Victorian Infectious Diseases Reference Laboratory Research Articles

Evaluation of 16 molecular assays for the detection of orthopox and mpox viruses

The current global mpox virus (MPXV) outbreak has been declared a Public Health Emergency of International Concern by WHO, with more than 80,000 cases confirmed across multiple continents. Diagnosis is confirmed by PCR of viral DNA from vesicle and other swabs. The aim of this study was to assess commercial RT PCR assays for Orthopoxvirus (OPX) and MPXV for analytical sensitivity, and percent agreements and compare them to primer/probe sets employed at the Victorian Infectious Diseases Reference Laboratory (VIDRL), Centers for Disease Control andPrevention (CDC) and US Army Medical Research Institute of Infectious Diseases (USAMRIID). Limits of detection (LOD), intra-run variability, cross-reactivity and performance on forty clinical samples was assessed on eleven commercial assays and five primer/probe combinations used at VIDRL, CDC and USAMRIID. All assays were able to detect OPX and MPXV (LOD 57 to 14,495 copies/mL) with intra-run coefficients of variation between Cycle thresholds of 0.58 and 3.44, and there was no unexpected cross-reactivity. All assays demonstrated 100% negative percent agreement with clinical samples and all but one yielded 100% positive percent agreement. Variations in LOD between assays may be dependent on the platform used and sample type. Despite the overall comparable performance of the assays assessed, it is important that routine laboratories perform in-house validations before implementing RT PCR for OPX and/or MPXV as reliable and accurate laboratory diagnosis of MPXV and isolation is crucial to containing the spread of this current outbreak and informing public health interventions and response.

Correlation between monkeypox viral load and infectious virus in clinical specimens

In the 2022 mpox outbreak, several studies have explored longitudinal DNA shedding of mpox virus (MPXV) using PCR. However, there are fewer studies assessing infectivity in cell culture, and, by inference, MPXV transmissibility. Such information could help inform infection control and public health guidelines. The aim of this study was to correlate cell culture infectivity of clinical samples with viral loads in clinical samples. Between May to October 2022, clinical samples from different body sites sent to the Victorian Infectious Diseases Reference Laboratory in Melbourne, Australia for MPXV PCR detection were cultured in Vero cells as a surrogate for infectivity. In the study period, 144 samples from 70 patients were tested by MPXV PCR. Viral loads in skin lesions were significantly higher than those in throat or nasopharyngeal samples (median Ct 22.0vs 29.0, p=0.0013 and median Ct 22.0vs 36.5, p=0.0001, respectively). Similarly, viral loads were significantly higher in anal samples compared to throat or nasopharyngeal samples (median Ct 20.0vs. 29.0, p=<0.0001 and median Ct 20.0vs. 36.5, p=<0.0001, respectively). Viral culture was successfully performed in 80/94 samples. Using logistic regression analysis, 50% of the samples were positive in viral culture at Ct 34.1 (95% confidence intervals 32.1-37.4). Our data further validate recent findings showing that samples with a higher MPXV viral load are more likely to demonstrate infectivity in cell culture. Although the presence of infectious virus in cell culture may not directly translate with clinical transmission risk, our data may be used as an adjunct help inform guidelines on testing and isolation policies in individuals with mpox.

Results from the second WHO external quality assessment for the molecular detection of respiratory syncytial virus, 2019-2020.

External quality assessments (EQAs) for the molecular detection of human respiratory syncytial virus (RSV) are necessary to ensure the standardisation of reliable results. The Phase II, 2019-2020 World Health Organization (WHO) RSV EQA included 28 laboratories in 26 countries. The EQA panel evaluated performance in the molecular detection and subtyping of RSV-A and RSV-B. This manuscript describes the preparation, distribution, and analysis of the 2019-2020 WHO RSV EQA. Panel isolates underwent whole genome sequencing and in silico primer matching. The final panel included nine contemporary, one historical virus and two negative controls. The EQA panel was manufactured and distributed by the UK National External Quality Assessment Service (UK NEQAS). National laboratories used WHO reference assays developed by the United States Centers for Disease Control and Prevention, an RSV subtyping assay developed by the Victorian Infectious Diseases Reference Laboratory (Australia), or other in-house or commercial assays already in use at their laboratories. An in silico analysis of isolates showed a good match to assay primer/probes. The panel was distributed to 28 laboratories. Isolates were correctly identified in 98% of samples for detection and 99.6% for subtyping. The WHO RSV EQA 2019-2020 showed that laboratories performed at high standards. Updating the composition of RSV molecular EQAs with contemporary strains to ensure representation of circulating strains, and ensuring primer matching with EQA panel viruses, is advantageous in assessing diagnostic competencies of laboratories. Ongoing EQAs are recommended because of continued evolution of mismatches between current circulating strains and existing primer sets.

