Evaluation of sample pooling for diagnosis of COVID-19 by real time-PCR: A resource-saving combat strategy.

BACKGROUND AND OBJECTIVES: Although about 80% of coronavirus disease-2019 (COVID-19) cases are reported to be mild, the remaining 20% of cases often result in severe disease with the potential of crushing already overstrained health care services. There has been sustainable growth of COVID-19 cases worldwide since mid-May 2020. To keep tabs on community transmission of COVID-19 infection screening of the samples from a large population is needed which includes asymptomatic/symptomatic individuals along with the migrant population. This requires extra resources, man power, and time for detection of severe acute respiratory syndrome coronavirus 2 by real-time polymerase chain reaction (RT-PCR). In the current scenario, the pooled sample testing strategy advocated by the Indian Council of Medical Research, New Delhi is a new approach that is very promising in resource-limited settings. In this study, we have evaluated the pooled strategy in terms of accurate testing results, utilization of consumables, and identification of borderline positive cases. MATERIALS AND METHODS: Between April and June 2020, we performed COVID-19 testing by RT-PCR from areas with varying prevalence of population referred to COVID laboratory, Dr Ram Manohar Lohia Institute of Medical Sciences, Lucknow. In the first step, the samples are collated into pools of 5 or 10. These pools are tested by RT-PCR. Negative pools were reported as negative whereas positive pools of 5 and 10 are then deconvoluted and each sample is tested individually. RESULTS: In the present study, we tested 4620 samples in 462 pools of 10 and 14 940 samples in 2990 pools of 5. Among 10 samples pool, 61 (13%) pools flagged positive in the first step. In the second step, among 61 pools (610 samples) deconvoluted strategy was followed in which 72 individual samples came positive. The pooled-sample testing strategy helps saves substantial resources and time during surge testing and enhanced pandemic surveillance. This approach requires around 76% to 93% fewer tests done in low to moderate prevalence settings and group sizes up to 5-10 in a population, compared to individual testing. CONCLUSIONS: Pooled-sample PCR analysis strategies can save substantial resources and time for COVID-19 mass testing in comparison with individual testing without compromising the resulting outcome of the test. In particular, the pooled-sample approach can facilitate mass screening in the early coming stages of COVID-19 outbreaks, especially in low- and middle-income settings, and control the spread by meticulous testing of all risk groups.

Molecular epidemiology & therapeutic options of carbapenem-resistant Gram-negative bacteria.

Background & objectives: The growing incidence and the wide diversity of carbapenemase-producing bacterial strains is a major concern as only a few antimicrobial agents are active on carbapenem-resistant bacteria. This study was designed to study molecular epidemiology of carbapenem-resistant Gram-negative bacterial (GNB) isolates from the community and hospital settings. Methods: In this study, non-duplicate GNB were isolated from clinical specimens, and phenotypic test such as modified Hodge test, metallo ß-lactamase E-strip test, etc. were performed on carbapenem-resistant bacteria. Multiplex PCR was performed to identify the presence of blaIMP, blaVIM, blaKPC, blaOXA48, blaOXA23, blaSPM, blaGIM, blaSIM and blaNDM. Minimum inhibitory concentration (MIC) of colistin, fosfomycin, minocycline, chloramphenicol and tigecycline was also determined. Results: Of the 3414 GNB studied, carbapenem resistance was 9.20 per cent and maximum resistance (11.2%) was present at tertiary care centre, followed by secondary care (4%) and primary centre (2.1%). Among the carbapenem-resistant bacteria, overall, the most common isolate was Pseudomonas aeruginosa (24%). On multiplex PCR 90.3 per cent carbapenem-resistant isolates were positive for carbapenemase gene. The blaNDM(63%) was the most prevalent gene followed by blaVIM(18.4%). MIC results showed that 88 per cent carbapenem-resistant Enterobacteriaceae were sensitive to fosfomycin, whereas 78 per cent of P. aeruginosa and 85 per cent Acinetobacter spp. were sensitive to colistin. Interpretation & conclusions: Carbapenem resistance in GNB isolates from the community and hospital settings was found to be on the rise and should be closely monitored. In the absence of new antibiotics in pipeline and limited therapeutic options, prudent use of antibiotics and strict infection control practices should be followed in hospital to limit the emergence and spread of multidrug-resistant bacteria.

