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.

Whole genome sequencing for the prediction of resistant tuberculosis strains from northern India.

PURPOSE: Tuberculosis is an important public health problem among infectious diseases. The problem becomes more concerning with the emergence of MDR-TB and pre-XDR-TB. Whole genome sequencing (WGS) detection of resistance has recently gained popularity as it has advantages over other commercial techniques. METHODS: We performed in-house WGS followed by detailed analysis by an in-house pipeline to identify the resistance markers. This was accompanied by Phenotypic DST, and Sanger sequencing on all the 12 XDR, 06 pre-XDR, and 06 susceptible M. tb isolates. These results were collated with online M. tb WGS pipelines (TB profiler, PhyResSE, Mykrobe predictor) for comparative analysis. RESULTS: Following our in-house analysis, we observed 64 non-synonymous SNPs, fifteen synonymous SNPs, and five INDELs in 25 drug resistance-associated genes/intergenic regions (IGRs) in M. tb isolates. Sensitivity for detecting XDR is 33%, 58%, 83%, and 83%, respectively, using Mykrobe predictor, PhyResSE, TB-profiler, and in-house pipeline for WGS analysis, respectively. TB-profiler detected a rare mutation H70R in the gyrA gene in one pre-XDR isolate. Lineage 2.2.1 East-Asian (Beijing sublineage type) predominated (60%) in WGS data analysis of the XDR isolates. CONCLUSIONS: Our findings suggest that in-house analysis of WGS data and TB-profiler sensitivity was better for the detection of second-line resistance as compared to other automated tested tools. Frequent upgradation of newer mutations associated with resistance needs to be updated, as it potentiates tailored treatment for patients.

Limits of antibacterial activity of triangular silver nanoplates and photothermal enhancement thereof for Bacillus subtilis.

Currently, nanoparticles are being actively explored for antimicrobial applications involving variety of pathogens. Bacillus subtilis is a major concern considering its sporulation and biofilm formation capability which involves high bacteria loadings. Also, there is natural ability of B subtilis to adapt and develop resistance to the silver nanoparticles alone. So, this study reports the limits of antibacterial activity of triangular silver nanoplates (∆AgNPs) and further photothermal enhancement for B. subtilis ATCC 6051 for considerably high bacterial load of 2.5 × 107 to 5 × 108 CFU/ml. Triangular silver nanoplates were synthesized using one pot synthesis method and showed significant photothermal response i.e., ∼36 °C temperature rise on near infrared irradiation as well as photothermal stability. Triangular silver nanoplates alone showed absolute destruction for 2.5 × 107 CFU/ml initial B. subtilis load in 5 min. Whereas, for further higher bacterial loads, the antibacterial efficacy of ∆AgNPs is observed to be insignificant. For higher initial bacterial loads of 5 × 107 CFU/ml and 5 × 108 CFU/ml, photothermally enhanced triangular silver nanoplates resulted in complete destruction of bacteria in about 5 and 10 min, respectively. Antibacterial efficacy and mechanism of the destruction assessed via scanning electron microscopy and LIVE/DEAD assay confirmed morphological deformities. Further the generation of higher levels of reactive oxygen species is also confirmed due to photothermal activation of ∆AgNPs. The study concludes that ∆AgNPs alone are effective only up to bacterial load of 2.5 × 107 CFU/ml. Whereas, for higher bacterial loads of B. subtilis, photothermally activated ∆AgNPs lead to irreversible damage due to multiple targeting mechanisms leading to absolute elimination in short span of 5-10 min for the chosen irradiation conditions. Ultimately, this study demonstrates photothermally enhanced silver nanoplates as a potential antimicrobial agent for considerably high bacterial loads of B. subtilis. Overall, the broader window of considered high bacterial loadings and its irradiation by this technique shows the full-proof nature of photothermal applications for scenarios involving high cell density such as biofilms and wound infections etc. Further, the concept may be useful for sterilization or decontamination of samples, devices, etc. because B. subtilis and its spores are the challenges during sterilization.

Prevalence of Trichomonas vaginalis by polymerase chain reaction-based molecular method among symptomatic women from Northern India.

Introduction: Trichomoniasis remains one of the most common sexually transmitted infections, which is curable. To prevent complications and transmission, prompt and correct diagnosis is essential to treat Trichomonas vaginalis. The present study was done to evaluate polymerase chain reaction (PCR) with other conventional techniques for the diagnosis of T. vaginalis infection and determine the prevalence of T. vaginalis in women with vaginal discharge based on PCR assay. Methods: Vaginal swabs were collected by the trained health-care professional using FLOQSwabs™ (Copan, Italy) during routine pelvic examinations among 1974 symptomatic females. The wet microscopy, culture, and PCR were performed. Results: The sensitivity of wet mount and culture in comparison to PCR was 60.87% and 56.52%, respectively. The kappa inter-rater agreement of T. vaginalis PCR showed substantial agreement with wet mount microscopy (κ = 0.742) and culture (κ = 0.707). The PCR detected an additional 17 cases that were missed by conventional techniques. Discussion: The study highlights the importance of PCR for T. vaginalis screening among symptomatic females.

