Rapid SARS-CoV-2 Variants Enzymatic Detection (SAVED) by CRISPR-Cas12a.

The continuous and rapid surge of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with high transmissibility and evading neutralization is alarming, necessitating expeditious detection of the variants concerned. Here, we report the development of rapid SARS-CoV-2 variants enzymatic detection (SAVED) based on CRISPR-Cas12a targeting of previously crucial variants, including Alpha, Beta, Gamma, Delta, Lambda, Mu, Kappa, and currently circulating variant of concern (VOC) Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5. SAVED is inexpensive (US$3.23 per reaction) and instrument-free. SAVED results can be read out by fluorescence reader and tube visualization under UV/blue light, and it is stable for 1 h, enabling high-throughput screening and point-of-care testing. We validated SAVED performance on clinical samples with 100% specificity in all samples and 100% sensitivity for the current pandemic Omicron variant samples having a threshold cycle (CT) value of ≤34.9. We utilized chimeric CRISPR RNA (crRNA) and short crRNA (15-nucleotide [nt] to 17-nt spacer) to achieve single nucleotide polymorphism (SNP) genotyping, which is necessary for variant differentiation and is a challenge to accomplish using CRISPR-Cas12a technology. We propose a scheme that can be used for discriminating variants effortlessly and allows for modifications to incorporate newer upcoming variants as the mutation site of these variants may reappear in future variants. IMPORTANCE Rapid differentiation and detection tests that can directly identify SARS-CoV-2 variants must be developed in order to meet the demands of public health or clinical decisions. This will allow for the prompt treatment or isolation of infected people and the implementation of various quarantine measures for those exposed. We report the development of the rapid SARS-CoV-2 variants enzymatic detection (SAVED) method based on CRISPR-Cas12a that targets previously significant variants like Alpha, Beta, Gamma, Delta, Lambda, Mu, and Kappa as well as the VOC Omicron and its subvariants BA.1, BA.2, BA.3, BA.4, and BA.5 that are currently circulating. SAVED uses no sophisticated instruments and is reasonably priced ($3.23 per reaction). As the mutation location of these variations may reoccur in subsequent variants, we offer a system that can be applied for variant discrimination with ease and allows for adjustments to integrate newer incoming variants.

Detection of SARS-CoV-2 VOC-Omicron using commercial sample-to-answer real-time RT-PCR platforms and melting curve-based SNP assays.