Mycobacterium tuberculosis Genotypes and Drug Susceptibility Test Results from Timor-Leste: A Pilot Study.

Tuberculosis (TB) is prevalent and a major public health problem in Timor-Leste. The government of Timor-Leste is prioritising the surveillance of TB and drug-susceptibility testing (DST) to understand the burden of TB and TB drug resistance in the country. Moreover, little is known about the origin of Mycobacterium tuberculosis (MTB) in Timor-Leste. This study reports MTB DST and sequencing for Timor-Leste. A pilot study was carried out in which a convenience sample of TB isolates from mucopurulent sputum collected from presumptive TB patients in the capital Dili between July and December 2016 was tested for phenotypic and genotypic evidence of drug resistance. Standard MTB culture was performed at the Timor-Leste National Health Laboratory (NHL). The MTB isolates were sent to the Victorian Infectious Diseases Reference Laboratory (VIDRL) in Australia for DST and sequencing. Overall, 36 MTB isolates were detected at the NHL; 20 isolates were recovered during sub-culturing at VIDRL. All 20 isolates were susceptible to rifampicin, isoniazid, pyrazinamide, and ethambutol, with no genotypic markers of resistance identified. On sequencing, lineage 4 was the most common. The results of this study provide a small snapshot of MTB diversity and resistance in an under-sampled region with very high TB incidence. Future investment in whole-genome sequencing capacity in Timor-Leste will make it possible to undertake further, more representative analyses that may be used to evaluate transmission dynamics and epidemiology of genotypic markers of resistance.

Factors associated with weak positive SARS-CoV-2 diagnosis by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR)

During the COVID-19 pandemic, the reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assay has been the primary method of diagnosis of SARS-CoV-2 infection. However, RT-qPCR assay interpretation can be ambiguous with no universal absolute cut-off value to determine sample positivity, which particularly complicates the analysis of samples with high Ct values, or weak positives. Therefore, we sought to analyse factors associated with weak positive SARS-CoV-2 diagnosis.We analysed sample data associated with all positive SARS-CoV-2 RT-qPCR diagnostic tests performed by the Victorian Infectious Diseases Reference Laboratory (VIDRL) in Melbourne, Australia, during the Victorian first wave (22 January 2020–30 May 2020). A subset of samples was screened for the presence of host DNA and RNA using qPCR assays for CCR5 and 18S, respectively.Assays targeting the viral RNA-dependent RNA polymerase (RdRp) had higher Ct values than assays targeting the viral N and E genes. Weak positives were not associated with the age or sex of individuals’ samples nor with reduced levels of host DNA and RNA. We observed a relationship between Ct value and time post-SARS-CoV-2 diagnosis.High Ct value or weak positive SARS-CoV-2 was not associated with any particular bias including poor biological sampling.

