Cycle threshold values of SARS-CoV-2 RNA in conjunctival swabs and nasopharyngeal secretions: a comparative study from a tertiary care center in India.

PURPOSE: To detect the viral RNA load of SARS-CoV-2 in conjunctival swabs of COVID-19 patients, and compare with nasopharyngeal swabs. METHODS: Conjunctival swabs of COVID-19 patients (with PCR positive nasopharyngeal swabs) were subjected to quantitative reverse transcription-polymerase chain reaction (RT-PCR) for detection of SARS-CoV-2 RNA. The cycle threshold (Ct) values of Open Reading Frame 1 (ORF 1 Ab gene) and nucleoprotein (N gene) PCRs were used to assess the viral RNA load, and compare them with the baseline values of nasopharyngeal swabs. RESULTS: Of 93 patients, 17 (18.27%) demonstrated SARS-CoV-2 RNA in conjunctival swabs. Baseline nasopharyngeal swabs were collected at a median of 2 days; while, the conjunctival swabs were collected at median 7 days, from onset of illness (p < 0.001). Despite a significant delay in conjunctival swab collection than nasopharyngeal swabs, the Ct values (ORF or N gene PCRs) were comparable between nasopharyngeal swab and conjunctival swab samples. Subsequently, during the recovery period, in four of these 17 patients (with conjunctival swab positivity), when the second nasopharyngeal swab was 'negative', the conjunctival swab was 'positive'. CONCLUSION: The conjunctival swabs demonstrated SARS-CoV-2 RNA in 17 (18.27%) of 93 COVID-19 patients. Our results may suggest a delayed or a prolonged shedding of the virus/viral RNA on the ocular surface than in nasopharyngeal mucosa.

Comparative genomics reveals the evolution of antimicrobial resistance in Bacteroides nordii.

Bacteroides nordii, is an understudied member of the pathogenic B. fragilis group which comprises several multidrug-resistant (MDR) strains. Thus, it is of great interest to study the genome biology of Bacteroides nordii. However, no detailed study is available that characterized B. nordii at the genetic level and explored its role as a potential pathogen. We isolated an MDR strain viz., B. nordii PGMM4098 from the pus sample and subjected it to whole genome sequencing using Illumina technology. The draft genome was de-novo assembled and annotated, followed by comprehensive comparative genomics analyses using the publicly available genome dataset of B. nordii. The pan-genome analysis revealed the open nature of B. nordii, indicating the continuous accumulation of novel genes in non-core components leading to the emergence of new strains of this species. The thirteen antimicrobial resistance (AMR) genes identified in the genomes of all B. nordii strains were part of the non-core component of the pan-genome. Of these, four AMR genes, nimE, aadS, mef(En2), and ermB/F/G were found to be acquired via the process of horizontal gene transfer (HGT) from anaerobic Bacteroidetes. Importantly, the nimE gene conferring metronidazole resistance was found to be present only in B. nordii PGMM4098, which harbors five other AMR genes encoded in its genome. Of these, nimE (metronidazole resistance), ermB/F/G (macrolide-lincosamide-streptogramin B resistance), and cfxA2/A3 (class A ß-lactam resistance) genes were further validated using targeted polymerase chain reaction assay. Notably, these three genes were also found to be under the operation of positive selective pressure suggesting the diversification of these genes, which might lead to the emergence of new MDR strains of B. nordii in the near future. Our study reported and characterized the genome of the first MDR strain of B. nordii and revealed the AMR evolution in this species using a comprehensive comparative genomics approach.

Diagnostic accuracy of the metagenomic next-generation sequencing (mNGS) for detection of bacterial meningoencephalitis: a systematic review and meta-analysis.

