Rapid detection of monkeypox virus using a CRISPR-Cas12a mediated assay: a laboratory validation and evaluation study.

BACKGROUND: The 2022 outbreak of mpox (formerly known as monkeypox) led to the spread of monkeypox virus (MPXV) in over 110 countries, demanding effective disease management and surveillance. As current diagnostics rely largely on centralised laboratory testing, our objective was to develop a simple rapid point-of-care assay to detect MPXV in clinical samples using isothermal amplification coupled with CRISPR and CRISPR-associated protein (Cas) technology. METHODS: In this proof-of-concept study, we developed a portable isothermal amplification CRISPR-Cas12a-based assay for the detection of MPXV. We designed a panel of 22 primer-guide RNA sets using pangenome and gene-agnostic approaches, and subsequently shortlisted the three sets producing the strongest signals for evaluation of analytical sensitivity and specificity using a fluorescence-based readout. The set displaying 100% specificity and the lowest limit of detection (LOD) was selected for further assay validation using both a fluorescence-based and lateral-flow readout. Assay specificity was confirmed using a panel of viral and bacterial pathogens. Finally, we did a blind concordance study on genomic DNA extracted from 185 clinical samples, comparing assay results with a gold-standard quantitative PCR (qPCR) assay. We identified the optimal time to detection and analysed the performance of the assay relative to qPCR using receiver operating characteristic (ROC) curves. We also assessed the compatibility with lateral-flow strips, both visually and computationally, where strips were interpreted blinded to the fluorescence results on the basis of the presence or absence of test bands. FINDINGS: With an optimal run duration of approximately 45 min from isothermal amplification to CRISPR-assay readout, the MPXV recombinase polymerase amplification CRISPR-Cas12a-based assay with the selected primer-guide set had an LOD of 1 copy per µL and 100% specificity against tested viral pathogens. Blinded concordance testing of 185 clinical samples resulted in 100% sensitivity (95% CI 89·3-100) and 99·3% specificity (95% CI 95·7-100) using the fluorescence readout. For optimal time to detection by fluorescence readout, we estimated the areas under the ROC curve to be 0·98 at 2 min and 0·99 at 4 min. Lateral-flow strips had 100% sensitivity (89·3-100) and 98·6% specificity (94·7-100) with both visual and computational assessment. Overall, lateral-flow results were highly concordant with fluorescence-based readouts (179 of 185 tests, 96·8% concordant), with discrepancies associated with low viral load samples. INTERPRETATION: Our assay for the diagnosis of mpox displayed good performance characteristics compared with qPCR. Although optimisation of the assay will be required before deployment, its usability and versatility present a potential solution to MPXV detection in low-resource and remote settings, as well as a means of community-based, on-site testing. FUNDING: Victorian Medical Research Accelerator Fund and the Australian Government Department of Health.

Laboratory assessment of a multi-target assay for the rapid detection of viruses causing vesicular diseases.

BACKGROUND: The recent mpox outbreak has highlighted the need to rapidly diagnose the causative agents of viral vesicular disease to inform treatment and control measures. Common causes of vesicular disease include Monkeypox virus (MPXV), clades I and II, Herpes simplex viruses Type 1 and Type 2 (HSV-1, HSV-2), human herpes virus 6 (HHV-6), Varicella-zoster virus (VZV) and Enteroviruses (EVs). Here, we assessed a syndromic viral vesicular panel for rapid and simultaneous detection of these 7 targets in a single cartridge. OBJECTIVE: The aim of this study was to evaluate the QIAStat-Dx ® viral vesicular (VV) panel and compare with laboratory developed tests (LDTs). Limit of detection, inter-run variability, cross-reactivity and specificity were assessed. Positive and negative percent agreement, and correlation between assays was determined using 124 clinical samples from multiple anatomical sites. RESULTS: The overall concordance between the QIAstat and LDTs was 96%. Positive percent agreement was 82% for HHV-6, 89% for HSV-1 and 100% for MPXV, HSV-2, EV and VZV. Negative percent agreement was 100% for all targets assessed. There was no cross-reactivity with Vaccinia, Orf, Molluscum contagiosum viruses, and a pooled respiratory panel. CONCLUSION: The QIAstat VV multi-target syndromic panel combine ease of use, rapid turnaround, good sensitivity and specificity for enhanced diagnosis, clinical care and public health responses.

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

BACKGROUND: 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. OBJECTIVE: 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. RESULTS: 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. CONCLUSIONS: 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.

Monkeypox infection presenting as genital rash, Australia, May 2022.