Genomics-Informed Responses in the Elimination of COVID-19 in Australia

BACKGROUND: A cornerstone of Australia’s ability to control COVID-19 has been effective border control, using an extensive supervised quarantine program. However, a rapid recrudescence in COVID-19 cases was observed in the state of Victoria in June 2020. Here, we describe the genomic findings that located the source of this second wave as a breach in supervised hotel quarantine and demonstrate the successful elimination of COVID-19 for a second time in Australia.METHODS: Genome sequencing was performed on all available SARS-CoV-2-positive samples in Victoria and integrated genomic and epidemiological investigation undertaken.RESULTS: At 31st January 2021, 20,451 COVID-19 cases were reported in Victoria; samples were sequenced from 75% of cases (15,431/20,451). Genomics revealed 98% (10,426/10,646) of locally-acquired cases during the second wave were derived from a single incursion from hotel quarantine, with the outbreak strain rapidly detected in other Australian states and territories. Phylodynamic analyses indicated an epidemic growth rate comparable to emerging variants, such as B.1.1.7 in the United Kingdom. Strict public health interventions resulted in the elimination of the outbreak strain by 29th October 2020. Subsequent cases represented independent international or interstate introductions, with limited local spread.CONCLUSIONS: Rapid escalation of clonal outbreaks can occur from even a single breach of control practices, as revealed through our genomic ‘enhanced outbreak-detection' system. The subsequent elimination and rapid control of new SARS-CoV-2 incursions reinforce that decisive public health responses to emergent cases are effective even with high epidemic growth rates, and “elimination” should be favored in settings where this is achievable.FUNDING STATEMENT: The Microbiological Diagnostic Unit Public Health Laboratory (MDU PHL) and the Victorian Infectious Diseases Reference Laboratory (VIDRL) at The Doherty Institute are funded by the Victorian Government. This work was supported by the National Health and Medical Research Council, Australia (NHMRC); Partnership Grant (APP1149991), Investigator Grant to BPH (APP1196103), Investigator Grant to DAW (APP1174555), Research Fellowship to TPS (APP1105525), MRFF COVID-19 Genomics Grant (MRF9200006).DECLARATION OF INTERESTS: None to declare. ETHICS APPROVAL STATEMENT: Data were collected in accordance with the Victorian Public Health and Wellbeing Act 2008. Ethical approval was received from the University of Melbourne Human Research Ethics Committee (study number 1954615.3).

Cross-contamination in Molecular Diagnostic Laboratories in Low- and Middle-income Countries: A Challenge to COVID-19 Testing

At the start of the pandemic, the Philippines had to send swab samples to the Victorian Infectious Diseases Reference Laboratory in Melbourne, Australia for COVID-19 confirmation. With the increasing number of suspected cases needing confirmatory diagnostic testing, there was a demand to rapidly expand the capacity for widescale testing. Remarkably, within 200 days from announcement of the first confirmed COVID-19 case in the Philippines in January 30, 2020, the country has been able to expand its testing capacity from one national reference laboratory, the Research Institute for Tropical Medicine (RITM), to more than 100 licensed reverse transcription-polymerase chain reaction (RT-PCR) and cartridge-based PCR laboratories across the country. Due to the shortage of a trained clinical laboratory workforce, diagnostic centers are forced to hire additional personnel who have limited experience and technical knowledge and skills of molecular assays, especially in processing specimens, interpreting the results, identifying errors, and troubleshooting, in order to meet the demand of increased testing. Thus, the vulnerability to diagnostic errors, including cross-contamination, is increased and with the tendency for generating false positive results that can compromise the health of the patient and disrupt the efficacy of public health policies and public health response, surveillance programs, and restrictive measures for containing the outbreak. Hence, this review article aims to present the different sources of contamination in the laboratory setting where RT-PCR assays are conducted, as well as provide efficient, effective and feasible solutions to address these issues, most especially in low- and middle-income countries (LMICs) like the Philippines.

We Are All Africans: A Highly Personal Migratory View of the History of Hepatitis B.

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A severe 2017 influenza season dominated by influenza A(H3N2), Victoria, Australia.

