The changing epidemiology of Neisseria gonorrhoeae genogroups and antimicrobial resistance in Queensland, Australia, 2010-15: a case series analysis of unique Neisseria gonorrhoeae isolates.

BACKGROUND: Neisseria gonorrhoeae (NG) can lead to serious reproductive and sexual health outcomes, and the annual number of NG notifications in Australia increased steadily from 10329 in 2010 to 29549 by 2020. Australian populations most affected are urban men who have sex with men and First Nations peoples living in remote areas, and a resurgence in urban heterosexuals has been observed since 2012. METHODS: A case series analysis of Queensland NG isolates (2010-15) exploring temporal trends and antimicrobial resistance by demographic and geographic distribution and genotype was performed. Proportions describe age, sex, strain, genogroup (NG multi-antigen sequence typing), region, swab site, antimicrobial sensitivity and isolate rates per 100000 population. Dominant genogroups were identified. RESULTS: Among 3953 isolates, the median age was 25years (IQR 20-34years) and most (n =2871/3915, 73%) were men. Brisbane city (68.8) and Far North Queensland (54.1) excluding Cairns showed the highest rates. Forty-six genogroups were documented, seven (G2992, G6876, G1415, G4186, G5, G1407 and G6937) comprised half of all isolates. The predominant male genogroup was G2992 (16%), and G6876 (20%) for females; G5 was predominantly male from 2010 to 2011, but equal in both sexes from 2012 to 2015. CONCLUSION: Considerable temporal, geographical and demographical diversity was observed in Queensland NG isolates, which has public health implications. Certain genogroups are more transient than others, and evidence suggests bridging from male-dominant networks to heterosexual networks. Molecular surveillance can enhance tracking the epidemiology and movement of NG in Australia, highlighting the necessity of genotyping to expose potentially prevalent strains circulating in undetected or underrepresented networks by current screening methods.

Optimized Method for Bacterial Nucleic Acid Extraction from Positive Blood Culture Broth for Whole-Genome Sequencing, Resistance Phenotype Prediction, and Downstream Molecular Applications.

The application of direct metagenomic sequencing from positive blood culture broth may solve the challenges of sequencing from low-bacterial-load blood samples in patients with sepsis. Forty prospectively collected blood culture broth samples growing Gram-negative bacteria were extracted using commercially available kits to achieve high-quality DNA. Species identification via metagenomic sequencing and susceptibility prediction via a machine-learning algorithm (AREScloud) were compared to conventional methods and other rapid diagnostic platforms (Accelerate Pheno and blood culture identification [BCID] panel). A two-kit method (using MolYsis Basic and Qiagen DNeasy UltraClean kits) resulted in optimal extractions. Taxonomic profiling by direct metagenomic sequencing matched conventional identification in 38/40 (95%) samples. In two polymicrobial samples, a second organism was missed by sequencing. Prediction models were able to accurately infer susceptibility profiles for 6 common pathogens against 17 antibiotics, with an overall categorical agreement (CA) of 95% (increasing to >95% for 5/6 of the most common pathogens, if Klebsiella oxytoca was excluded). The performance of whole-genome sequencing (WGS)-antimicrobial susceptibility testing (AST) was suboptimal for uncommon pathogens (e.g., Elizabethkingia) and some ß-lactamase inhibitor antibiotics (e.g., ticarcillin-clavulanate). The time to pathogen identification was the fastest with BCID (1 h from blood culture positivity). Accelerate Pheno provided a susceptibility result in approximately 8 h. Illumina-based direct sequencing methods provided results in time frames similar to those of conventional culture-based methods. Direct metagenomic sequencing from blood cultures for pathogen detection and susceptibility prediction is feasible. Additional work is required to optimize algorithms for uncommon species and complex resistance genotypes as well as to streamline methods to provide more rapid results.

Emergence and spread of ciprofloxacin-resistant Neisseria gonorrhoeae in New South Wales, Australia: lessons from history.