Rapid diagnosis and whole genome sequencing confirmed a case of monkeypox in an HIV-positive individual receiving antiretroviral therapy. The patient had a normal CD4+ T-cell count and suppressed HIV viral load and presented with a genital rash in Melbourne, Australia after return from Europe in May 2022. He subsequently developed systemic illness and disseminated rash and 11 days after symptom onset, he was hospitalised to manage painful bacterial cellulitis of the genital area.

Prolonged Detection of Japanese Encephalitis Virus in Urine and Whole Blood in a Returned Short-term Traveler.

We describe a fatal case of Japanese encephalitis virus infection following short-term travel to Thailand. Viral RNA was detected in urine and whole blood out to 26 and 28 days, respectively, after the onset of symptoms. Live virus was isolated from a urine specimen from day 14.

Parechovirus Genotype 3 Outbreak among Infants, New South Wales, Australia, 2013-2014.

From October 2013 through February 2014, human parechovirus genotype 3 infection was identified in 183 infants in New South Wales, Australia. Of those infants, 57% were male and 95% required hospitalization. Common signs and symptoms were fever >38°C (86%), irritability (80%), tachycardia (68%), and rash (62%). Compared with affected infants in the Northern Hemisphere, infants in New South Wales were slightly older, both sexes were affected more equally, and rash occurred with considerably higher frequency. The New South Wales syndromic surveillance system, which uses near real-time emergency department and ambulance data, was useful for monitoring the outbreak. An alert distributed to clinicians reduced unnecessary hospitalization for patients with suspected sepsis.

Sepsis-like disease in infants due to human parechovirus type 3 during an outbreak in Australia.

BACKGROUND: Infections with human parechoviruses (HPeVs) are associated with a wide range of clinical presentations in children, ranging from mild or asymptomatic infections to severe sepsis-like presentations or meningoencephalitis. METHODS: We reviewed medical records of infants admitted to 5 hospitals in New South Wales, Australia, during an outbreak of HPeV-3 infection. Data were collected on clinical presentation, laboratory markers, and outcome of infants with HPeV infection confirmed by reverse transcription polymerase chain reaction. RESULTS: We identified 118 infected infants. Most presented with an acute sepsis-like syndrome with high fever, tachycardia, poor perfusion, and severe irritability. Other common features were erythrodermic rash, abdominal distension, edema, and hepatitis. The age range of infants was 4 days to 9.5 months; 75% were <2 months old, including all but 1 of the 30 infants (25%) admitted to intensive care units (ICUs), who as a group, were significantly younger than infants not admitted to ICUs. Only 4% of evaluable cerebrospinal fluid samples had pleocytosis, but HPeV was detected in 95%. Brain magnetic resonance imaging on a small number of children demonstrated white matter changes and diffusion restriction. Sequencing of the VP1 gene confirmed HPeV-3 in all samples tested. All children recovered without ongoing complications at last follow-up. CONCLUSIONS: We report the largest series of HPeV-3 infection in infants, and the first outbreak in Australia. Infants presented with a severe sepsis-like syndrome with a high rate of ICU admissions, but all recovered from the acute infection without complications. Long-term sequelae are unknown.

Detection and genotyping of enteroviruses in cerebrospinal fluid in patients in Victoria, Australia, 2007-2013.

Genotyping by VP1 fragment polymerase chain reaction (PCR) and nucleic acid sequencing to detect enterovirus (EV) genotypes was performed directly on 729 EV PCR positive cerebrospinal fluid (CSF) samples collected between 2007 and 2012 from Victorian hospital inpatients. The overall genotype identification rate from CSF-positive material was 43%. The four most common genotypes identified were Echovirus 6 (24%), Echovirus 30 (17%), Echovirus 25 (10%), and Coxsackievirus A9 (10%), together comprising 61% of all EVs typed. The seasonal distribution of all EVs identified followed the recognized pattern of mainly summer epidemics. Three of the four predominant genotypes were present in each of the 6 years in which the study was conducted, with 20 other EV genotypes also detected, often in only a single year. Genotyping of EVs directly in CSF is faster, simpler and more sensitive than traditional virus neutralization assays performed on EV positive samples.

Influenza vaccine effectiveness during the 2012 influenza season in Victoria, Australia: influences of waning immunity and vaccine match.