Surveillance for influenza-like illness (ILI) and laboratory-confirmed influenza in Victoria, Australia is undertaken jointly by the Victorian Infectious Diseases Reference Laboratory and the Victorian Government Department of Health and Human Services from May to October each year. Surveillance data comprise notifiable laboratory-confirmed influenza and ILI reporting from from two sources – a general practice sentinel surveillance programme and a locum service.The magnitude of the 2017 influenza season was high in Victoria with widespread circulation of influenza type A(H3N2), which peaked in September. A record number of laboratory-confirmed influenza cases were notified, and the proportion of ILI cases to total consultations from both the general practice and locum service were higher than previous years. Notified cases of influenza A were older than influenza B cases with 25% compared to 17% aged more than 65 years, respectively. The proportion of swabs that were positive for influenza peaked at 58%. Antigenic characterization suggested a good match between the circulating and vaccine strains of influenza A(H3N2).Most of the increases observed in notified cases of laboratory-confirmed influenza in recent years in Victoria have been attributed to increases in testing. However, that cases of ILI also increased in Victoria in 2017 is suggestive that 2017 was a relatively severe season. The dominance of influenza type A(H3N2), the extended duration of elevated activity, and a potential phylogenetic mismatch of vaccine to circulating strains are likely to have contributed to the relative severity of the 2017 season.Victoria is Australia’s second most populous state and is the mainland’s southernmost state. It has a temperate climate with an influenza season usually occurring in the cooler months between May and October. The Victorian Infectious Diseases Reference Laboratory (VIDRL), in partnership with the Victorian Government Department of Health and Human Services (DHHS), coordinates influenza-like illness (ILI) and laboratory-confirmed influenza surveillance in Victoria. There are three data sources included in the influenza surveillance system.

Feline leprosy due to Candidatus 'Mycobacterium lepraefelis': Further clinical and molecular characterisation of eight previously reported cases and an additional 30 cases.

This paper, the last in a series of three on 'feline leprosy', provides a detailed description of disease referable to the previously unnamed species, Candidatus 'Mycobacterium lepraefelis', a close relative of the human pathogens Mycobacterium leprae and Mycobacterium lepromatosis. Cases were sourced retrospectively and prospectively for this observational study, describing clinical, geographical and molecular microbiological data for cats definitively diagnosed with Candidatus 'M lepraefelis' infection. A total of 145 cases of feline leprosy were scrutinised; 114 'new' cases were sourced from the Victorian Infectious Diseases Reference Laboratory (VIDRL) records, veterinary pathology laboratories or veterinarians, and 31 cases were derived from six published studies. Thirty-eight cats were definitively diagnosed with Candidatus 'M lepraefelis' infection. Typically, cats tended to be middle-aged or older when first infected, with a male predilection. Affected cats typically had widespread cutaneous lesions, in some cases after initially localised disease. Advanced cases were often systemically unwell. All cats had outdoor access. The histological picture was lepromatous in the majority of patients, although two cases had tuberculoid disease. In one case that underwent necropsy, lesions were evident in the liver, spleen and lungs. Treatment was varied, although most cats received a combination of oral clarithromycin and rifampicin. Prognosis for recovery was variable, but typically poor. Candidatus 'M lepraefelis' typically causes high bacterial index (lepromatous) feline leprosy that in some cases progresses to systemic mycobacteriosis. The disease has a variable clinical course and prognosis. Many cases either died or were euthanased due to the infection. Multilocus sequence analysis reveals a heterogeneous picture and further analysis of draft genome sequencing may give clues to the taxonomy and epidemiology of this organism. Prospective treatment trials and/or additional drug susceptibility testing in specialised systems would further inform treatment recommendations. Comparative aspects: This paper finishes with a discussion of comparative aspects of infection caused by the three feline leproid disease agents that have been the subject of this series: Candidatus 'Mycobacterium tarwinense', Mycobacterium lepraemurium and Candidatus 'M lepraefelis'.

Feline leprosy due to Mycobacterium lepraemurium.

This paper, the second in a series of three on 'feline leprosy', provides a detailed description of disease referable to Mycobacterium lepraemurium, the most common cause of feline leprosy worldwide. Cases were sourced retrospectively and prospectively for this observational study, describing clinical, geographical and molecular microbiological data for cats definitively diagnosed with M lepraemurium infection. A total of 145 cases of feline leprosy were scrutinised; 114 'new' cases were sourced from the Victorian Infectious Diseases Reference Laboratory records, veterinary pathology laboratories or veterinarians, and 31 cases were derived from six published studies. Sixty-five cats were definitively diagnosed with M lepraemurium infection. Typically, cats were 1-3 years of age when first infected, with a male gender predilection. Affected cats were generally systemically well. All had outdoor access. Lesions tended to consist of one or more cutaneous/subcutaneous nodules, typically located on the head and/or forelimbs, possibly reflecting the most likely locations for a rodent bite as the site of inoculation for organisms. Nodules had the propensity to ulcerate at some stage in the clinical course. The cytological and histological picture varied from tuberculoid, with relatively low bacterial numbers, to lepromatous with moderate to high bacterial numbers. Treatment was varied, although most cats underwent surgical resection of lesions with adjunctive medical therapy, most often using a combination of oral clarithromycin and rifampicin. Prognosis for recovery was generally good, and in two cases there was spontaneous remission without the requirement for medical intervention. Untreated cats continued to enjoy an acceptable quality of life despite persistence of the disease, which extended locally but had no apparent tendency to disseminate to internal organs. M lepraemurium causes high bacterial index (lepromatous) or low bacterial index (tuberculoid) feline leprosy. The infection typically causes nodules of the skin and/or subcutis (which tend towards ulceration) on the head and/or forelimbs. The disease usually has an indolent clinical course and infected cats have a generally favourable response to therapeutic interventions, with rare cases undergoing spontaneous resolution. Genomic analysis may yield clues as to the environmental niche and culture requirements of this elusive organism. Prospective treatment trials and/or additional drug susceptibility testing in specialised systems would further inform treatment recommendations.