Association of intermediate Nugent Score and bacterial vaginosis with sexually transmitted infections and vulvovaginal candidiasis.

Background Bacterial vaginosis is a common vaginal syndrome among females, which leads to significant morbidity and complications, if left untreated. The association of bacterial vaginosis with various sexually transmitted infections has been mentioned in previous literature. However, studies on the intermediate Nugent Score are lacking. This study was planned to examine the association of sexually transmitted infections with the intermediate Nugent Score. Materials and Methods The study included was conducted to include females presenting with vaginal discharge, burning micturition, itching, lower abdominal pain and infertility. The Nugent scoring was used to categorize patients into those having normal flora, intermediate or bacterial vaginosis. Conventional and molecular techniques targeting Trichomonas vaginalis, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma hominis, Syphilis, Neisseria gonorrhoeae and vulvovaginal candidiasis were performed. Results A total of 3,531 clinical samples were collected from females with a median age of 28.0 years. The number of patients with bacterial vaginosis and intermediate Nugent Score and positive cases were significantly higher in the 21-35 years age group (P < 0.0001). We observed that the likelihood of test results being positive for Trichomonas vaginalis was higher (P < 0.05), as the abnormality of the vaginal flora increased. Mycoplasma hominis was observed to be significantly higher in the intermediate Nugent Score group than the BV-positive patients (0.6 vs 0.2, P = 0.002). The number of vulvovaginal candidiasis cases in both the bacterial vaginosis-negative and bacterial vaginosis-positive groups were nearly the same (9.3 vs 9.8%). Limitation Individual follow-up couldn't be performed on the patients. Conclusion We observed that the dysbiosis in vaginal microbiota, with an increase in Nugent scoring, was significantly associated with an increased risk for the acquisition of sexually transmitted infections and vulvovaginal candidiasis.

Stable isotope labeling as a promising tool for rapid drug susceptibility testing in Neisseria gonorrhoeae.

The world is heading towards an era of intractable and impending untreatable N. gonorrhoeae, thereby underlining the significance of rapid and accurate prediction of drug resistance as an indispensable need of the hour. In the present study, we optimized and evaluated a stable isotope labeling-based approach using the MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry) for rapid and reliable detection of ciprofloxacin and azithromycin resistance in N. gonorrhoeae. All the isolates were cultured under three varied condition setups viz. medium supplemented with normal lysine, heavy lysine (isotope), and heavy lysine along with the antibiotics (ciprofloxacin/azithromycin), respectively. After incubation, spectra were acquired using the MALDI-TOF MS which were further screened for unique patterns (media-specific spectra) to differentiate drug-susceptible and resistant isolates. The results of the stable isotope labeling assay were comparable to the results of phenotypic methods used for susceptibility testing.

A retrospective analysis of sexually transmitted infections among males presenting to a tertiary care hospital of India.

Context: Sexually transmitted infections (STIs) are one of the most neglected diseases, leading to a high percentage of morbidity and mortality in India. The World Health Organization estimated that 20% of persons living with human immunodeficiency virus/acquired immunodeficiency syndrome are in their 20s and one out of twenty adolescents contract an STI each year. Aims: The present study was conducted to study the characteristics of the pattern of STI in adult males and study the prevalence of various STIs among them. Settings and Design: This retrospective study was conducted by retrieving records of males presenting to STI laboratory of our tertiary care hospital between (April 2018 and December 2019). Subjects and Methods: The patients comprised high-risk group males, approached through nongovernmental organizations (NGOs) and slum population visiting the dispensary attached to our institute. The age group of the patient included was between 0 and 85 years. Results: A total of 1023 males presented to our STI laboratory out of which 124 (12.12%) were symptomatic. The most common complaint was urethral irritation seen in 22.5%, followed by discharge in 9.6%. The most common sexually transmitted disease among symptomatic (34/124) as well as asymptomatic (172/899) men was syphilis showing a combined prevalence of 20% (206/1023). Out of 124 symptomatic patients, 29 (23.38%) complained of urethritis due to gonococcal infection. The association between the two was found to be significant (i.e., P < 0.05). Conclusion: STIs are a serious health problem in our country. Approximately 6% of the adult population have one or the other STI amounting to 30-35 million cases per year. An intensive study is the need of the hour which could help clinicians as well as microbiologists to control the spread of these infections.

Prevalence of nonviral reproductive tract infections/sexually transmitted infections in female patients with cervicovaginal discharge: Excerpts from a regional reference center in North India.