OBJECTIVES: Our aim was to investigate the emergence and spread of ciprofloxacin resistance in clinical Neisseria gonorrhoeae isolates in New South Wales, Australia, from the first reported case in 1991 until ciprofloxacin resistance was sustained at or above the WHO threshold for treatment change of 5% (1999), to inform future strategies for controlling gonococcal antimicrobial resistance. METHODS: The index isolate and all subsequent clinical isolates of ciprofloxacin-resistant N. gonorrhoeae in New South Wales from 1991 to 1999 were genotyped using a previously described method on the Agena MassARRAY iPLEX platform. Region of acquisition data, where available, were used to determine whether cases were travel associated. RESULTS: In New South Wales, of the 325 ciprofloxacin-resistant N. gonorrhoeae isolates reported from 1991 to 1999, 98% (320/325) were able to be recovered and 100% (320/320) were genotyped. There were 66 different genotypes, comprising 1-99 isolates each. Notably no single clone was found to account for ciprofloxacin resistance being sustained in the population, with considerable variability in genotype prevalence observed throughout the study period. A total of 65% (209/320) of genotyped isolates had information regarding the likely place of acquisition; of these, 44% (93/209) were associated with overseas travel or sexual contact with an overseas visitor. The first ciprofloxacin-resistant N. gonorrhoeae in New South Wales was associated with travel to Thailand. Index cases of each resistant genotype were significantly more likely to have been acquired overseas (51.5%), predominantly in Asia (45%, 30/66). CONCLUSIONS: The continued importation of multiple genotypes, rather than the expansion of a single genotype, led to ciprofloxacin-resistant N. gonorrhoeae being established in New South Wales.

Evaluation of the SpeeDx Carba (beta) multiplex real-time PCR assay for detection of NDM, KPC, OXA-48-like, IMP-4-like and VIM carbapenemase genes.

BACKGROUND: Carbapenemase-producing organisms (CPOs) have emerged as antibiotic-resistant bacteria of global concern. Here we assessed the performance of the Carba (beta) assay, a multiplex real-time PCR assay developed by SpeeDx for the detection of key carbapenemase-encoding genes: KPC, NDM, OXA-48-like, IMP-4-like, and VIM. METHODS: DNA extracts of 180 isolates were tested with the Carba (beta) assay, using previously validated in-house TaqMan probe assays for the relevant carbapenemase genes as the reference standard. The Carba (beta) assay was then directly used to screen 460 DNA extracts of faecal specimens, with positive results subjected to the aforementioned in-house assays plus Sanger sequencing. RESULTS: The Carba (beta) assay correctly identified the presence of the respective carbapenemase genes in 154 of 156 isolates and provided negative results for all 24 non-CPO isolates. Two isolates provided positive results for OXA-48-like carbapenemase by the Carba (beta) assay only. The Carba (beta) assay had sensitivities of 100% for all targets, and specificities of 100% for KPC, NDM, IMP-4-like, and VIM targets, and 98.5% for OXA-48-like targets. When applied directly to faecal specimens, eight samples were positive by the Carba (beta) assay, two of which were confirmed by in-house TaqMan probe PCR or DNA sequencing. CONCLUSIONS: The Carba (beta) assay is highly sensitive and specific for detecting key carbapenemase genes in isolates. Further testing is required to assess this assay's suitability for direct screening of clinical specimens.

Whole-genome sequencing as an improved means of investigating Neisseria gonorrhoeae treatment failures.