The early diagnosis of bacterial meningoencephalitis (BM/E) is difficult, and delay in diagnosis can cause complications leading to neurological impairment/death. In cases of unexplained BM/E, the metagenomic NGS (mNGS) offers an advantage over conventional methods, especially when a rare pathogen is implicated or the patient is on antibiotics. This study aims to evaluate and compare the diagnostic efficacy of mNGS for the diagnosis of BM/E using cerebrospinal fluid (CSF) specimens versus a composite reference standard (CRS). The electronic databases (Embase, PubMed, and Web of Science) were searched up to 15 June 2021. Studies such as cohort, case-control, prospective, or retrospective studies that assessed the diagnostic efficacy of mNGS in suspected bacterial meningitis/encephalitis cases were included. Ten studies met the inclusion criteria, including three retrospective and seven prospective studies. The sensitivity of mNGS for diagnosis of BM/E from CSF samples ranged from 33 (95% CI: 13-62) to 98% (95% CI: 76-99). The specificity of mNGS ranged from 67 (95% CI: 55-78) to 98% (95% CI: 95-99). The estimated AUC (area under curve) by hierarchical summary receiver operating characteristic (HSROC) of the studies being analyzed was 0.912. The meta-regression analysis demonstrated that the different types of studies (single-center vs. multi-center) had an effect on the specificity of mNGS for BM/E compared with CRS (90% vs. 96%, meta-regression P < 0.05). The current analysis revealed moderate diagnostic accuracy of mNGS. This approach can be helpful, especially in cases of undiagnosed BM/E by identification of organism and subsequently accelerating the patient management.

Antimicrobial susceptibility testing of anaerobic bacteria: In routine and research.

To date, antimicrobial susceptibility testing (AST) in anaerobes is highly disarrayed. The testing is not routinely performed in many laboratories to direct appropriate antimicrobial therapy, and literature on AST remains scarce. This review aims to present the current methods employed for the AST of anaerobic bacteria both in routine and research. Anaerobes are potential human pathogens and predominate the human microbiota. Despite their significant role in human health and disease, they are not paid enough heed. The educated experience-based treatment has often been instituted with anaerobic infections due to the challenging AST and relatively predictable patterns of antimicrobial resistance (AMR). However, the AMR in anaerobes remains no more predictable, especially in Gram-negative anaerobes like Bacteroides spp., where multi-drug resistance is also emerging. The changing antibiograms and the emergence of resistance determinants in anaerobes necessitate routine AST, periodic monitoring, and literature review of these organisms. The present scenario dictates to limit the empirical management of anaerobic infections and encourage consistent AST practice in routine by employing economical, faster, and more pragmatic approaches, especially in tertiary care setups that cater to a vast patient population and are held accountable for producing accurate clinical and surveillance data. The article may provide a broad perspective on the topic and different AST solutions.

Clinical anaerobic infections in an Indian tertiary care hospital: A two-year retrospective study.

OBJECTIVE: To study the spectrum of anaerobic infections and the frequency of clinically relevant anaerobes in various infections in a tertiary care hospital in North India. METHOD: In this two-year (2018-2019) retrospective study, a total of 22,177 samples were processed both anaerobically and aerobically. The samples included blood, pus, body fluids, necrotic tissues, abscess, peritoneal fluids, and other specimens of conditions predisposing to anaerobic infections. The recovered bacterial isolates were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). RESULT: Of the 22,177 samples processed anaerobically, 1094 showed significant anaerobic growth yielding 1464 anaerobes. Bacteroides fragilis (12.7%) and Peptoniphilus harei (12.2%) were the most predominant anaerobes, whereas Escherichia coli (32.6%) and Staphylococcus aureus (13.2%) were the most frequently recovered facultative anaerobes in polymicrobial growth. Both aerobic and anaerobic data were available for 673/1094 samples, of which 68% represented a polymicrobial etiology and 32% as monomicrobial. Of the cases where complete clinical information was available (504/1094), the majority of the anaerobes were recovered from the skin and soft tissue infections (36.3%) and intra-abdominal infections (17.1%). The clinical specimen most frequently growing anaerobes was abscess (29.1%), followed by diabetic foot ulcers (14.1%). CONCLUSION: The study documents the frequency of clinically significant anaerobic bacteria in various infections, and their associations with aerobes in polymicrobial growth. The present study may aid us in devising better therapeutic strategies against both aerobes and anaerobes in anaerobic infections, which is often empirical. Besides, the data can update clinicians with the changing patterns of anaerobic infections, which remains a neglected concern.

Factors affecting decontamination of N95 masks for reuse: Feasibility & practicality of various methods.