Feline leprosy due to Candidatus 'Mycobacterium tarwinense':Further clinical and molecular characterisation of 15 previously reported cases and an additional 27 cases

This paper, the first in a series of three on 'feline leprosy', provides a detailed description of disease referable to Candidatus 'Mycobacterium tarwinense', the most common cause of feline leprosy in Victoria, Australia. Cases were sourced retrospectively and prospectively for this observational study, describing clinical, geographical and molecular microbiological data for cats definitively diagnosed with Candidatus 'M tarwinense' infection. A total of 145 cases of feline leprosy were scrutinised; 114 'new' cases were sourced from the Victorian Infectious Diseases Reference Laboratory records, veterinary pathology laboratories or veterinarians, and 31 cases were derived from six published studies. Forty-two cats were definitively diagnosed with Candidatus 'M tarwinense' infection. Typically, cats were between 3 and 11 years of age, with no gender predilection, and were generally systemically well. All had outdoor access. Most cats underwent surgical resection of lesions with adjunctive medical therapy, often utilising a combination of oral clarithromycin and rifampicin for at least 3 months. Prognosis for recovery was generally good. Resolution of lesions was not observed in the absence of treatment, but a number of untreated cats continued to enjoy an acceptable quality of life despite persistence of the disease, which extended locally but did not appear to disseminate to internal organs. Preliminary results of draft genome sequencing confirmed that the species is a member of the Mycobacterium simiae complex. Candidatus 'M tarwinense', a fastidious member of the M simiae complex, is capable of causing feline leprosy with a tendency to produce lesions on the head, particularly involving the eyes and periocular skin. The disease has an indolent clinical course and generally responds favourably to therapy despite lesions often containing large numbers of organisms. Detailed genomic analysis may yield clues as to the environmental niche and culture requirement of this elusive organism. Prospective treatment trials and/or drug susceptibility testing in specialised systems would further inform treatment recommendations.

Evidence and policy for influenza control

H Kelly1,2, B J Cowling (bcowling@hku.hk)3 1. Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia 2. National Centre for Epidemiology and Population Health, Australian National University, Canberra, Australia 3. Division of Epidemiology and Biostatistics, School of Public Health, Li Ka Shing Faculty of Medicine, the University of Hong Kong, Hong Kong Special Administrative Region, China

Evaluation of the World Health Organization Global Measles and Rubella Quality Assurance Program, 2001–2008

During 2001-2008, the Victorian Infectious Diseases Reference Laboratory (VIDRL) prepared and provided a measles and rubella proficiency test panel for distribution to the World Health Organization (WHO) measles and rubella network laboratories as part of their annual laboratory accreditation assessment. Panel test results were forwarded to VIDRL, and results from 8 consecutive years were analyzed. We assessed the type of assays used and results achieved on the basis of the positive and negative interpretation of submitted results, by year and WHO region, for measles and rubella. Over time, there has been a noticeable increase in laboratory and WHO regional participation. For all panels, the proportion of laboratories in all WHO regions using the WHO-validated Dade Behring assay for measles and rubella-specific IgM antibodies ranged from 35% to 100% and 59% to 100%, respectively. For all regions and years, the proportion of laboratories obtaining a pass score ranged from 87% to 100% for measles and 93% to 100% for rubella. During 2001-2008, a large proportion of laboratories worldwide achieved and maintained a pass score for both measles and rubella. Measles and rubella proficiency testing is regarded as a major achievement for the WHO measles and rubella laboratory program.