Background Although rare, Neisseria gonorrhoeae treatment failures associated with ceftriaxone have been reported. The World Health Organization (WHO) recommends standardised protocols to verify these cases. Two cases from Australia were previously investigated using N. gonorrhoeae multiantigen sequence typing (NG-MAST), which has been used extensively to assess treatment failures. Case 1 pharyngeal isolates were indistinguishable, whereas Case 2 pharyngeal isolates were distinguished based on an 18-bp deletion in the major outer membrane porin encoded by the porB gene, questioning the reliability of NG-MAST results. Here we used whole-genome sequencing (WGS) to reinvestigate Cases 1 and 2, with a view to examining WGS to assess treatment failures. METHODS: Pre- and post-treatment isolates for each case underwent Illumina sequencing, and the two post-treatment isolates underwent additional long-read sequencing using Pacific Biosciences. Sequence data were interrogated to identify differences at single nucleotide resolution. RESULTS: WGS identified variation in the pilin subunit encoded by the pilE locus for both cases and the specific 18-bp porB deletion in Case 2 was confirmed, but otherwise the isolates in each case were indistinguishable. CONCLUSIONS: The WHO recommends standardised protocols for verifying N. gonorrhoeae treatment failures. Case 2 highlights the enhanced resolution of WGS over NG-MAST and emphasises the immediate effect that WGS can have in a direct clinical application for N. gonorrhoeae. Assessing the whole genome compared with two highly variable regions also provides a more confident predictor for determining treatment failure. Furthermore, WGS facilitates rapid comparisons of these cases in the future.

Azithromycin-resistant Neisseria gonorrhoeae spreading amongst men who have sex with men (MSM) and heterosexuals in New South Wales, Australia, 2017.

Objectives: To identify the genetic basis of resistance as well as to better understand the epidemiology of a recent surge in azithromycin-resistant Neisseria gonorrhoeae in New South Wales, Australia. Methods: Azithromycin-resistant N. gonorrhoeae isolates (n = 118) collected from 107 males, 10 females and 1 transsexual between January and July 2017 were genotyped using a previously described iPLEX method. The results were compared with phenotypic resistance profiles and available patient data. Results: The iPLEX results revealed 10 different N. gonorrhoeae genotypes (designated AZI-G1 to AZI-G10) of which three were responsible for the majority of infections; AZI-G10 (74.6%, 88 isolates; 87 males and 1 transsexual), AZI-G4 (11.0%, 13 isolates; 7 males and 6 females) and AZI-G7 (6.8%, 8 isolates; 7 males and 1 female). The observed resistance was attributable to one of two different azithromycin resistance mechanisms; the 23S rRNA C2611T mutation was identified in 24% of isolates, whereas the majority of resistance (76%) was associated with a meningococcal-type mtrR variant. Additionally, one isolate was found to harbour both the 23S rRNA C2611T mutation and a type XXXIV mosaic penA sequence associated with cephalosporin resistance. Conclusions: These data indicate outbreaks of azithromycin-resistant gonococci amongst networks of MSM and heterosexuals in New South Wales. The results also provide further evidence that azithromycin may soon be an ineffective treatment option for gonococcal infection and highlight the urgent need to explore alternative therapies.

Molecular Antimicrobial Resistance Surveillance for Neisseria gonorrhoeae, Northern Territory, Australia.

Neisseria gonorrhoeae antimicrobial resistance (AMR) is a globally recognized health threat; new strategies are needed to enhance AMR surveillance. The Northern Territory of Australia is unique in that 2 different first-line therapies, based primarily on geographic location, are used for gonorrhea treatment. We tested 1,629 N. gonorrhoeae nucleic acid amplification test-positive clinical samples, collected from regions where ceftriaxone plus azithromycin or amoxicillin plus azithromycin are recommended first-line treatments, by using 8 N. gonorrhoeae AMR PCR assays. We compared results with those from routine culture-based surveillance data. PCR data confirmed an absence of ceftriaxone resistance and a low level of azithromycin resistance (0.2%), and that penicillin resistance was <5% in amoxicillin plus azithromycin regions. Rates of ciprofloxacin resistance and penicillinase-producing N. gonorrhoeae were lower when molecular methods were used. Molecular methods to detect N. gonorrhoeae AMR can increase the evidence base for treatment guidelines, particularly in settings where culture-based surveillance is limited.

Changes in the rates of Neisseria gonorrhoeae antimicrobial resistance are primarily driven by dynamic fluctuations in common gonococcal genotypes.