Objectives: The World Health Organization (WHO) had designated the SARS-CoV-2 lineage B.1.1.529 as the new Variant of Concern Omicron (VOC-Omicron) on 26th November 20211. Real-time reverse transcription polymerase chain reaction (RT-PCR), single nucleotide polymorphisms (SNP) and whole genome sequencing (WGS) tests were widely employed to detect SARS-CoV-2 and its variant. Yet, the SARS-CoV-2 Omicron detection performance of commercial real-time RT-PCR platforms and SARS-CoV-2 spike SNP assays remain to be elucidated. Methods: In the first part of this study, we evaluated the VOC-Omicron detection performance of three commercial RT-PCR sample-to-answer platforms i.e. Roche cobas® 6800/8800, Roche cobas® Liat®, and Cepheid GeneXpert® systems. The detection performances were compared to one commercial conventional real-time RT-PCR assay (TIB MOLBIOL LightMix Modular SARS and Wuhan CoV E-gene) and one in-house real-time RT-PCR assay targeting RNA-dependent RNA polymerase (RdRP) gene of SARS-CoV-2 in the WHO COVID-19 Reference Laboratory at Public Health Laboratory Services Branch, Centre for Health Protection, Department of Health, The Government of the Hong Kong Special Administrative Region. In the second part of this study, we evaluated the SNP detection performance of four TIB MOLBIOL melting curve-based assays (1. Spike S371L/S373P, 2. Spike E484A, 3. Spike E484K and 4. Spike N501Y) in clinical samples obtained from hospitalized COVID-19 patients in Hong Kong. The SNP results were compared to whole genome sequences generated by Illumina platform. Results: The VOC-Omicron detection limits of three commercial sample-to-answer assays were tested to be ≤ 2.35 Log10 dC/ml. The detection performances of the sample-to-answer platforms were comparable to the two tested conventional real-time RT-PCR assays. The test sensitivities of TIB MOLBIOL VirSNiP SARS-CoV-2 Spike S371L/S373P assay and the Spike E484A assays were 100% and 96.6% respectively and the test specificities of both assays were 100%. An aberrant melting peak at Tm 42-44°C was observed when the specimens with Omicron variant were tested with the TIB MOLBIOL VirSNiP SARS-CoV-2 Spike E484K assay. Notably, the TIB MOLBIOL VirSNiP SARS-CoV-2 Spike N501Y assay failed to detect the spike N501Y mutation of Omicron variant in the tested specimens. Conclusions: The SARS-CoV-2 detection sensitivity of three commercial platforms, Roche cobas® 6800/8800, Roche cobas® Liat®, and Cepheid GeneXpert® systems were shown not to be impacted by the large number of mutations of VOC-Omicron. Also, the signature mutations i.e. Spike S371L/Spike S373P and Spike E484A in VOC-Omicron were correctly identified by the TIB MOLBIOL VirSNiP SARS-CoV-2 Spike S371L/S373P and VirSNiP SARS-CoV-2 Spike E484A assays. Unexpected findings including a shifted melting peak or absence of amplification curve/melting peak were observed when specimens with Omicron variant were tested with the TIB MOLBIOL VirSNiP SARS-CoV-2 Spike E484K assay and Spike N501Y assay, suggesting a potential alert for Omicron variant, prior confirmation by whole genome sequencing.

Risk of air and surface contamination of SARS-CoV-2 in isolation wards and its relationship with patient and environmental characteristics.

Air and surface contamination of the SARS-CoV-2 have been reported by multiple studies. However, the evidence is limited for the change of environmental contamination of this virus in the surrounding of patients with COVID-19 at different time points during the course of disease and under different conditions of the patients. Therefore, this study aims to understand the risk factors associated with the appearance of SARS-CoV-2 through the period when the patients were staying in the isolation wards. In this study, COVID-19 patients admitted to the isolation wards were followed up for up to 10 days for daily collection of air and surface samples in their surroundings. The positivity rate of the environmental samples at different locations was plotted, and multiple multi-level mixed-effect logistic regressions were used to examine the association between the positivity of environmental samples and their daily health conditions and environmental factors. It found 6.6 % of surface samples (133/2031 samples) and 2.1 % of air samples (22/1075 samples) were positive, and the positivity rate reached to peak during 2-3 days after admission to the ward. The virus was more likely to present at bedrail, patients' personal items and medical equipment, while less likely to be detected in the air outside the range of 2 m from the patients. It also revealed that higher positivity rate is associated with lower environmental temperature, fever and cough at the day of sampling, lower Ct values of latest test for respiratory tract samples, and pre-existing respiratory or cardiovascular conditions. The finding can be used to guide the hospital infection control strategies by identifying high-risk areas and patients. Extra personal hygiene precautions and equipment for continuously environmental disinfection can be used for these high-risk areas and patients to reduce the risk of hospital infection.

Effects of Echinocandins in Combination with Nikkomycin Z against Invasive Candida albicans Bloodstream Isolates and the fks Mutants.

We evaluated the in vitro and in vivo effects of nikkomycin Z combined with an echinocandin (anidulafungin or micafungin) against two Candida albicans isolates and their lab-derived echinocandin-resistant fks mutants with FKS1 S645Y and FKS1 S645P. Synergistic effects were observed in all tested strains (fractional inhibitory concentration index, <0.5). Enhanced survival was observed in an immunocompromised murine model (log-rank test, P < 0.02). Our study demonstrated the therapeutic potential of nikkomycin Z-echinocandin combinations in managing echinocandin resistance.