Evaluation of the Rapidec Carba NP Test Kit for Detection of Carbapenemase-Producing Gram-Negative Bacteria.

Recently, bioMérieux, France, introduced the Rapidec Carba NP test kit for rapid detection of carbapenemase-producing Gram-negative bacteria. This kit was evaluated in this study, and we report sensitivity, specificity, and positive and negative predictive values of 92.6%, 96.2%, 95.83%, and 92.6%, respectively. The test was easy to perform and interpret and relatively inexpensive ($5/Rs 300 per test) and provides a practical solution for early detection of carbapenemase-producing, multidrug-resistant Gram-negative bacteria.

Hepatitis C virus core antigen: A diagnostic and treatment monitoring marker of hepatitis C virus in Indian population.

BACKGROUND: The diagnosis and treatment monitoring of hepatitis C is quite challenging. The screening test, i.e. antibody assay, is unable to detect acute cases, while the gold standard hepatitis C virus (HCV) reverse transcriptase polymerase chain reaction (RTPCR) assay is not feasible in resource-limited countries such as India due to high cost and infrastructure requirement. European Association for the Study of the Liver and World Health Organization have approved a new marker, i.e. HCV core antigen (HCVcAg) assay, as an alternative to molecular assay. In this study, we have evaluated HCVcAg assay for diagnosis and treatment monitoring follow-up in Indian population infected with hepatitis C. METHODS: Blood specimen of 90 clinically suspected cases of acute hepatitis C were tested simultaneously for anti-HCV antibody assay via ELISA (enzyme-linked immunoassay), HCVcAg assay by chemiluminescence immune assay (CLIA) and HCV RTPCR VL (viral load) assay. Thirty-four HCV RTPCR positive patients were further enrolled in treatment monitoring group whose blood samples were tested at the beginning of treatment, two weeks, four weeks and 12 weeks via HCV core Ag assay and HCV RTPCR Viral Load assay. RESULTS: Considering HCV RTPCR as gold standard, diagnostic performance of HCV core Ag assay and anti-HCV antibody assay was evaluated. The sensitivity and specificity of HCV core Ag assay were higher than that of anti-HCV Antibody assay, i.e. 88.3% and 100% vs. 23.3% and 83.3%, respectively. The overall diagnostic accuracy of HCV core Ag assay was 92.20%. Among treatment follow-up group, HCV core Ag levels correlated well with HCV viral load levels, at the beginning of treatment (baseline) till 12 weeks showing highly significant Spearman rank correlation coefficient of > 0.9 with HCV viral load levels. CONCLUSIONS: HCV core Ag assay is a cost-effective, practically feasible substitute of HCV RTPCR viral load assay for diagnosis as well as long duration treatment monitoring of hepatitis C infection in resource-limited settings.

A Road Less Traveled: E-test Method for Antifungal Susceptibility Testing in Trichophyton mentagrophyte Isolates Among Patients Presenting With Dermatophytosis at a Tertiary Healthcare Center in North India.

Introduction Dermatophytosis is a common infection of the skin, hair, and nails caused by dermatophytes, a group of filamentous fungi capable of digesting and obtaining nutrients from keratin. Dermatophytes comprise three important genera: Epidermophyton, Microsporum,and Trichophyton. This study aimed to analyze the antifungal susceptibility patterns of Trichophyton mentagrophytes isolates using the epsilometer test (E-test) method. Material and methods This prospective observational study was conducted on clinically suspected cases of dermatophytosis. All samples, including skin scrapings, hair, and nails, were subjected to potassium hydroxide (KOH) examination followed by fungal culture. The Trichophyton mentagrophytes isolates were then subjected to antifungal susceptibility testing using the E-test method for the two most prescribed antifungals: itraconazole and fluconazole. Results In this study, one-third of the patients who tested positive for dermatophytosis belonged to the same family, with spouses being the most commonly affected. Tinea corporis was the most common clinical presentation, with Trichophyton mentagrophytes identified as the most common etiological agent. Itraconazole was more effective than fluconazole. Conclusion The current study demonstrated that antifungal susceptibility testing of dermatophytes using the E-test is easier and can be applied in routine laboratories as a screening method, serving as an alternative to broth microdilution.

