [ABSTRACT FROM PUBLISHER]