Photodynamic inactivation of bacteria and viruses using two monosubstituted zinc(II) phthalocyanines.

A zinc(II) phthalocyanine substituted with a triamino moiety and its tri-N-methylated analogue have been prepared and characterized with various spectroscopic methods. Both compounds remain non-aggregated in N,N-dimethylformamide and in water containing 0.05% Cremophor EL (v/v), and can generate singlet oxygen effectively. The photodynamic activities of these compounds have been examined against a range of bacterial strains, including the Gram-positive methicillin-sensitive Staphylococcus aureus ATCC 25923 and methicillin-resistant Staphylococcus aureus ATCC BAA-43, and the Gram-negative Escherichia coli ATCC 35218 and Pseudomonas aeruginosa ATCC 27853. Both photosensitizers are highly cytotoxic, particularly for the two Gram-positive strains, for which as low as 5 nM of dye is required to induce a 4-log reduction of their viability. The tri-N-methylated derivative has also been shown to be able to effectively inhibit the growth of a series of clinical strains of Staphylococcus aureus and Escherichia coli, and biofilms of methicillin-resistant Staphylococcus aureus ATCC 67928 and ATCC 68507, and Staphylococcus epidermidis ATCC 35984. In addition, the photodynamic inactivation of a range of viruses using these two compounds has also been investigated. Both compounds are highly photocytotoxic against the enveloped viruses influenza A virus (H1N1) and herpes simplex virus type 1 (HSV1), but exhibit no significant cytotoxicity toward the non-enveloped viruses adenovirus type 3 (Ad3) and coxsackievirus (Cox B1).

Oligolysine-conjugated zinc(II) phthalocyanines as efficient photosensitizers for antimicrobial photodynamic therapy.

A series of zinc(II) phthalocyanines conjugated with an oligolysine chain (n=2, 4, and 8) were synthesized and characterized by using various spectroscopic methods. As shown by using UV/Vis and fluorescence spectroscopic methods, these compounds were nonaggregated in N,N-dimethylformamide, and gave a weak fluorescence emission and high singlet oxygen quantum yield (Φ(Δ) =0.86-0.89) as a result of their di-α-substitution. They became slightly aggregated in water with 0.05 % Cremophor EL, but they could still generate singlet oxygen effectively. The antimicrobial photodynamic activities of these compounds were then examined against various bacterial strains, including the Gram-positive methicillin-sensitive Staphylococcus aureus ATCC 25923 and methicillin-resistant Staphylococcus aureus ATCC BAA-43, and the Gram-negative Escherichia coli ATCC 35218 and Pseudomonas aeruginosa ATCC 27853. Generally, the dyes were much more potent toward the Gram-positive bacteria. Only 15 to 90 nM of these photosensitizers was required to induce a 4 log reduction in the cell viability of the strains. For Escherichia coli, the photocytotoxicity increased with the length of the oligolysine chain. The octalysine derivative showed the highest potency with a 4 log reduction concentration of 0.8 µM. Pseudomonas aeruginosa was most resistant to the photodynamic treatment. The potency of the tetralysine derivative toward a series of clinical strains of Staphylococcus aureus was also examined and found to be comparable with that toward the nonclinical counterparts. Moreover, the efficacy of these compounds in photodynamic inactivation of viruses was also examined. They were highly photocytotoxic against the enveloped viruses influenza A virus (H1N1) and herpes simplex virus type 1 (HSV1), but exhibited no significant cytotoxicity against the nonenveloped viruses adenovirus type 3 (Ad3) or coxsackievirus (Cox B1). The octalysine derivative also showed the highest potency with an IC(50) value of 0.05 nM for the two enveloped viruses.

Highly conjugative IncX4 plasmids carrying blaCTX-M in Escherichia coli from humans and food animals.

