CLEVER assay: A visual and rapid RNA extraction-free detection of SARS-CoV-2 based on CRISPR-Cas integrated RT-LAMP technology.

AIM: The current scenario of COVID-19 pandemic has presented an almost insurmountable challenge even for the most sophisticated hospitals equipped with modern biomedical technology. There is an urgency to develop simple, fast and highly accurate methods for the rapid identification and isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected patients. To address the ongoing challenge, the present study offers a CLEVER assay (CRISPR-Cas integrated RT-LAMP Easy, Visual and Extraction-free RNA) which will allow RNA extraction-free method to visually diagnose COVID-19. RNA extraction is a major hurdle in preventing rapid and large-scale screening of samples particularly in low-resource regions because of the logistics and costs involved. METHOD AND RESULT: Herein, the visual SARS-CoV-2 detection method consists of RNA extraction-free method directly utilizing the patient's nasopharyngeal and oropharyngeal samples for reverse transcription loop-mediated isothermal amplification (RT-LAMP). Additionally, the assay also utilizes the integration of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12-based system using different guide RNAs of N, E and an internal control POP7 (human RNase P) genes along with visual detection via lateral flow readout-based dip sticks with unaided eye (~100 min). Overall, the clinical sensitivity and specificity of the CLEVER assay were 89.6% and 100%, respectively. CONCLUSION: Together, our CLEVER assay offers a point-of-care tool with no equipment dependency and minimum technical expertise requirement for COVID-19 diagnosis. SIGNIFICANCE AND IMPACT OF THE STUDY: To address the challenges associated with COVID-19 diagnosis, we need a faster, direct and more versatile detection method for an efficient epidemiological management of the COVID-19 outbreak. The present study involves developing a method for detection of SARS-CoV-2 in human body without RNA isolation step that can visually be detected with unaided eye. Taken together, our assay offers to overcome one major defect of the prior art, that is, RNA extraction step, which could limit the deployment of the previous assays in a testing site having limited lab infrastructure.

Convergence of Minds: For Better Patient Outcome in Intensive Care Unit Infections.

BACKGROUND: There is emergence of resistance to the last-line antibiotics such as carbapenems in Intensive Care Units (ICUs), leaving little effective therapeutic options. Since there are no more newer antibiotics in the armamentarium in the near future, it has become imperative that we harness the interdisciplinary knowledge for the best clinical outcome of the patient. AIMS: The aim of the conference was to utilize the synergies between the clinical microbiologists and critical care specialists for better patient care and clinical outcome. MATERIALS AND METHODS: A combined continuing medical education program (CME) under the aegis of the Indian Association of Medical Microbiologists - Delhi Chapter and the Indian Society of Critical Care Medicine, Delhi and national capital region was organized to share their expertise on the various topics covering epidemiology, diagnosis, management, and prevention of hospital-acquired infections in ICUs. RESULTS: It was agreed that synergy between the clinical microbiologists and critical care medicine is required in understanding the scope of laboratory tests, investigative pathway testing, hospital epidemiology, and optimum use of antibiotics. A consensus on the use of rapid diagnostics such as point-of-care tests, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and molecular tests for the early diagnosis of infectious disease was made. It was agreed that stewardship activities along with hospital infection control practices should be further strengthened for better utilization of the antibiotics. Through this CME, we identified the barriers and actionables for appropriate antimicrobial usage in Indian ICUs. CONCLUSIONS: A close coordination between clinical microbiology and critical care medicine opens up avenues to improve antimicrobial prescription practices.

Misidentification of Brucella melitensis as Bergeyella zoohelcum by MicroScan WalkAway®: a case report.