Vaccine effectiveness may wane with increasing time since vaccination. This analysis used the Victorian sentinel general practitioner (GP) network to estimate vaccine effectiveness for trivalent inactivated vaccines in the 2012 season. A test-negative design was used where patients presenting to GPs with influenza-like illness who tested positive for influenza were cases and noncases were those who tested negative. Vaccination status was recorded by GPs. Vaccine effectiveness was calculated as (1-odds ratio) × 100%. Estimates were compared early versus late in the season and by time since vaccination. Virus isolates were assessed antigenically by hemagglutination inhibition assay in a selection of positive samples and viruses from healthy adults who experienced a vaccine breakthrough were analyzed genetically. The adjusted vaccine effectiveness estimate for any type of influenza was 45% (95% CI: 8,66) and for influenza A(H3) was 35% (95% CI: -11,62). A non-significant effect of waning effectiveness by time since vaccination was observed for A(H3). For those vaccinated <93 days of presentation vaccine effectiveness was 37% (95% CI: -29,69), while for those vaccinated ≥93 days before presentation it was 18% (95% CI: -83,63). Comparison of early versus late in the season estimates was very sensitive to the cut off week chosen for analysis. Antigenic data suggested that low vaccine effectiveness was not associated with poor vaccine match among the A(H3) viruses. However, genetic analysis suggested nucleotide substitutions in antigenic sites. In 2012, the trivalent influenza vaccine provided moderate protection against influenza and showed limited evidence for waning effectiveness. Antigenic and genetic data can provide additional insight into understanding these estimates.

Epidemiology of the 2012 influenza season in Victoria, Australia.

OBJECTIVE: To assess the magnitude and severity of the 2012 influenza season in Victoria, Australia using surveillance data from five sources. METHODS: Data from influenza notifications, sentinel general practices, a sentinel hospital network, a sentinel locum service and strain typing databases for 2012 were descriptively analysed. RESULTS: Influenza and influenza-like illness activity was moderate compared to previous years, although a considerable increase in notified laboratory-confirmed influenza was observed. Type A influenza comprised between 83% and 87% of cases from the general practitioners, hospitals and notifiable surveillance data. Influenza A/H3 was dominant in July and August, and most tested isolates were antigenically similar to the A/Perth/16/2009 virus used in the vaccine. There was a smaller peak of influenza type B in September. No tested viruses were resistant to any neuraminidase inhibitor antivirals. Higher proportions of type A/H3, hospitalized cases and those with a comorbid condition indicated for influenza vaccination were aged 65 years or older. Influenza vaccination coverage among influenza-like illness patients was 24% in sentinel general practices and 50% in hospitals. DISCUSSION: The 2012 influenza season in Victoria was average compared to previous years, with an increased dominance of A/H3 accompanied by increases in older and hospitalized cases. Differences in magnitude and the epidemiological profile of cases detected by the different data sources demonstrate the importance of using a range of surveillance data to assess the relative severity of influenza seasons.

Evaluation of swabs, transport media, and specimen transport conditions for optimal detection of viruses by PCR.

Depletion of swabs and viral transport medium during epidemics may prompt the use of unvalidated alternatives. Swabs collected and transported dry or in saline were compared to commercially available swab/medium combinations for PCR detection of influenza, enterovirus, herpes simplex virus, and adenovirus. Each was detected at an ambient temperature (22°C) and 4°C for 7 days. Detection of influenza on dry or saline swabs is important because of its capacity to cause outbreaks involving large numbers of cases.

Respiratory virus laboratory pandemic planning and surveillance in central Viet Nam, 2008-2010.

INTRODUCTION: Laboratory capacity is needed in central Viet Nam to provide early warning to public health authorities of respiratory outbreaks of importance to human health, for example the outbreak of influenza A(H1N1) pandemic in 2009. Polymerase chain reaction (PCR) procedures established as part of a capacity-building process were used to conduct prospective respiratory surveillance in a region where few previous studies have been undertaken. METHODS: Between October 2008 and September 2010, nose and throat swabs from adults and children (approximately 20 per week) presenting with an acute respiratory illness to the Ninh Hoa General Hospital were collected. Same-day PCR testing and result reporting for 13 respiratory viruses were carried out by locally trained scientists. RESULTS: Of 2144 surveillance samples tested, 1235 (57.6%) were positive for at least one virus. The most common were influenza A strains (17.9%), with pandemic influenza A(H1N1) 2009 and seasonal H3N2 strain accounting for 52% and 43% of these, respectively. Other virus detections included: rhinovirus (12.4%), enterovirus (8.9%), influenza B (8.3%), adenovirus (5.3%), parainfluenza (4.7%), respiratory syncytial virus (RSV) (3.9%), human coronavirus (3.0%) and human metapneumovirus (0.3%). The detection rate was greatest in the 0-5 year age group. Viral co-infections were identified in 148 (6.9%) cases. DISCUSSION: The outbreak in 2009 of the influenza A(H1N1) pandemic strain provided a practical test of the laboratory's pandemic plan. This study shows that the availability of appropriate equipment and molecular-based testing can contribute to important individual and public health outcomes in geographical locations susceptible to emerging infections.