This study investigated the prevalence of IncX plasmid subtypes in commensal and pathogenic Escherichia coli isolates and the biological features of the IncX4 subtype. Two hundred and twenty-five E. coli isolates from multiple sources (47 chickens, 41 pigs, 30 cattle and 107 humans) obtained during the period 2006-2012 were tested for the presence of IncX1 to IncX5. Overall, the prevalence of IncX plasmids in chicken, pig, cattle and human isolates were 21.2 % (10/47), 19.5 % (8/41), 3.3 % (1/30) and 4.8 % (5/107), respectively. IncX4 was the most common subtype, followed by IncX1 and IncX3, while no IncX2 or IncX5 were found. Seven out of 16 (43.8 %) IncX4 plasmids were found to carry blaCTX-M genes and six of them originating from different host sources (four chickens, one pig and one human) had identical or highly similar RFLP patterns. Three IncX4 plasmids carrying blaCTX-M from different host sources were investigated further. It was found that the IncX4 plasmids had little effect on bacterial host growth parameters after their introduction to J53 recipients. Conjugation experiments demonstrated that the IncX4 plasmids could be efficiently transferred at 30-42 °C at rates which were generally 10(2)-10(5)-fold higher than those for the epidemic IncFII plasmid carrying blaCTX-M (pHK01). In conclusion, the IncX plasmids are more common than previously recognized. The efficient transfer of IncX4 plasmid at different temperatures and the lack of fitness burden on bacterial hosts highlight the ability of this plasmid replicon to be an important vehicle for dissemination of antimicrobial resistance.

pIMP-PH114 carrying bla IMP-4 in a Klebsiella pneumoniae strain is closely related to other multidrug-resistant IncA/C2 plasmids.

The IncA/C plasmids are broad host-range vehicles which have been associated with wide dissemination of CMY-2 among Enterobacteriaceae of human and animal origins. Acquired metallo-ß-lactamases (MBLs) such as the IMP-type enzymes are increasingly reported in multidrug-resistant Gram-negative bacteria worldwide, particularly in Enterobacteriaceae. We described the complete sequence of the first IMP-4-encoding IncA/C2 plasmid, pIMP-PH114 (151,885 bp), from a sequence type 1 Klebsiella pneumoniae strain that was recovered from a patient who was hospitalized in the Philippines. pIMP-PH114 consists of a backbone from the IncA/C2 plasmids, with the insertion of a novel Tn21-like class 1 integron composite structure (containing the cassette array bla IMP-4-qacG-aacA4-catB3, followed by a class C ß-lactamase bla DHA-1 and the mercury resistance operon, merRTPCADE) and a sul2-floR encoding region. Phylogenetic analysis of the IncA/C repA sequences showed that pIMP-PH114 formed a subgroup with other IncA/C plasmids involved in the international spread of CMY-2, TEM-24 and NDM-1. Identical bla IMP-4 arrays have been described among different Enterobacteriaceae and Acinetobacter spp. in China, Singapore and Australia but the genetic context is different. The broad host range of IncA/C plasmids may have facilitated dissemination of the bla IMP-4 arrays among different diverse groups of bacteria.

Prevalence and molecular epidemiology of plasmid-mediated fosfomycin resistance genes among blood and urinary Escherichia coli isolates.

A total of 1878 non-duplicate clinical Escherichia coli isolates (comprising 1711 urinary isolates and 167 blood-culture isolates), which were collected from multiple centres in Hong Kong during 1996-2008, were used to investigate the prevalence and molecular epidemiology of plasmid-mediated fosfomycin (fos) resistance genes. Eighteen of the 1878 clinical E. coli isolates were fosfomycin resistant, of which six were fosA3 positive and two were positive for another fosA variant (designated fosKP96). No isolates had the fosC2 gene. The clones of the eight isolates were diverse: sequence type (ST) 95 (n = 2), ST118 (n = 1), ST131 (n = 1), ST617 (n = 1), ST648 (n = 1), ST1488 (n = 1) and ST2847 (n = 1). In the isolates, fosA3 and blaCTX-M genes were co-harboured on conjugative plasmids with F2:A-:B- (n = 2), N (n = 1), F-:A-:B1 and N (n = 1) and untypable (n = 2) replicons. Both fosKP96-carrying plasmids belonged to replicon N. RFLP analysis showed that the two F2:A-:B- plasmids carrying fosA3 and blaCTX-M-3 genes shared the same pattern. Complete sequencing of one of the two F2:A-:B- plasmids, pFOS-HK151325 (69 768 bp) demonstrated it to be >99 % identical to the previously sequenced plasmid pHK23a originating from a pig E. coli isolate in the same region. This study demonstrated the dissemination of fosA3 genes in diverse E. coli clones on multiple blaCTX-M-carrying plasmid types, of which F2:A-:B- plasmids closely related to pHK23a were shared by isolates from human and animal sources.