Objectives: To examine how gonococcal genotypes and associated changes over time influence rates of Neisseria gonorrhoeae antimicrobial resistance. Methods: All available N. gonorrhoeae isolates collected in New South Wales, Australia in the first half of both 2012 and 2014 were genotyped using the Agena MassARRAY iPLEX platform. Genotypic data were compared with phenotypic antimicrobial resistance profiles over time. We focused on penicillin and ciprofloxacin as significant increases in resistance to both antibiotics were observed over this time period. Results: Genotyping data were obtained for 760 and 782 isolates in 2012 and 2014, respectively. A total of 162 distinct genotypes were identified in the study, including 36 (22.2%) genotypes present in both years ( persisting genotypes), 54 (33.3%) observed in 2012 only and 72 (44.4%) observed in 2014 only (s ingle-year genotypes). Overall, persisting genotypes comprised 15 of the 20 most common genotypes, 8 of which showed a significant change in proportion from 2012 to 2014. Persisting genotypes also comprised the majority (>70%) of ciprofloxacin- and penicillin-resistant isolates in both years. Significant fluctuations in the most common persisting genotypes accounted for the majority of observed increases in both ciprofloxacin and penicillin resistance. Single-year genotypes contributed to ∼20% of ciprofloxacin and penicillin resistance in each year. Conclusions: The results show that the gonococcal genotypes persisting in the study population fluctuated significantly within a 3 year period, with numerous other genotypes appearing or disappearing. It is the net effect of these changes that determines N. gonorrhoeae antimicrobial resistance levels within the population.

Mixed gonococcal infections in a high-risk population, Sydney, Australia 2015: implications for antimicrobial resistance surveillance?

OBJECTIVES: Previous studies have shown that mixed-strain gonococcal infections can occur. However, it remains unclear whether such infections impact upon the reliability of Neisseria gonorrhoeae antimicrobial resistance (AMR) surveillance. In this study, we aimed to resolve this question by intensively sampling isolates from gonorrhoea-positive specimens in a high-risk population in Sydney, Australia. METHODS: A total of 615 N. gonorrhoeae isolates, originating from 63 clinical samples (31 rectal swabs and 32 throat swabs), were characterized. All isolates were subject to N. gonorrhoeae identification, antimicrobial susceptibility testing and genotyping by SNP-based MLST. RESULTS: Only 2 of the 63 (3.2%) samples provided evidence of mixed-strain infections. These comprised two rectal swabs that harboured isolates of different SNP-based MLST genotypes; however, the AMR susceptibility profiles of the different genotypes from these samples were indistinguishable. Within-sample differences in the AMR susceptibility profiles were observed for a further seven samples; however, the differences were not considered significant; MIC values were typically within a 2-fold difference or were close to test breakpoints. CONCLUSIONS: Results of this study provide further evidence that mixed-strain gonococcal infections do occur, although at low prevalence. Our data indicate that at a population level such infections are unlikely to impact significantly upon N. gonorrhoeae AMR surveillance.

Antibiotic perturbation of mixed-strain Pseudomonas aeruginosa infection in patients with cystic fibrosis.

BACKGROUND: Pulmonary exacerbations in cystic fibrosis (CF) remain poorly understood and treatment is usually targeted at Pseudomonas aeruginosa. Within Australia a predominant shared P. aeruginosa strain (AUST-02) is associated with greater treatment needs. This single centre study assessed temporal shared strain population dynamics during and after antibiotic treatment of exacerbations. METHODS: Sputum was collected from 12 adult patients with a history of chronic AUST-02 infection at four time-points during and after treatment of an exacerbation. Forty-eight P. aeruginosa isolates within each sample underwent AUST-02 allele-specific PCR and SNP-based strain genotyping. RESULTS: Various commonly shared Australian strains (AUST-01, 0.1%; AUST-02, 54.3%; AUST-06, 36.6%; AUST-07, 4.6%; AUST-11, 4.3%) and two unique strains (0.1%) were identified from 45 sputum samples (2160 isolates). Based on within-patient relative abundance of strains, a "single-strain infection" (n = 7) or "mixed-strain infection" (n = 5) was assigned to each patient. A significant temporal variation in the P. aeruginosa population composition was found for those with mixed-strain infection (P < 0.001). Patients with mixed-strain infections had more long-term treatment requirements than those with single-strain infection. Moreover, despite both groups having similar lung function at study entry, patients with single-strain infection had greater improvement in FEV1% predicted following their exacerbation treatment (P = 0.02). CONCLUSION: Pulmonary exacerbations may reveal multiple, unrelated P. aeruginosa strains whose relative abundance with one another may change rapidly, in a sustained and unpredictable manner.