OBJECTIVE: To describe the misidentification of Brucella melitensis as Bergeyella zoohelcum by MicroScan WalkAway®, a commonly used bacterial identification system. CLINICAL PRESENTATION AND INTERVENTION: A 35-year-old man was admitted to the Intensive Care Unit with sepsis syndrome. Three sets of aerobic blood culture samples were positive after 48 h of incubation. The isolated organism was identified as B. zoohelcum using the MicroScan WalkAway (Siemens Healthcare Diagnostics Inc., West Sacramento, Calif., USA). However, due to the rareness of the pathogen, the isolate was reidentified as B. melitensis with Vitek® 2 system and later 16S ribosomal sequence analysis confirmed the isolate as B. melitensis having 100% match. CONCLUSION: This case showed that Brucella can be misidentified using MicroScan WalkAway. Countries where brucellosis is endemic need to be careful while using such automated identification systems in order not to miss the diagnosis of Brucella.

Rationalizing antibiotic use to limit antibiotic resistance in India.

Antibiotic resistance, a global concern, is particularly pressing in developing nations, including India, where the burden of infectious disease is high and healthcare spending is low. The Global Antibiotic Resistance Partnership (GARP) was established to develop actionable policy recommendations specifically relevant to low- and middle-income countries where suboptimal access to antibiotics - not a major concern in high-income countries - is possibly as severe a problem as is the spread of resistant organisms. This report summarizes the situation as it is known regarding antibiotic use and growing resistance in India and recommends short and long term actions. Recommendations aim at (i) reducing the need for antibiotics; (ii) lowering resistance-enhancing drug pressure through improved antibiotic targeting, and (iii) eliminating antibiotic use for growth promotion in agriculture. The highest priority needs to be given to (i) national surveillance of antibiotic resistance and antibiotic use - better information to underpin decisions on standard treatment guidelines, education and other actions, as well as to monitor changes over time; (ii) increasing the use of diagnostic tests, which necessitates behavioural changes and improvements in microbiology laboratory capacity; (iii) setting up and/or strengthening infection control committees in hospitals; and (iv) restricting the use of antibiotics for non-therapeutic uses in agriculture. These interventions should help to reduce the spread of antibiotic resistance, improve public health directly, benefit the populace and reduce pressure on the healthcare system. Finally, increasing the types and coverage of childhood vaccines offered by the government would reduce the disease burden enormously and spare antibiotics.

Emergence and molecular characterization of extensively drug-resistant Mycobacterium tuberculosis clinical isolates from the Delhi Region in India.

We screened 194 Mycobacterium tuberculosis strains isolated from tuberculosis (TB) patients in Delhi and neighboring regions in India to identify the prevalence of extensive drug resistance (XDR) in clinical isolates. Among these, 104 isolates were found to be multidrug resistant (MDR), and 6 were identified as XDR isolates, which was later confirmed by antimicrobial susceptibility testing against the respective drug screening panel. Genotyping was carried out by amplifying and sequencing the following genes: rpoB (rifampin), katG (isoniazid), gyrA (fluoroquinolones), and rrs (amikacin, kanamycin, and capreomycin). Our analyses indicated that mutations at the hot spots of these genes were positively correlated with drug resistance in clinical isolates. The key mutation observed for rpoB was in the codon for amino acid position 531 (S531L), and other mutations were seen in the hot spot, including those encoding Q510P, L511H, D516V, and H526Y mutations. We identified S315T and R463L substitutions encoded in the katG locus. An S95T substitution encoded in the gyrA locus was the most common mutation observed in fluoroquinolone-resistant isolates. In addition, we saw D94G and D94N mutations encoded in the QRDR region. The 16S rRNA (rrs) gene encoded mainly the A1401G mutation and an additional mutation, G1484T, resulting in ribosomal modifications. Taken together, the data in this report clearly establish the presence of phenotypically distinct XDR strains in India by molecular profiling and further identify specific mutational hot spots within key genes of XDR-TB strains.

WITHDRAWN: Can quantitative RT-PCR for SARS-coV-2 help in better management of patients and control of coronavirus disease 2019 pandemic.

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Can quantitative RT-PCR for SARS-CoV-2 help in better management of patients and control of coronavirus disease 2019 pandemic.