Recurrent COVID-19 infection in a health care worker: a case report.

BACKGROUND: Recurrent coronavirus disease 2019 (COVID-19) infection is an emerging problem and may prove to be one of the greatest problems in controlling the pandemic in the future. Recurrent infections can be due to reactivation of dormant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or reinfection with similar or different strains of SARS-CoV-2. CASE PRESENTATION: Here we present an interesting case of a health care worker working as a laboratory assistant at a COVID-19 laboratory who developed recurrent COVID-19 infection. He did not develop an immune response after the first episode of COVID-19; however, immunoglobulin G (IgG) antibodies were detected after the second episode. CONCLUSIONS: Through this case, we discuss the concept of reactivation and reinfection in the post-COVID period. We suggest that standard guidelines should be established to check for viral shedding and immune response among cured cases of COVID-19 after discharge via serial real-time polymerase chain reaction (RT-PCR) testing and IgG antibody detection. Further, strict hygiene practices should be stressed to these patients with possibility of COVID-19 recurrence.

"David vs. Goliath": A simple antigen detection test with potential to change diagnostic strategy for SARS-CoV-2.

INTRODUCTION: As regard to all pandemics, the current COVID-19 pandemic, could also have been better managed with prudent use of preventive measures coupled with rapid diagnostic tools such as rapid antigen tests, but their efficacy is under question because of projected lower sensitivity as compared to Real Time Reverse Transcriptase Polymerase Chain Reaction, which although considered gold standard has its own limitations. METHODOLOGY: A prospective, single centre study was carried out to evaluate the performance of Standard Q COVID-19 Ag, a rapid immuno-chromatographic assay for antigen detection, against TrueNat, a chip-based, point-of-care, portable, Real-Time PCR analyzer for diagnosis of COVID-19; on 467 nasal swab samples from suspected subjects at a fever clinic in North India in month of July 2020. RESULTS: Of the 467 specimens tested, TrueNat showed positive result in 29 (6.2%), majority of whom were asymptomatic (72.4%) while 4/29 (13.9%) had influenza like illness and 2/29 (6.8%) presented with severe acute respiratory illness. Compared to TrueNat, Rapid antigen test gave concordance for 26 samples, while for 2 samples the result was false positive; giving an overall sensitivity of 89.7% (95% CI = 72.6- 97.8) and a specificity of 99.5%, indicating strong agreement between two methods. CONCLUSION: Community prevalence plays an important role is choosing the laboratory test and result interpretation. Rapid antigen detection tests definitely have a big role to play, especially in resource limited setting, for early diagnosis as well as for source control to halt the spread.

Can rapid dengue diagnostic kits be trusted? A comparative study of commercially available rapid kits for serodiagnosis of dengue fever.