A real-time PCR assay for direct characterization of the Neisseria gonorrhoeae GyrA 91 locus associated with ciprofloxacin susceptibility.

OBJECTIVES: The objective of this study was to develop a real-time PCR method for specific detection of the gonococcal GyrA amino acid 91 locus directly in clinical samples so as to predict Neisseria gonorrhoeae ciprofloxacin susceptibility. METHODS: The real-time PCR assay, GyrA91-PCR, was designed using two probes, one for detection of the WT S91 sequence and the other for detection of the S91F alteration. The performance of the assay was initially assessed using characterized N. gonorrhoeae isolates (n = 70), a panel of commensal Neisseria and Moraxella species (n = 55 isolates) and clinical samples providing negative results by a commercial N. gonorrhoeae nucleic acid amplification test (NAAT) method (n = 171). The GyrA91-PCR was then applied directly to N. gonorrhoeae NAAT-positive clinical samples (n = 210) from the year 2014 for which corresponding N. gonorrhoeae isolates with susceptibility results were also available. RESULTS: The GyrA91-PCR accurately characterized the GyrA 91 locus of all 70 N. gonorrhoeae isolates (sensitivity = 100%, 95% CI = 94.9%-100%), whereas all non-gonococcal isolates and N. gonorrhoeae NAAT-negative clinical samples gave negative results by the GyrA91-PCR (specificity = 100%, 95% CI = 98.4%-100%). When applied to the 210 N. gonorrhoeae NAAT-positive clinical samples, the GyrA91-PCR successfully characterized 195 samples (92.9%, 95% CI = 88.5%-95.9%). When compared with the corresponding bacterial culture results, positivity by the GyrA91-PCR WT probe correctly predicted N. gonorrhoeae susceptibility to ciprofloxacin in 161 of 162 (99.4%, 95% CI = 96.6%-99.9%) samples. CONCLUSIONS: The use of a PCR assay for detection of mutation in gyrA applied directly to clinical samples can predict ciprofloxacin susceptibility in N. gonorrhoeae.

Real-time PCR detection of Neisseria gonorrhoeae susceptibility to penicillin.

OBJECTIVES: The objective of this study was to develop a real-time PCR assay targeting the gonococcal porB gene (PorB-PCR) for predicting susceptibility of Neisseria gonorrhoeae to penicillin. This complements a previously described PCR assay for detecting penicillinase-producing N. gonorrhoeae (PPNG) developed by our laboratory (PPNG-PCR). METHODS: The PorB-PCR assay was designed using six probes to characterize various combinations of amino acids at positions 101 and 102 of the PorB1b class protein, including the WT G101/A102 and mutant G101K/A102D, G101K/A102N and G101K/A102G sequences, as well as the PorB1a sequence. The ability of these sequences to predict penicillin susceptibility was initially assessed using 2307 N. gonorrhoeae isolates from throughout Australia for which phenotypic susceptibility data were available. The assay was then applied to N. gonorrhoeae-positive clinical specimens (n = 70). Specificity was assessed by testing commensal Neisseria strains (n = 75) and N. gonorrhoeae-negative clinical specimens (n = 171). RESULTS: Testing of the 2307 N. gonorrhoeae isolates using PorB-PCR to detect G101/A102 and PorB1a sequences identified a total of 78.4% (61.2% and 17.2%, respectively) of penicillin-susceptible isolates with specificities of 97.4% and 99.3% and positive predictive values of 98.8% and 98.9%, where PPNG strains were simultaneously identified and excluded. Similar performance data were obtained when the PorB-PCR assay was applied to the N. gonorrhoeae-positive clinical specimens. No false-positive results were observed for the N. gonorrhoeae-negative samples and no cross-reactions were observed with the non-gonococcal species. CONCLUSIONS: When used in parallel with the previously described PPNG-PCR, the PorB-PCR approach has the potential to facilitate individualized treatment of gonorrhoea using penicillin.