The emergence of SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), represents a public health emergency of unprecedented proportion. The global containment efforts have been focused on testing, tracing of contacts and treatment (isolation) of those found COVID-19 positive. Since the whole genome sequences of a number of strains of this novel RNA virus were available in the public domain by early January 2020, a number of real-time polymerase chain reaction (RT-PCR) protocols were designed and used for diagnosis of this infection. Most RT-PCRs are designed for qualitative COVID-19 reporting (SARS-CoV-2 detected or not detected), but have been used for semi-quantitative estimation of viral load based on cycle threshold value. Our manuscript discusses the utility of quantitative PCR testing for COVID-19 and its patient management benefits.

How to Treat Sepsis in the Background of Resistance?: Role of Pharmacodynamics / Pharmacokinetics in Treating Sepsis.

Though a decline has been seen in child mortality and morbidity over the last decades, sepsis in neonates and infants remains a major cause of death. Optimal use of antibiotics in sepsis management is a key factor which can further reduce the number of poor clinical outcomes. Selecting the right antibiotic to which the offending bacteria is susceptible and administrating the antibiotic within the first hour can save many lives. However, the pharmacokinetic profile of an antibiotic is affected by developmental changes such as capacity of drug metabolizing enzymes and maturation of organ function. This can affect antibiotic exposure and response in neonates and infants. While suspecting sepsis, the primary focus of empiric treatment during the initial phase is to assure efficacy and it must be broad based to cover all suspected pathogens. Once the bacterial etiology is confirmed as a cause of sepsis and the in vitro antibiotic susceptibility is established, targeted treatment can be started which ensures optimal balance between efficacy and safety.

Mode of action of Ranbezolid against staphylococci and structural modeling studies of its interaction with ribosomes.

Oxazolidinones are known to inhibit protein biosynthesis and act against a wide spectrum of gram-positive bacteria. A new investigational oxazolidinone, ranbezolid, inhibited bacterial protein synthesis in Staphylococcus aureus and Staphylococcus epidermidis. In S. epidermidis, ranbezolid showed inhibition of cell wall and lipid synthesis and a dose-dependent effect on membrane integrity. A kill-kinetics study showed that ranbezolid was bactericidal against S. epidermidis. In vitro translation of the luciferase gene done using bacterial and mammalian ribosomes indicated that ranbezolid specifically inhibited the bacterial ribosome. Molecular modeling studies revealed that both linezolid and ranbezolid fit in similar manners the active site of ribosomes, with total scores, i.e., theoretical binding affinities after consensus, of 5.2 and 6.9, respectively. The nitrofuran ring in ranbezolid is extended toward C2507, G2583, and U2584, and the nitro group forms a hydrogen bond from the base of G2583. The interaction of ranbezolid with the bacterial ribosomes clearly helps to elucidate its potent activity against the target pathogen.

Synthesis and biological activity of novel oxazolidinones.

A number of 5-substituted derivatives of Ranbezolid, a novel oxazolidinone were synthesized. Antibacterial activity of the compounds against a number of sensitive and resistant bacteria showed promising results.

Ciprofloxacin-resistant Neisseria meningitidis, Delhi, India.

Decreased susceptibility of Neisseria meningitidis isolates to ciprofloxacin emerged from an outbreak in Delhi, India. Results of antimicrobial susceptibility testing of the meningococcal isolates to ciprofloxacin and further sequencing of DNA gyrase A quinolone-resistance-determining region confirmed the emergence of ciprofloxacin resistance in the outbreak.

Synthesis and antibacterial activity of potent heterocyclic oxazolidinones and the identification of RBx 8700.

Several potent oxazolidinone antibacterial agents were obtained by systematic modification of the linker between the five-membered heterocycle and the piperazinyl ring of RBx 7644 (Ranbezolid, 1) and its thienyl analogue 2, leading to the identification of an expanded spectrum compound RBx 8700 (6b).

Antifungal potential of disulfiram.

