Browsing by Author "Alka Shukla"

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Assessment of Successful qRT-PCR of SARS-CoV-2 Assay in Pool Screening Using Isopropyl Alcohol Purification Step in RNA Extraction
(Hindawi Limited, 2021) Mayank Gangwar; Alka Shukla; Virendra Kumar Patel; Pradyot Prakash; Gopal Nath
The study is aimed at establishing the optimal parameters for RNA purification of pooled specimens, in SARS-CoV-2 assay. This research work evaluates the difference of extracted RNA purity of pooled samples with and without treatment with isopropyl alcohol and its effect on real-time RT-PCR. As per the protocol of the Indian Council of Medical Research (ICMR), 5 sample pools were analysed using qRT-PCR. A total of 100 pooled samples were selected for the study by mixing 50 μL of one COVID-19 positive nasopharyngeal/oropharyngeal (NP/OP) specimen and 50 μL each of 4 known negative specimens. Pool RNA was extracted using the column-based method, and 1 set of pooled extracted RNA was tested as such, while RNA of the second set was treated additionally with chilled isopropyl alcohol (modified protocol). Further, the purity of extracted RNA in both the groups was checked using Microvolume Spectrophotometers (Nanodrop) followed by RT-PCR targeting E-gene and RNaseP target. The results showed that the purity index of extracted RNA of untreated pooled specimens was inferior to isopropyl alcohol-treated templates, which was observed to be 85% sensitivity and 100% specificity. The average Cq (E gene) in the unpurified and purified pool RNA group was 34.66 and 31.48, respectively. The nanodrop data suggested that purified RNA concentration was significantly increased with an average value of 24.73±1.49 ng/uL, which might be the reason for high sensitivity and specificity. Thus, this group testing of SARS-CoV-2 cases using pools of 5 individual samples would be the best alternative for saving molecular reagents, personnel time, and can increase the overall testing capacity. However, purity of RNA is one of the important determinants to procure unfailing results, thus, this additional purification step must be included in the protocol after RNA has been extracted using commercially available kit before performing qRT-PCR. © 2021 Mayank Gangwar et al.
Bacteriophage therapy a promising alternative treatment approach for infections caused by multidrug-resistant (MDR) Enterococcus species
(Elsevier B.V., 2025) Minakshi Sahu; Sudhir Kumar Singh; Meenakshi Singh; Alka Shukla; Gopal Nath
The 21st-century real threat is the upsurge of microbial resistance and the ineffectiveness of drugs to even a minor injury and common infection, which further leads to an increase in mortality rate. Enterococci bacterial species, such as Enterococcus faecalis and Enterococcus faecium, are common human pathogens that cause many diseases, such as bacteremia, urinary tract infections (UTI), meningitis, and endocarditis. The higher occurrence of MDR bacteria and the ineffectiveness of available antibiotics in treating infectious diseases have prompted a search for viable alternatives, such as bacteriophage therapy and combinational therapy of bacteriophage with standard antibiotics. The current review focuses on the role of Enterococcus spp. as the causative agent of life-threatening infections and how the bacteriophages are effective against them in reference to different clinical settings and their ability to thwart biofilm formation. Furthermore, combining antibiotics and bacteriophage improves killing efficacy and prevents phage resistance development. Compared to conventional antibiotics alone, the synergy between the antibiotics and phages disrupts the biofilm efficiently in MDR biofilm infections. Finally, the application of bacteriophage endolysin has been discussed, gathering significant attention worldwide due to their cell wall lysing propensity and potential therapeutic usage for treating enterococci-associated infectious diseases. © 2025
Epidemiological Distribution and Potential Risk Factors of Orientia tsutsugamushi Infection in Eastern Uttar Pradesh, India
(Hamadan University of Medical Sciences, 2021) Alka Shukla; Mayank Gangwar; Akanksha Srivastava; Sonam Rastogi; Deepak Kumar; Digvijay Singh; Rajesh Kumar; Pradyot Prakash; Gopal Nath
Background: Scrub typhus (ST) is a rickettsial infection caused by Orientia tsutsugamushi, which presents with flu like symptoms. This disease has been reported from all over India but with slight variations in its pattern. For decreasing the prevalence, preventing new incidences, and predicting the course of the ST, therefore, it is crucial to gain knowledge and perception of local risk components associated with the disease. The present study aimed to investigate the epidemiological distribution and potential risk factors of O. tsutsugamushi Infection in Eastern Uttar Pradesh (EUP), India. Methods: The serums of 211 samples were collected from the suspected cases along with the detailed information about the participants such as age, location, and place recorded in case history form (CRF). IgM estimation was performed using enzyme-linked immunosorbent assay (ELISA) assay. Results: A total of 58 samples (27.4%) out of 211 ones were found to be positive for IgM antibodies against O. tsutsugamushi bacterium. Furthermore, the results were correlated with epidemiological data such as gender, rural or urban background, pets, and occupation. The results showed that 76.7% of the study participants were from rural areas or had bushes around their houses, 88.3% of them had pets/cattle or frequent encounter with rodents at their houses, and 30.3% of them had no toilet facilities at home. Conclusions: It was concluded that the proximity to pets/cattle, having rodents in closer vicinity, residing in places surrounded by vegetation/farm/bushy areas, and following occupations involving field work increased the chances of getting bitten by mites/chiggers. Overall, Orientia tsutsugamushi prevalence increased in EUP, with respect to clinical features, disease presentation, and laboratory diagnosis can help our community to reduce the mortality caused by this infectious disease. © 2021 The Author(s); Published by Hamadan University of Medical Sciences.