The SARS-CoV-2 pandemic has led to an enormous increase in cases worldwide in a short time. The potential shortage might call for the reuse of personal protective equipment especially N95 masks. In this review, the methods available for decontamination of N95 masks have been compared to highlight the advantages and efficacies of different methods. Studies conducted to evaluate the biocidal efficacy, effect on filtration efficacy of the decontamination method, and maintenance of structural integrity of masks, were reviewed. Ultraviolet germicidal irradiation (UVGI) and hydrogen peroxide (H2O2) vapour were the most commonly evaluated interventions and showed good germicidal activity without significant deleterious effects on mask performance. Vapourous H2O2 was the best method as it maintained NIOSH (The National Institute for Occupational Safety and Health) recommendations of the mask on re-use and additionally, one mask could be decontaminated and reused 30 times. Ethylene oxide (EtO) preserved the maximum filtration efficacy and flow resistance. Chemical and heat-based methods had the advantages of being cost-effective and feasible but affected the structural integrity and fit of the masks. For the decontamination of N95 masks, among the heat-based methods steam was found to be the best for low middle-income countries setting. H2O2-based methods, UVGI, and EtO all exhibited both adequate biocidal efficacies and functionality (fit testing and structural integrity). Further studies on logistics, healthcare worker acceptability of reuse, and actual efficacy of protection against SARS-CoV-2 infection should be carried out to validate the use of decontamination in the real-life settings.

Comparative evaluation of agar dilution and broth microdilution by commercial and in-house plates for Bacteroides fragilis group: An economical and expeditious approach for resource-limited settings.

OBJECTIVE: To compare the performance of agar dilution and broth microdilution by commercial and in-house prepared plates for the Bacteroides fragilis group. The cost analysis was performed to demonstrate that in-house prepared BMD plates were a suitable alternative to agar dilution given the high cost and low feasibility of incorporating commercial BMD plates in routine, particularly in the tertiary care institutes of many low- and middle-income countries. METHODS: Thirty B. fragilis group isolates were tested against six antibiotics, frequently used as empirical therapy for anaerobic infections including metronidazole, clindamycin, imipenem, piperacillin-tazobactam, cefoxitin, and chloramphenicol. The running consumable expenditure for all methodologies was calculated. RESULTS: The results demonstrated essential and categorical agreement of >90% for all antibiotics except cefoxitin, which showed <90% categorical agreement. No major or very major errors were observed. We observed a high agreement and strong concordance for MIC values between both methods and inter-rate reliability of >0.9 by Cohen's kappa analysis, indicating almost perfect agreement between both methods using either of the plates. In contrast to agar dilution, a 20.5 fold cost reduction was seen in BMD using in-house plates and a 5.8 fold reduction using commercial plates to test a single isolate. However, when testing 30 isolates concurrently the cost significantly increased for commercial BMD plates by 8.4 folds, and only 1.03 fold cost reduction was seen with in-house BMD plates. CONCLUSION: BMD gives comparable results to agar dilution and can be considered a method of choice to test a small number of samples. The technique is an economical option when plates are standardized in-house and could be employed for susceptibility testing of the B. fragilis group.

Detection of co-infection of Gardnerella vaginalis and Atopobium vaginae using qualitative PCR: A better predictor of bacterial vaginosis.

The present study aimed to determine the utility of detection of co-infection of Gardnerella vaginalis and Atopobium vaginae using qualitative PCR for diagnosing bacterial vaginosis (BV). Vaginal samples (n = 385) categorized as positive (n = 108) or negative (n = 208) for bacterial vaginosis based on the Nugent scoring system, were analyzed for the presence of G. vaginalis and A. vaginae by conventional PCR. We compared the sensitivity, specificity, positive predictive value, negative predictive value and odds ratio for the detection of each bacterium alone with the combination of the two bacteria for diagnosing BV. The detection of co-infection of the two bacteria demonstrated a sensitivity of 96%, a specificity of 82.9%, a positive predictive value of 68.5%, a negative predictive value of 98.2% with an odds ratio of 116 (CI -32 - 409). In our study, we found a high sensitivity, specificity, negative predictive value and odds ratio for the detection of co-infection of A. vaginae and G. vaginalis for the diagnosis of BV.

Bacterial coinfections and secondary infections in COVID-19 patients from a tertiary care hospital of northern India: Time to adhere to culture-based practices.