Multitarget PCR Assay for Direct Detection of Penicillinase-Producing Neisseria gonorrhoeae for Enhanced Surveillance of Gonococcal Antimicrobial Resistance.

A multitarget PCR was developed for the direct detection of penicillinase-producing Neisseria gonorrhoeae (PPNG). The assay was validated by testing 342 PPNG isolates and 415 clinical samples. The method is suitable for routine detection of PPNG strains. Its multitarget approach reduces the potential for false-negative results caused by sequence variations.

Direct real-time PCR-based detection of Neisseria gonorrhoeae 23S rRNA mutations associated with azithromycin resistance.

OBJECTIVES: Surveillance for Neisseria gonorrhoeae azithromycin resistance is of growing importance given increasing use of ceftriaxone and azithromycin dual therapy for gonorrhoea treatment. In this study, we developed two real-time PCR methods for direct detection of two key N. gonorrhoeae 23S rRNA mutations associated with azithromycin resistance. METHODS: The real-time PCR assays, 2611-PCR and 2059-PCR, targeted the gonococcal 23S rRNA C2611T and A2059G mutations, respectively. A major design challenge was that gonococcal 23S rRNA sequences have high sequence homology with those of commensal Neisseria species. To limit the potential for cross-reaction, 'non-template' bases were utilized in primer sequences. The performance of the methods was initially assessed using a panel of gonococcal (n = 70) and non-gonococcal (n = 28) Neisseria species. Analytical specificity was further assessed by testing N. gonorrhoeae nucleic acid amplification test (NAAT)-negative clinical samples (n = 90), before being applied to N. gonorrhoeae NAAT-positive clinical samples (n = 306). RESULTS: Cross-reactions with commensal Neisseria strains remained evident for both assays; however, cycle threshold (Ct) values were significantly delayed, indicating reduced sensitivity for non-gonococcal species. For the N. gonorrhoeae NAAT-negative clinical samples, 7/21 pharyngeal samples provided evidence of cross-reaction (Ct values >40 cycles); however, the remaining urogenital and rectal swab samples were negative. In total, the gonococcal 2611 and 2059 23S rRNA nucleotides were both successfully characterized in 266/306 (87%) of the N. gonorrhoeae NAAT-positive clinical specimens. CONCLUSIONS: Real-time PCR detection of gonococcal 23S rRNA mutations directly from clinical samples is feasible and may enhance culture- and non-culture-based N. gonorrhoeae resistance surveillance.

Penicillinase-producing plasmid types in Neisseria gonorrhoeae clinical isolates from Australia.

Penicillinase-producing Neisseria gonorrhoeae (PPNG) carrying the blaTEM-135 gene is of particular concern, as it is considered a stepping stone toward resistance to extended-spectrum cephalosporins. Here, we sought to characterize plasmid types and the occurrence of the blaTEM-135 gene for N. gonorrhoeae clinical isolates from Australia. We found that blaTEM-135 was prevalent in Australian PPNG and was detected on all three major plasmid types.

Evaluation of Illumina® COVIDSeq™ as a tool for Omicron SARS-CoV-2 characterisation.

The continued emergence and transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants requires ongoing genetic surveillance to support public health responses. The expansion of reliable next generation sequence (NGS) platforms has enabled the rapid characterisation of the constant emergence of new SARS-CoV-2 variants using nasopharyngeal swab specimens. Several studies have assessed the ability of COVIDSeq to type earlier SARS-CoV-2 strains (pre-Delta) rapidly and successfully, however, there is limited data showing suitability against Omicron variants. In the present study, we evaluated the performance of the Illumina COVIDSeq Assay as a streamlined amplicon-based NGS platform for detection and typing of Omicron variants. Our results demonstrate the high performance of SARS-CoV-2 sequencing using the COVIDSeq approach, with good repeatability, reproducibility and sensitivity for samples approaching CT 31. The COVIDSeq approach was 100% concordant with samples previously characterized by sequencing methods. The quick library preparation process and high throughput kit made it ideal for reflex testing, with a total time required for sequencing and analysis of approximately two days. This study demonstrates the effectiveness and versatility of the amplicon-based NGS characterisation method for SARS-CoV-2, providing a foundation for further research and development of custom-designed amplicon panels targeting different microorganisms.