Immunological and safety profile of bacteriophage therapy: A pre-clinical study
(John Wiley and Sons Inc, 2022) Mayank Gangwar; Sonam Rastogi; Digvijay Singh; Alka Shukla; Neeraj Dhameja; Deepak Kumar; Rajesh Kumar; Gopal Nath
Aims: Numerous pre-clinical and clinical studies have recently demonstrated the significant role of phage therapy in treating multidrug-resistant bacterial infections. However, only a few researchers have focused on monitoring the phage-mediated adverse reactions during phage therapy. The present study aimed to demonstrated the oral acute and sub-acute toxicity of bacteriophages (Klebsiella pneumoniae XDR strain) in Charles Foster rats with special reference to immunological response and adverse effects. Methods and Results: Bacteriophages were orally administered in dosages of 1010 PFU/ml and a 1015 PFU/ml to Charles Foster rats as a single dose (in acute toxicity study) and daily dosage for 28 days (in sub-acute toxicity study). One millilitre suspension of bacteriophages was administered through the oral gavage feeding tube. No adverse effect was observed in any of the experimental as well as in the control animals. Furthermore, an insignificant change in food and water intake and body weight was observed throughout the study period compared with the control group rats. On the 28th day of phage administration, blood was collected to estimate haematological, biochemical and cytokines parameters. The data suggested no difference in the haematological, biochemical and cytokine profiles compared to the control group. No significant change in any of the treatment groups could be observed on the gross and histopathological examinations. The cytokines estimated, interleukin-1 beta (IL-1β), IL-4, IL-6 and IFN-gamma, were found within the normal range during the experiment. Conclusions: The results concluded that no adverse effect, including the severe detrimental impact on oral administration of high (1010 PFU/ml) and very high dose (1015 PFU/ml) of the bacteriophages cocktail. Significance and Impact of Study: The high and long-term oral administration of bacteriophages did not induce noticeable immunological response as well. © 2022 Society for Applied Microbiology.
Study on the Effect of Oral Administration of Bacteriophages in Charles Foster Rats With Special Reference to Immunological and Adverse Effects
(Frontiers Media S.A., 2021) Mayank Gangwar; Sonam Rastogi; Digvijay Singh; Alka Shukla; Neeraj Dhameja; Deepak Kumar; Rajesh Kumar; Gopal Nath
Numerous pre-clinical and clinical studies have recently demonstrated the significant role of phage therapy in treating multidrug-resistant bacterial infections. However, only a few researchers have focused on monitoring the phage-mediated adverse reactions during phage therapy. Besides adverse reactions, immunological response after short- and long-term oral administration of bacteriophages is also lacking. In this study, we administered the bacteriophages orally against Klebsiella pneumoniae XDR strain in dosages of 1015 PFU/ml and a 1020 PFU/ml (still higher) to Charles Foster rats as a single dose (in acute toxicity study) and daily dosage for 28 days (in sub-acute toxicity study). One milliliter suspension of bacteriophages was administered through the oral gavage feeding tube. No adverse effect was observed in any of the experimental as well as in the control animals.Further, an insignificant change in food and water intake and body weight was observed throughout the study period compared with the control group rats. On the 28th day of phage administration, blood was collected to estimate hematological, biochemical, and cytokines parameters. The data suggested no difference in the hematological, biochemical, and cytokine profile compared to the control group. No significant change in any of the treatment groups could be observed on the gross and histopathological examinations. The cytokines estimated, interleukin-1 beta (IL-1β), IL-4, IL-6, and INF-gamma, were found within the normal range during the experiment. The results suggested no adverse effect, including the severe detrimental impact on oral administration of high (1015 PFU/ml) and very high dose (1020 PFU/ml) of the bacteriophages cocktail. The high and long-term oral administration of bacteriophages did not induce noticeable immunological response as well. © Copyright © 2021 Gangwar, Rastogi, Singh, Shukla, Dhameja, Kumar, Kumar and Nath.
Vitality of Proteinase K in rRTPCR Detection of SARS-CoV2 Bypassing RNA Extraction
(Frontiers Media S.A., 2021) Alka Shukla; Mayank Gangwar; Gaurav Sharma; Pradyot Prakash; Gopal Nath
This study aimed to detect the SARS-COV2 viral component directly from inoculated VTM without RNA extraction. Inoculated VTMs of already tested 50 positive and 50 negative samples were divided into three groups. Group I was treated with Proteinase K (PK) followed by 3-step-heat treatment at different temperatures (25°C, 60°C, and 98°C) and stored at 4°C. Group II was directly subjected to 3-step-heat treatment without PK exposure and stored at 4°C. And group III was set-up as standard group; it was processed using Qiagen’s column based QIAamp Nucleic Acid kit and the obtained nucleic acids were stored at 4°C. These stored samples were used as a template to execute real-time polymerase chain reaction, and results were noted. Group I demonstrated 96% and 88% sensitivity for N and ORF1ab genes respectively, whereas group II demonstrated 78% and 60% when compared to the results of standard group III. Overall group I showed better results than group II when compared to group III. Thus, in situations where gold-standard reagents are not available, PK exposure and heat treatment can be employed to carry out molecular detection of SARS-CoV2 viral component. Copyright © 2021 Shukla, Gangwar, Sharma, Prakash and Nath.