P1-S1.01 Trends in chlamydia and gonorrhoea positivity among heterosexual men and men who have sex with men (MSM) attending a large urban sexual health service in Australia, 2002–2009

BackgroundNotifications for Sexually transmitted infections cannot be used to measure changing incidence or prevalence of infection if a substantial proportion of infections are asymptomatic and screening rates are low. This...

Reality Check of Laboratory Service Effectiveness during Pandemic (H1N1) 2009, Victoria, Australia

Abstract In Australia, the outbreak of pandemic (H1N1) 2009 began in Melbourne, Victoria; in the first 17 days, the Victorian Infectious Diseases Reference Laboratory detected 977 cases. Although the laboratory had a pandemic plan in place, a retrospective evaluation found 3 major variations from plan assumptions: 1) higher peak demand not limited by a case definition, 2) prolonged peak demand because containment attempts continued despite widespread influenza, and 3) unexpected influence of negative test results on public health actions. Although implementation of the plan was generally successful, the greatest challenges were limited availability of skilled staff and test reagents. Despite peak demand of 1,401 tests per day, results were provided within the usual 24 hours of specimen receipt; however, turnaround time seemed slower because of slow transport times (>3 days for 45% of specimens). Hence, effective laboratory capability might be enhanced by speeding transport of specimens and improving transmission of clinical data.

Reality Check of Laboratory Service Effectiveness during Pandemic (H1N1) 2009, Victoria, Australia

In Australia, the outbreak of pandemic (H1N1) 2009 began in Melbourne, Victoria; in the first 17 days, the Victorian Infectious Diseases Reference Laboratory detected 977 cases. Although the laboratory had a pandemic plan in place, a retrospective evaluation found 3 major variations from plan assumptions: 1) higher peak demand not limited by a case definition, 2) prolonged peak demand because containment attempts continued despite widespread influenza, and 3) unexpected influence of negative test results on public health actions. Although implementation of the plan was generally successful, the greatest challenges were limited availability of skilled staff and test reagents. Despite peak demand of 1,401 tests per day, results were provided within the usual 24 hours of specimen receipt; however, turnaround time seemed slower because of slow transport times (>3 days for 45% of specimens). Hence, effective laboratory capability might be enhanced by speeding transport of specimens and improving transmission of clinical data.

All-Oral Antibiotic Treatment for Buruli Ulcer: A Report of Four Patients

1Department of Infectious Diseases, Austin Health, Melbourne, Australia, 2Department of Surgery, Box Hill Hospital, Melbourne, Australia, 3Department of Surgery,Monash University, Melbourne, Australia, 4Department of Anatomy and Developmental Biology, Monash University, Melbourne, Australia, 5WHO Collaborating Centrefor Mycobacterium ulcerans (Western Pacific Region) and Victorian Infectious Diseases Reference Laboratory, Melbourne, Australia, 6Department of AnatomicalPathology, Box Hill Hospital, Melbourne, Australia, 7Department of Infectious Diseases, Royal Children’s Hospital, Melbourne, Australia

Influenza surveillance in Australia: we need to do more than count

Laboratory-confirmed influenza is a nationally notifiable disease in Australia. According to notification data, Queensland has experienced more severe influenza seasons than other states and territories. However, this method ignores available denominator data: the number of laboratory tests performed. We propose that negative results of laboratory tests for influenza should be made notifiable, alongside laboratory-confirmed disease, and used to calculate the proportion of positive test results in real-time. Using data from the public health pathology services of three Australian states - Queensland Health laboratories, the Victorian Infectious Diseases Reference Laboratory and Western Australia's PathWest - for 2004 to 2008, we show that incorporating laboratory-negative test data into national surveillance data would add to and improve our understanding of influenza epidemiology.

Indeterminate Interferon-γ Release Assay Results in Children

Indeterminate Interferon-γ Release Assay Results in Children