Disulfiram, an alcohol antagonistic drug has been on the market since 1949 with 80% bioavailability and an established safety profile. Recently it has been reported as a P-glycoprotein efflux pump modulator. Herein we report its antifungal potential. The MIC50 and MIC90 of disulfiram for yeast isolates is 4 and 8 microg/ml, respectively, and the MIC range is 1-16 micro g/ml for both fluconazole sensitive and resistant strains. Interestingly, disulfiram also showed fungicidal activity on Aspergillus spp. with MIC50 and MIC90 of 2 and 8 microg/ml, respectively.

Characterisation of laboratory-generated vancomycin intermediate resistant Staphylococcus aureus strains.

Vancomycin has been the drug of choice for 30 years for the treatment of methicillin-resistant Staphylococcus aureus (MRSA). Emergence of decreased vancomycin susceptibility in MRSA strains presents a significant clinical problem with few therapeutic options. This study was performed to generate and characterise S. aureus strains with reduced susceptibility to vancomycin. Eighteen S. aureus strains were subjected to serial passaging on vancomycin to generate vancomycin intermediate resistant S. aureus (VISA) strains. Minimum inhibitory concentration (MIC) determination was performed for the parent and the passaged cultures with 13 different antibiotics. The strains were tested by the following five methods: simplified population analysis; CDC method; modified vancomycin agar screen; population analysis profile (PAP); and modified population analysis (PAP-area under the curve (AUC) ratio). Phenotypic changes such as doubling time, synergy with beta-lactam antibiotics and effect on norA efflux pumps were also studied for these strains. The result indicated that 8 VISA mutants (vancomycin MICs, 8-16 microg/mL) were generated in vitro from the 18 S. aureus strains. The CDC and modified agar methods proved to be the most sensitive and specific methods for detection of VISA strains. The PAP for all the VISA strains ranged from 12 microg/mL to > 16 microg/mL, with a PAP-AUC ratio of > 1.3. All mutants showed increased doubling time compared with their parent isolate. Synergism of the vancomycin and beta-lactam combinations was observed for all methicillin-resistant mutants. Upon acquisition of vancomycin resistance, a few mutants showed decreased oxacillin resistance. Two VISA strains were chosen for molecular characterisation of the mecA gene and one mutant showed genotypic changes with deletion of mecA. Loss of norA efflux pumps leading to fluoroquinolone sensitivity was also observed in four mutants.

Activity of RBx 7644 and RBx 8700, new investigational oxazolidinones, against Mycobacterium tuberculosis infected murine macrophages.

The decision to develop a new chemical entity should not only be based on its ability to inhibit multidrug-resistant tuberculosis (MDR-TB) strains but also on its ability to enter macrophages and be active against intracellular bacteria. RBx 7644 and RBx 8700, two novel extended spectrum oxazolidinones, were investigated for their activity against sensitive and MDR isolates of Mycobacterium tuberculosis and for activity against bacteria within a macrophage cell line. RBx 8700 showed excellent in vitro activity against sensitive as well as MDR M. tuberculosis strains with MIC(50) and MIC(90) values of 0.032 and 0.25mg/L (sensitive) and 0.25 and 1.0mg/L (MDR) strains. The corresponding MIC(50) and MIC(90) values of RBx 7644, linezolid, rifampicin and isoniazid were 8 and 16; 32 and 64; 64 and 64; 64 and 64 mg/L, respectively. RBx 8700 and rifampicin were bactericidal at 0.5 and 0.25mg/L when tested intracellularly whereas linezolid reduced the count by 100-fold at a concentration of 8 mg/L. In combination studies with standard antimycobacterial drugs, RBx 8700 did not show any antagonistic effect.

Adverse effect of staphylococci slime on in vitro activity of glycopeptides.