OBJECTIVE: Bacterial co-pathogens are common in various viral respiratory tract infections, leading to increased disease severity and mortality. Still, they are understudied during large outbreaks and pandemics. This study was conducted to highlight the overall burden of these infections in COVID-19 patients admitted to our tertiary care hospital, along with their antibiotic susceptibility patterns. MATERIAL AND METHODS: During the six-month study period, clinical samples (blood samples, respiratory samples, and sterile body fluids, including cerebrospinal fluid [CSF]) of COVID-19 patients with suspected bacterial coinfections (at presentation) or secondary infections (after 48 hours of hospitalization) were received and processed for the same. RESULTS: Clinical samples of 814 COVID-19 patients were received for bacterial culture and susceptibility. Out of the total patient sample, 75% had already received empirical antibiotics before the samples were sent for analysis. Overall, 17.9% of cultures were positive for bacterial infections. Out of the total patients with bacterial infection, 74% (108/146) of patients had secondary bacterial infections (after 48 hours of hospitalization) and 26% (38/146) had bacterial coinfections (at the time of admission). Out of the 143 total isolates obtained, the majority (86%) were gram-negative organisms, of which Acinetobacter species was the commonest organism (35.6%), followed by Klebsiella pneumoniae (18.1%). The majority (50.7%) of the pathogenic organisms reported were multidrug resistant. CONCLUSION: The overall rate of secondary bacterial infections (SBIs) in our study was lower (7.9%) than reported by other studies. A rational approach would be to adhere to the practice of initiating culture-based guidance for antibiotics and to restrict unnecessary empirical antimicrobial therapy.

A child with recurrent otitis media due to Bacteroides thetaiotaomicron.

Brain abscess remains a life-threatening condition. Here, we are reporting a case of brain abscess due to Bacteroides thetaiotaomicron in a previously known case of recurrent otitis media. A 15 years old boy with a history of recurrent otitis media presented with the complaints of right otalgia, headache and fever. Computed Tomography (CT) brain and neck revealed fluid filled right middle ear cavity with bony destruction along the inner cortex of right temporal bone. The abscess was drained and culture showed growth of Bacteroides thetaiotaomicron. This report illustrates the importance of MALDI-TOF MS in the species level identification of anaerobes thereby facilitating the selection of appropriate and prompt adjuvant antibiotic therapy. This timely identification thus led to a favourable outcome in an era of increasing antimicrobial resistance.

Fusobacterial brain abscess caused by paradoxical embolization associated with Ebstein's anomaly in an adult patient: A case report.

Brain abscess remains a life-threatening condition. Here, we are reporting a case of brain abscess due to Fusobacterium nucleatum in a previously known case of Ebstein anomaly. A 44-year-old male presented with the complaints of headache, and fever. Cerebral imaging revealed parieto-occipital (PO) abscess. The abscess was drained and culture showed growth of Fusobacterium nucleatum. This report illustrates the importance of considering anaerobes as the cause of brain abscess, underscores the usefulness of MALDI, which facilitated the selection of appropriate and prompt adjuvant antibiotic therapy and a favourable outcome.

Clostridium septicum arthritis in a young infant: A case report.

Septic arthritis is one of the most serious conditions leading to grave consequences in all age groups, with a number of cases seen in infancy and childhood. Although septic arthritis in neonates is a known clinical entity, it is a diagnostic dilemma for the paediatricians. Of the reported causes of bacterial arthritis, anaerobic bacteria account for only 1%. Anaerobic bacterial septic arthritis may be more common than appreciated and therefore anaerobic as well as aerobic cultures should be done in all cases of septic arthritis. We report the first documented case of spontaneous septic arthritis due to Clostridium septicum in an infant with successful outcome.

Changing spectrum of primary pyomyositis in North India.

Recent data have demonstrated the changing epidemiology of primary pyomyositis worldwide. Our hospital-based retrospective study investigated the clinical and microbiological spectrum of primary pyomyositis between 2013 and 2021 in PGIMER (Chandigarh), India. Over a quarter had predisposing conditions, mainly diabetes mellitus and immunosuppressive therapy. Fever, muscle pain, local swelling and breathlessness were the usual presentations, with quadriceps, iliopsoas and gluteal muscles commonly affected. Staphylococcus aureus was the predominant cause, with c.50% methicillin-resistant strains. Almost two-thirds presented with metastatic infection (stage 3 pyomyositis), frequently with septic lung emboli. Patients with methicillin-sensitive and resistant Staphylococcus aureus had a similar incidence of metastatic infection. In-hospital mortality was c.10% and was strongly associated with a high international normalised ratio. Primary pyomyositis remains a significant problem, with a dramatic increase in community-associated methicillin-resistant Staphylococcus aureus.

Salmonella enterica serovar Give causing brain abscess and bacteremia: A case report.