Background: To study the prevalence of common nonviral reproductive tract infections/sexually transmitted infections (RTI/STI) prevailing among females who presented to our regional STI reference center and to ascertain the association of various symptoms with different RTI/STIs. Materials and Methods: A retrospective analysis of female patients presenting to our STI Regional center located in the Department of Medical Microbiology in PGIMER, Chandigarh, was done between April 2018 and December 2019 for patients presenting with cervico-vaginal discharge. Two to three swabs were collected from each patient. The first swab was subjected to wet mount, gram stain, Potassium hydroxide (KOH) test, and culture on blood agar, the colonies obtained were identified by matrix-assisted laser desorption time of flight mass spectrometer (MALDI TOF-MS). Second swab was used for DNA extraction and detection of Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), and Chlamydia trachomatis (CT) by polymerase chain reaction. The third swab, when available, was inoculated onto pleuropneumonia-like organisms (PPLO) broth. Results: One thousand and thirteenth of 1472 (69%) female patients were symptomatic and the most common presenting symptoms were vaginal discharge (707/1013 [69.8%]), infertility (266/1013 [26.2%]), genital itching (60/1013 [5.9%]), lower abdomen pain (47/1013 [4.6%]) and burning micturition (16/1013 [1.6%]). The most prevalent RTI/STI was bacterial vaginosis (BV) 18.2% (269/1472), followed by vulvovaginal candidiasis (VVC) 6.8% (100/1472) and trichomoniasis (TV) 1.9% (28/1472). Five cases each of Mycoplasma genitalium and Ureaplasma urealyticum, three of NG and one of CT were also identified. Coinfections were seen in 40 (2.7%) cases. The most common causative agent responsible for VVC in our study was Candida albicans (65%). Conclusion: RTI/STIs were common among women and 69% were symptomatic. BV was the most common STI present in 18.2%, followed by VVC (6.8%) and trichomoniasis (1.9%).

COVID diagnostics by molecular methods: A systematic review of nucleic acid based testing systems.

BACKGROUND: The selection of appropriate kit and PCR equipment for the detection of SARS CoV-2 is critically important in view of many options available in the diagnostic market. Since last year many molecular products are available for COVID-19 diagnostics., some of these diagnostics have become commercially available for healthcare workers and clinical laboratories. However, the diagnostic technologies have specific limitations and reported several false-positive and false-negative cases, especially during the early stages of kit development and use. The current article addresses these and other relevant questions important to the medical microbiologists running or aspiring to run COVID diagnostic services using PCR and related technologies. METHODS: In this Systematic Review we follow Preferred Reporting Items for a Systematic Review and Meta-analysis of Diagnostic Test Accuracy Studies (PRISMA-DTA). A total of 258 citations retrieved, among those 77 peer reviewed articles was assessed for eligibility, and 181 studies were excluded. Based on inclusion criteria final data extraction was done. RESULTS: The question of diagnostic dilemma has also been addressed in view of discordant results between assays, inter-test variability, repeat testing requirements in specific settings and inconclusive or indeterminate results. Kit efficiency was satisfactory for all assays and the estimates varied within sample types and technology. Using clinical samples, we observed some variations in detection rate between kits. Importantly, none of the assays showed cross-reactivity with other respiratory (corona) viruses, except as expected for the SARS-CoV-1 E-gene. CONCLUSIONS: We conclude SARS CoV-2 related molecular assays differed considerably in performance. Hence we need to understand importance of molecular diagnostics test interpretation in light of the latest pandemic virus.

Rapid detection of ERG11 polymorphism associated azole resistance in Candida tropicalis.

Increasing reports of azole resistance in Candida tropicalis, highlight the development of rapid resistance detection techniques. Nonsynonymous mutations in the lanosterol C14 alpha-demethylase (ERG11) gene is one of the predominant mechanisms of azole resistance in C. tropicalis. We evaluated the tetra primer-amplification refractory mutation system-PCR (T-ARMS-PCR), restriction site mutation (RSM), and high-resolution melt (HRM) analysis methods for rapid resistance detection based on ERG11 polymorphism in C. tropicalis. Twelve azole-resistant and 19 susceptible isolates of C. tropicalis were included. DNA sequencing of the isolates was performed to check the ERG11 polymorphism status among resistant and susceptible isolates. Three approaches T-ARMS-PCR, RSM, and HRM were evaluated and validated for the rapid detection of ERG11 mutation. The fluconazole MICs for the 12 resistant and 19 susceptible isolates were 32-256 mg/L and 0.5-1 mg/L, respectively. The resistant isolates showed A339T and C461T mutations in the ERG11 gene. The T-ARMS-PCR and RSM approaches discriminated all the resistant and susceptible isolates, whereas HRM analysis differentiated all except one susceptible isolate. The sensitivity, specificity, analytical sensitivity, time, and cost of analysis suggests that these three methods can be utilized for the rapid detection of ERG11 mutations in C. tropicalis. Additionally, an excellent concordance with DNA sequencing was noted for all three methods. The rapid, sensitive, and inexpensive T-ARMS-PCR, RSM, and HRM approaches are suitable for the detection of azole resistance based on ERG11 polymorphism in C. tropicalis and can be implemented in clinical setups for batter patient management.