Rapid inactivation and sample preparation for SARS-CoV-2 PCR-based diagnostics using TNA-Cifer Reagent E.

RT-qPCR remains a key diagnostic methodology for COVID-19/SARS-CoV-2. Typically, nasal or saliva swabs from patients are placed in virus transport media (VTM), RNA is extracted at the pathology laboratory, and viral RNA is measured using RT-qPCR. In this study, we describe the use of TNA-Cifer Reagent E in a pre-clinical evaluation study to inactivate SARS-CoV-2 as well as prepare samples for RT-qPCR. Adding 1 part TNA-Cifer Reagent E to 5 parts medium containing SARS-CoV-2 for 10 min at room temperature inactivated the virus and permitted RT-qPCR detection. TNA-Cifer Reagent E was compared with established column-based RNA extraction and purification methodology using a panel of human clinical nasal swab samples (n = 61), with TNA-Cifer Reagent E showing high specificity (100%) and sensitivity (97.37%). Mixtures of SARS-CoV-2 virus and TNA-Cifer Reagent E could be stored for 3 days at room temperature or for 2 weeks at 4°C without the loss of RT-qPCR detection sensitivity. The detection sensitivity was preserved when TNA-Cifer Reagent E was used in conjunction with a range of VTM for saliva samples but only PBS (Gibco) and Amies Orange for nasal samples. Thus, TNA-Cifer Reagent E improves safety by rapidly inactivating the virus during sample processing, potentially providing a safe means for molecular SARS-CoV-2 testing outside traditional laboratory settings. The reagent also eliminates the need for column-based and/or automated viral RNA extraction/purification processes, thereby providing cost savings for equipment and reagents, as well as reducing processing and handling times.

The impact of COVID-19 epidemic phase and changes in mean viral loads: implications for SARS-CoV-2 testing strategies.

The sensitivity of SARS-CoV-2 diagnostic tests is inherently linked to viral load. We explored whether average viral loads changed at a population level in Queensland, Australia during the early phase of the pandemic. RT-PCR threshold cycle (CT) values, a crude marker for viral load, were compared for samples collected in February/March-2020 to those collected in April/May-2020, noting that the major public health interventions began in late-March 2020. Positive detections peaked mid-March, which coincided with the highest detection numbers and lowest CT values. However, this changed from April where the later CT samples (CT > 30) predominated. Overall, in February/March 29% (267/922) of samples had CT values >30 cycles compared to 88% (559/636) in April/May. Our study shows that SARS-CoV-2 viral loads in patients may vary at a population level over time. This needs considering when assessing suitability of diagnostic methods, particularly when methods in question are known to have reduced sensitivity.

SARS-CoV-2 RT-PCR to Screen for B.1.617.2 (Delta) Variant of Concern.

The continuous transmission and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has required that diagnostic capabilities be constantly monitored and updated as new variants emerge and prior variants disappear. Although whole genome sequencing provides full characterisation of SARS-CoV-2 directly from patient samples, this has limited throughput and requires sufficient resources. To enhance screening for circulating variants, we designed a rapid in-house RT-PCR assay to target a spike mutation (D950N) in Delta variants, which is not detected in the remaining variants of concern (VOCs). Assay sensitivity for detecting Delta variants was 93% and specificity was 100% using a sequenced sample bank of several lineages. As the D950N mutation is prevalent in >95% of the global Delta variant sequences deposited in GISAID, this assay has the potential to provide rapid results to determine if the samples are presumptively Delta variants and can support clinicians in timely clinical decision-making for effective treatments and surveillance.