Adhesion to biomaterial is assumed to be a crucial step in the development of staphylococcal foreign body infections. Production of extracellular slime has major implications for the development and implementation of therapeutic strategies. The effect of extracted slime was investigated on the activity of vancomycin, teicoplanin, linezolid, quinupristin/dalfopristin, rifampicin and ranbezolid against 10 clinical and 4 ATCC staphylococcal isolates. The slime extract caused a 2- to 16-fold increase in the MICs of vancomycin and teicoplanin, with a shift in the MIC(90) from 2 to 32 (vancomycin) and 2 to 16 (teicoplanin), whereas the MICs of linezolid and quinupristin/dalfopristin were only moderately affected. In time-kill studies, a significant decrease in bacterial killing (>3 log(10) cfu/ml) was observed with vancomycin and teicoplanin (4 x MIC) after addition of slime (5 and 20 mg/ml), whereas the effect of killing by linezolid and quinupristin/dalfopristin was very modest. The rifampicin and ranbezolid MICs and kill curves were not influenced by the addition of slime. The present study thus indicated that slime interferes with the antimicrobial effect of glycopeptide drugs (vancomycin, teicoplanin), and that for effective prevention and treatment of prosthetic device-related infections, appropriate and newer antibiotics such as ranbezolid should be considered.

In vitro activity of RBx 7644 (ranbezolid) on biofilm producing bacteria.

Biofilm associated infections are becoming more common. Treatment outcome of device related infections cannot be predicted by the results of a standard susceptibility test such as the MIC. Microorganisms involved in device related infections have a slow growth rate and adhere to surfaces. Activity against glass adherent bacteria has been shown to be correlated with treatment outcome in animal models of catheter related infections. Drug efficacy can be predicted if glass adherent staphylococci are killed by low drug concentration. The eradication of bacteria adhering to glass beads and inhibition of biofilm formation by ranbezolid and three other antibiotics (quinupristin/dalfopristin, vancomycin and linezolid) was studied. The results indicated that ranbezolid required only (2-4 x MIC) for total clearance of glass-adherent MRSA 562 and MRSE 879, compared with vancomycin (8 x), quinupristin/dalfopristin (1-4 x) and linezolid (4-16 x MIC). In addition ranbezolid inhibited biofilm formation to a greater extent at sub MIC and MIC level. In conclusion, this study indicated that ranbezolid had potent activity against adherent staphylococci isolates and may prove useful in the prevention and treatment of device related infections caused by staphylococci.

Anti-anaerobic potential of ranbezolid: insight into its mechanism of action against Bacteroides fragilis.

This study reports the anti-anaerobic properties of ranbezolid, a new investigational oxazolidinone. A time-kill kinetics study against anaerobes showed that ranbezolid was superior to linezolid and killed the anaerobic pathogens at 4-8h, except for Bacteroides fragilis where killing was observed at 24h. In addition, the time-kill kinetics study showed a concentration-dependent bactericidal potential of ranbezolid against anaerobes. Ranbezolid showed 5.39log(10) reduction and linezolid showed 1.15log(10) reduction in murine disk implant infection with B. fragilis ATCC 25285. Ranbezolid was very potent and showed fast protein synthesis inhibition against B. fragilis, a Gram-negative anaerobe. In addition, non-specific cell wall synthesis inhibition was also observed with ranbezolid. The potent and fast protein synthesis inhibition along with an additional mode of action of cell wall synthesis inhibition could be responsible for the cidal effect of ranbezolid against Gram-negative anaerobes.

Synthesis and antibacterial activity of novel oxazolidinones with methylene oxygen- and methylene sulfur-linked substituents at C5-position.

Novel oxazolidinone derivatives of the lead compound RBx 8700, containing methylene oxygen- and methylene sulfur-linked substituents at the C5-position, were synthesized. Antibacterial screening of these compounds against a panel of resistant and susceptible Gram-positive and fastidious Gram-negative bacteria gave compounds 2 and 4 as new antibacterial agents.

Synthesis and SAR of novel oxazolidinones: discovery of ranbezolid.

Novel oxazolidinones were synthesized containing a number of substituted five-membered heterocycles attached to the 'piperazinyl-phenyl-oxazolidinone' core of eperezolid. Further, the piperazine ring of the core was replaced by other diamino-heterocycles. These modifications led to several compounds with potent activity against a spectrum of resistant and susceptible gram-positive organisms, along with the identification of ranbezolid (RBx 7644) as a clinical candidate.