Molecular Characterization of an Atypical IncX3 Plasmid pKPC-NY79 Carrying bla KPC-2 in a Klebsiella pneumoniae.

The IncX family of plasmids has recently been expanded to include at least four subtypes, IncX1-IncX4. The revised classification provides an opportunity for improving our understanding of the sequence diversity of the IncX plasmids and the resistance genes they carried. We described the complete nucleotide sequence of a novel IncX3 plasmid, pKPC-NY79 (42,447 bp) from a sequence-type 258 Klebsiella pneumoniae strain that was isolated from a patient who was hospitalized in New York, United States. In pKPC-NY79, the plasmid scaffold and genetic load region were highly similar to homologous regions in pIncX-SHV (IncX3, JN247852) and the bla KPC carrying pKpQIL (IncFIIk, GU595196), respectively, indicating that it has possibly arisen through recombination of plasmids. The bla KPC-2 gene, as part of a transposon Tn4401a, was found within the genetic load region. The backbone of pKPC-NY79 differs from pIncX-SHV by a deletion involving the gene tandem hns-topB (encoding H-NS protein and topoisomerase III, respectively) and a putative ATPase gene. Unexpectedly, the impact of the hns-topB deletion on host fitness and plasmid stability was found to be small. In conclusion, the findings contribute to a better understanding of the plasmid platforms carrying bla KPC and of variations in the backbone of the IncX3 plasmids.

Identification and characterization of a novel incompatibility group X3 plasmid carrying bla NDM-1 in Enterobacteriaceae isolates with epidemiological links to multiple geographical areas in China.

The New Delhi metallo-ß-lactamase (NDM-1) is one of the most important resistance traits in Enterobacteriaceae. We characterized nine bla NDM-1 producing Enterobacteriaceae recovered from seven patients who have recently travelled or been treated in India (n=1) or mainland China (n=6) during December 2010-May 2012. All the China-linked patients had no links to the Indian subcontinent. The bla NDM-1 carrying plasmids belonged to the novel IncX3 (∼50 kb, in seven isolates including two Escherichia coli, two Klebsiella pneumoniae, one Citrobacter freundii, one Enterobacter aerogenes and one E. cloacae), IncA/C2 (∼140 kb, in one E. coli) or FII-F1B groups (∼110 kb, in one E. coli). Restriction fragment length polymorphism analysis of the seven IncX3 plasmids revealed identical pattern in six and two bands difference in the remaining one. The IncX3 plasmids carrying bla NDM-1 were epidemiologically linked to Guangzhou (n=1), Hunan (n=4), Haifeng (n=1) and Dongguan (n=1) in mainland China. Complete sequencing of the IncX3 plasmid pNDM-HN380 revealed that it was 54 035 bp long and encoded 52 open reading frames. The bla NDM-1 gene was found in a transposon-like structure flanked by ISAba125 and IS26, inserted into the plasmid genetic load region. The sequences of the bla NDM-1 containing module within the two IS elements were identical to those previously described for bla NDM-1-positive Tn125 in the plasmids or chromosome of Acinetobacter isolates. In summary, this is the first description of IncX3 plasmids carrying bla NDM-1. The findings indicate the worrisome involvement of an epidemic plasmid in the dissemination of NDM-1 in China.