Non-typhoidal Salmonellosis are an important cause of gastroenteritis and invasive disease in developing countries, with increase resistance and mortality in paediatric age group. We report here, a rare case of bacteremia and brain abscess in a 3year old female child with Salmonella enterica serovar Give as a causative organism.

Novel diagnostic approach for amoebic liver abscess using cell free (cf) DNA: a prospective study.

BACKGROUND: Amoebic liver abscess (ALA) is commonly seen in tropical countries and diagnosis of ALA relies mainly on non-specific serological and imaging techniques as well as PCR from pus. OBJECTIVE: This study evaluated the potential of using cell free DNA (cfDNA) from serum and urine for diagnosing ALA. METHODS: We prospectively evaluated quantitative PCR (qPCR) for detection of cf DNA in serum and urine sample in all liver abscess patients. The samples were collected from patients reporting to emergency ward of Postgraduate Institute of Medical Education and Research, Chandigarh, India with symptoms suggestive of liver abscess. Real time PCR was done to detect cf DNA in serum and urine by targeting 99-bp unit of small subunit rRNA of Entamoeba histolytica and conventional PCR for pus. RESULTS: A total 113 samples (serum and urine) and 100 pus samples were analysed. A total of 62 ALA patients were confirmed; with maximum 57 patients detected by qPCR for cfDNA in the serum, 55 patients by PCR on pus aspirate and 50 ALA patients by qPCR for cfDNA in urine sample. Therefore, the sensitivity of qPCR for detection of cf DNA in serum was 91.94% and for urine was 80.65%. CONCLUSION: A total of 11.2% of ALA patients were diagnosed only through detection of E. histolytica cf DNA in their serum and urine. Detection of cfDNA from serum, urine of ALA has a potential role in future especially for developing countries as it is a rapid, sensitive and patient friendly diagnostic approach.

Otogenic Cerebellar Abscess with Polymicrobial Anaerobic Infection in a Young Female- A Rare Presentation.

Brain abscess is a serious clinical condition caused by a localized collection of pus within the brain tissue. This typically occurs as a result of an infection that originates from a nearby area, such as an ear, sinus, or dental infection, or an infection in the bloodstream. Streptococcus and Staphylococcus species are the most common organisms implicated in brain abscesses. Apart from aerobic growth, cases of mixed infections of both aerobic and anaerobic organisms are also commonly reported in the literature. Herein we report a 23-year-old immunocompetent female with chronic otitis media who presented with cerebellar abscess where the aerobic growth was sterile and anaerobic culture revealed pure growth of dual anaerobes viz… Peptostreptococcus and Bacteroides thetaiotaomicron. This case highlights the importance of prompt diagnosis and management of polymicrobial anaerobic infection in cases of brain abscess.

Comparative evaluation of antimicrobial efficacy of chitosan nanoparticles and calcium hydroxide against endodontic biofilm of Enterococcus faecalis: An in vitro study.

Aim: The aim of the study was to assess and evaluate the antimicrobial effectiveness of chitosan nanoparticles (CSNPs) with calcium hydroxide in the elimination of Enterococcus faecalis. Materials and Methods: Using the broth microdilution method, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of calcium hydroxide and CSNPs were measured. The antibiofilm effect of calcium hydroxide and CSNPs against E. faecalis biofilm was qualitatively analyzed using a crystal violet assay. A 7-day-old biofilms of E. faecalis grown on dentine discs were assigned to the following three groups (n = 11 dentine discs), normal saline (group I), calcium hydroxide (group II), and CSNPs (group III). Quantification of live and dead cells using confocal microscopy was done to evaluate the antibiofilm efficacy of the medicaments included in the study. Results: MIC of calcium hydroxide and CSNPs against E. faecalis was observed at 2.5 mg/mL and 0.31 mg/mL, respectively. MBC of calcium hydroxide and CSNPs was observed at 2.5 mg/mL and 0.31 mg/mL, respectively. Using Crystal Violet (CV) assay, calcium hydroxide and CSNPs showed biofilm inhibition at concentrations of 2.5 mg/mL and 0.625 mg/mL, respectively. Confocal laser scanning microscopy analysis found that both calcium hydroxide and CSNPs showed a significant decrease in viable cells at their MBC values compared to the control group's normal saline. CSNPs showed a significantly lower percentage of live cells than calcium hydroxide (P < 0.05). Conclusion: The study results reveal that the antimicrobial efficacy of CSNPs is better than calcium hydroxide and normal saline against E. faecalis biofilm.