Estimation of type- and subtype-specific influenza vaccine effectiveness in Victoria, Australia using a test negative case control method, 2007-2008.

BACKGROUND: Antigenic variation of influenza virus necessitates annual reformulation of seasonal influenza vaccines, which contain two type A strains (H1N1 and H3N2) and one type B strain. We used a test negative case control design to estimate influenza vaccine effectiveness (VE) against influenza by type and subtype over two consecutive seasons in Victoria, Australia. METHODS: Patients presenting with influenza-like illness to general practitioners (GPs) in a sentinel surveillance network during 2007 and 2008 were tested for influenza. Cases tested positive for influenza by polymerase chain reaction and controls tested negative for influenza. Vaccination status was recorded by sentinel GPs. Vaccine effectiveness was calculated as [(1--adjusted odds ratio) × 100%]. RESULTS: There were 386 eligible study participants in 2007 of whom 50% were influenza positive and 19% were vaccinated. In 2008 there were 330 eligible study participants of whom 32% were influenza positive and 17% were vaccinated. Adjusted VE against A/H3N2 influenza in 2007 was 68% (95% CI, 32 to 85%) but VE against A/H1N1 (27%; 95% CI, -92 to 72%) and B (84%; 95% CI, -2 to 98%) were not statistically significant. In 2008, the adjusted VE estimate was positive against type B influenza (49%) but negative for A/H1N1 (-88%) and A/H3N2 (-66%); none was statistically significant. CONCLUSIONS: Type- and subtype-specific assessment of influenza VE is needed to identify variations that cannot be differentiated from a measure of VE against all influenza. Type- and subtype-specific influenza VE estimates in Victoria in 2007 and 2008 were generally consistent with strain circulation data.

Clinical and microbiologic features associated with novel swine-origin influenza A pandemic 2009 (H1N1) virus in children: a prospective cohort study.

BACKGROUND: Novel swine-origin influenza A pandemic 2009 (H1N1) virus (S-OIV) infection in the context of other respiratory viruses circulating in winter has not been studied. METHODS: Clinical and microbiologic data were collected prospectively from 444 consecutive patients presenting with an influenza-like illness (ILI) to a large pediatric hospital at the beginning of the S-OIV outbreak in Australia. RESULTS: Of 444 patients, 119 had polymerase chain reaction-confirmed S-OIV. Influenza A virus was detected by direct immunofluorescence in only 69 of these. Overall, inadequate respiratory samples were more common with rayon than flocked swabs (P = 0.01). The mean age of patients with S-OIV was higher than those with another cause of an ILI (10.2 vs. 6.4 years; P < 0.0001). The commonest symptoms in S-OIV were fever (93%) and cough (92%), followed by coryza (78%), sore throat (72%), headache (59%), myalgia (49%), vomiting (23%), and diarrhea (16%). Clinical features did not discriminate between patients with S-OIV and those with another ILI, except headache and myalgia, which were more common in children younger than 5 years who had S-OIV than those who did not (headache: P < 0.0001; myalgia: P = 0.0004). More patients with S-OIV had contact with a confirmed case but contact history had insufficient positive predictive value (44%) and negative predictive value (78%) for identifying S-OIV. Only 2% of the patients had a history of travel, and only 1 of these had S-OIV. CONCLUSIONS: A clinical case definition is unlikely to be useful for discriminating patients with S-OIV from those with another cause of an ILI during winter. Direct immunofluorescence for influenza A cannot be used alone to reliably detect S-OIV.

Higher than expected seasonal influenza activity in Victoria, 2007.

In 2007, the Victorian influenza season exceeded normal seasonal activity thresholds. The average rate of influenza-like illness (ILI) reported by general practitioners (GPs) participating in sentinel surveillance was 9.0 cases per 1,000 consultations, peaking at 22 cases per 1,000 consultations in mid-August. The average ILI rate reported by the Melbourne Medical Locum Service (MMLS) was 11.5 per 1,000 consultations over the season. The MMLS ILI rate peaked at 30 per 1,000 consultations at the same time as peak rates were reported by GPs, with a secondary peak observed three weeks later (22 cases per 1,000 consultations). Influenza cases notified to the Victorian Department of Human Services peaked in mid-August with a secondary peak of influenza A in early September. Of the influenza positive swabs collected by GPs and among those collected throughout the state, 92% were type A and 8% were type B. The most common strains identified in Victoria in the 2007 influenza season were A/ Brisbane/10/2007-like followed by A/Solomon Islands/3/2006-like. While neither virus strain was specifically included in the 2007 Australian influenza vaccine, reasonable cross protection was afforded by the strains in the vaccine.