BACKGROUND: Dengue virus infection is an important emerging disease of the tropical and subtropical regions and is mainly diagnosed by serological detection of NS1 antigen and IgM antidengue antibodies. Since enzyme-linked immunosorbent assay (ELISA) facilities are not easily available at most diagnostic centers, so most of them use various commercially available rapid diagnostic tests (RDTs) kits. AIMS AND OBJECTIVES: This study was designed to access the diagnostic accuracy of four commercially available and widely used RDTs for serodiagnosis of dengue virus infection in Indian laboratories. SUBJECTS AND METHODS: The study was conducted at Department of Microbiology, G.S.V.M Medical College, Kanpur, India, to estimate the sensitivity and specificity of following RDTs: (1) Dengue Cassette (Panbio, Australia), (2) Bioline Dengue Duo (SD Diagnostics, Korea), (3) Dengue Day 1 test (J Mitra and Co., India), and (4) Dengucheck Duo (Tulip Diagnostics, India) on 72 confirmed dengue serum samples that were positive by dengue reverse transcription-polymerase chain reaction, dengue NS1, and IgM ELISA along with 80 serum samples from nondengue febrile illness patients. RESULTS: The majority of the RDTs demonstrated low sensitivity but good specificity for detecting NS1 antigen. Detection of antidengue IgM antibodies by RDTs demonstrated low sensitivity ranging from 27.8% to 77.7%. However, specificity was generally higher (50%-86.2%) and more consistent across the assays. CONCLUSION: The study results differed markedly from the RDTs manufacturers' claimed performance characteristics. Therefore, the RDT results should be interpreted cautiously and ELISA should be performed as far as possible for serodiagnosis of dengue virus infection.

Nosocomial cutaneous zygomycosis in a patient with diabetic ketoacidosis.

Zygomycosis is an opportunistic fungal infection with a fulminant course. Varying clinical forms have been described, including cutaneous zygomycosis, which is mainly observed in diabetic and burns patients. We report herein a case of cutaneous zygomycosis of the nose in a 26-year-old female patient with diabetic ketoacidosis, developing secondary to the application of non-elasticized adhesive tape probably contaminated with fungal spores.

Wound infections secondary to snakebite.

BACKGROUND: The study was performed to identify the important bacterial pathogens responsible for wound infections secondary to snakebite and to determine their antimicrobial susceptibility. METHODOLOGY: All cases of wound infection secondary to snakebite were included in this retrospective study. Infected tissues were surgically debrided and inoculated on blood agar and MacConkey agar for aerobic bacterial culture, followed by antimicrobial susceptibility testing of the isolates by Kirby-Bauer disk diffusion method. RESULTS: Staphylococcus aureus (32%) was the most common isolate followed by Escherichia coli (15%); monomicrobial infections were more frequent than polymicrobial infections. The majority of the isolates were antibiotic sensitive. Ciprofloxacin, an oral drug covering both Gram-positive and Gram-negative isolates, was the most frequently prescribed antibiotic. The patients responded well to the treatment. CONCLUSION: The results of this study will be helpful in deciding the empirical antibiotic therapy in cases of wound infection secondary to snakebite in regions of Southeast Asia.

Rapid detection of dermatophytes from skin and hair.

BACKGROUND: Dermatophytes are a group of closely related keratinophilic fungi that can invade keratinized humans and animals tissues such as skin, hair and nails causing dermatophytosis. They are an important cause of superficial fungal infection. FINDINGS: Conventional methods like potassium hydroxide (KOH) microscopy and fungal culture lacks the ability to make an early and specific diagnosis. In this study we have evaluated nested Polymerase chain reaction (PCR) using primers targeting dermatophyte specific sequence of chitin synthase 1 (CHS1) gene and compared with conventional test. A total of 155 patients clinically suspected with dermatophytosis were included in the study. Of which 105 specimens were skin scrapings and 50 were hair. KOH microscopy, fungal culture and first round and nested PCR were done on clinical specimens, and results compared. Nested PCR for dermatophytes was positive in 83.8% specimens, followed by KOH microscopy (70%), first round PCR (50.8) and fungal culture (25.8). CONCLUSION: Results indicate that nested PCR may be considered as gold standard for the diagnosis of dermatophytosis and can aid the clinician in initiating prompt and appropriate antifungal therapy.

Evaluation of pan-dermatophyte nested PCR in diagnosis of onychomycosis.

In this study, nested PCR using novel primers targeting the pan-dermatophyte-specific sequence of the chitin synthase 1 gene (CHS1) was compared with KOH microscopy, culture isolation, and single-round PCR for diagnosis of 152 patients with clinically suspected onychomycosis. Results indicate that nested PCR may be considered the gold standard for the diagnosis of cases of onychomycosis for which the etiological agents are dermatophytes.