Evaluation of droplet digital qRT-PCR (dd qRT-PCR) for quantification of SARS CoV-2 RNA in stool and urine specimens of COVID-19 patients

IntroductionThere have been a few reports of viral load detection in stool and urine samples of patients with coronavirus disease 2019 (COVID-19), and the transmission of the virus through faecal oral route. For clinical diagnosis and treatment, the widely used reverse transcription-polymerase chain reaction (qRT-PCR) method has some limitations.MethodsThe aim of our study to assess the presence and concentration of SARS CoV-2 RNA in stool and urine samples from COVID-19 patients with mild, moderate, and severe disease, we compared a traditional qRT-PCR approach with a ddPCR. ddPCR and qRT-PCR-based target gene analysis were performed on 107 COVID-19-confirmed patients paired samples (N1 and N2). The MagMax magnetic beads base method was used to isolate RNA. Real-time qRT-PCR and dd PCR were performed on all patients.Results and DiscussionThe average cycle threshold (Ct) of qRT-PCR was highly correlated with the average copy number of 327.10 copies/l analyzed in ddPCR. In ddPCR, urine samples showed 27.1% positivity while for stool it was 100%.ConclusionThis study’s findings not only show that SARS CoV-2 is present in urine and faeces, but also suggest that low concentrations of the viral target ddPCR make it easier to identify positive samples and help resolve for cases of inconclusive diagnosis

jefA (Rv2459), a drug efflux gene in Mycobacterium tuberculosis confers resistance to isoniazid & ethambutol

Background & objectives: Drug efflux pumps have been contributing factor(s) in the development of multidrug resistance in various clinically relevant bacteria. During efflux pump gene expression studies on mycobacteria, we have found a previously uncharacterized open reading frame (ORF) Rv2459 to be overexpressed in drug stressed conditions. The objective of the present study was to investigate the role of this ORF as a drug efflux pump, which might add new information in our understanding about the alternative mechanisms of drug resistance in mycobacteria. Methods: The open reading frame Rv2459 of Mycobacterium tuberculosis encoding a probable drug efflux protein has been cloned using pSD5 E.coli-Mycobacterium shuttle vector and overexpressed in M. tuberculosis H37Rv. This ORF was named as jefA. Overexpression of this gene in clones has been verified by real-time reverse transcription PCR. Minimum inhibitory concentrations (MICs) of recombinant as well as non-recombinant clones were determined by resazurin microtitre assay plate method (REMA) with and without efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP) and verapamil. Results: In recombinant strains of M. tuberculosis, the overexpression of this gene led to an increase in MIC of anti-tubercular drugs isoniazid and ethambutol when tested by REMA. In the presence of CCCP and verapamil, the recombinant strains showed decrease in MIC for these drugs. Bioinformatic analysis has shown a close relation of JefA protein with drug efflux pumps of other clinically relevant bacteria. In homology derived structure prepared from nearest available model, it was observed that amino acids forming TMH 1, 8 and 11 participated in ethambutol specificity and those forming TMH 2, 7 and 10 participated in isoniazid specificity in JefA. Interpretation & conclusion: The increased transcription of jefA leads to increased resistance to ethambutol and isoniazid in M. tuberculosis via efflux pump like mechanism and contributes in the development of resistance to these drugs. JefA amino acid sequence is well conserved among clinically important bacterial genera, which further provides evidence of being a potent drug efflux pump. The involvement in drug resistance and very little homology with any of the human proteins makes JefA important to be included in the list of potential drug targets

Detection of Echovirus-18 in Children Suspected With SARS-CoV-2 Infection With Multisystem Inflammatory Syndrome: A Case Report From India

There have been several reports across the globe regarding the presentation of a severe multi-system hyperinflammatory syndrome, resembling Kawasaki disease (KD), in the pediatric population during the SARS-CoV-2 pandemic. The exact pathophysiology is still unclear; however, children typically demonstrate multi-organ dysfunction and less respiratory system involvement compared to adults. The limited literature is available at present for the identification and management of such patients. In this study, we investigated four cases in children ages 11–15 years that fulfilled the case definition for the pediatric multi-system inflammatory syndrome. All were found negative for SARS-CoV-2 from oropharyngeal swabs and stool. As they were having symptoms of diarrhea, tests for bacterial and enteric viral infections were performed after SARS-CoV-2 testing. Molecular analysis revealed that all the children were infected with enterovirus (Echovirus-18). Early and exact diagnosis is vital for timely, effective, and potentially life-saving management of such cases

Hand, foot, and mouth disease (HFMD) in India: A review on clinical manifestations, molecular epidemiology, pathogenesis, and prevention

HFMD is a childhood viral disease initiated by enteroviruses (EVs). Symptoms are initiated with mild-to-moderate fever of short duration followed by oral and skin lesions. Skin lesions are papulovesicular which appears on palms/soles of feet, hands, knees, and elbows. Oral lesions appear as vesicles producing multiple small superficial ulcers. Disease is usually mild illness but sometimes progresses in severe form as meningitis, encephalitis, and polio-like paralysis. Etiological agents of the disease belong to Picornaviridae family. The causative viral agents are from genus human enterovirus (HEV) such as enterovirus-A 71 (EV-A71), coxsackievirus –A6 (CV-A6), CV-A10, CV-A16. Coxsackievirus A-16 (CV-A16) and enterovirus A-71 (EV-A71) are the major etiological agents of this disease, among children reported globally. In India, studies conducted on HFMD cases revealed CV-A16 as a major EV type and under circulation over a period of time. Molecular studies of different CV-A16 isolates and the viral kinetic studies conducted on organ tissues of experimental mouse model with complete VP1 gene sequencing revealed presence of B1c sub genotype which is currently in circulation. Genetic changes observed at nucleotide and amino acid level in vital organs of experimental infected mice model might predict some targets and can act as markers of virulence. Mice infected with CV-A16 strains revealed progressive pathological changes in mice organs. Major affected organs were to be as brain, heart, intestine, and skeletal muscles. The present review focuses on HFMD caused by CV-A16 with epidemiological, molecular, pathogenesis and need of antivirals against the disease

Detection of Mycobacterium gilvum first time from the bathing water of leprosy patient from Purulia, West Bengal

In this present study for the first time the authors are reporting the isolation of Mycobacterium gilvum from the accumulated water in the drain connected to the bathing place of leprosy patients residing in an endemic region. The identification and characterization of this isolate was carried out by various conventional and molecular tests, including 16S rDNA sequencing. These findings might shed further light and association with amoeba in the leprosy endemic area of this rare Mycobacterium species

Detection and Molecular Characterization of Animal Adenovirus and Astrovirus from Western Maharashtra, India

Astroviruses (AstV) and adenoviruses (AdV) are associated with diarrhoea in young animals. However, the epidemiology and genetic diversity of AstVs and AdVs in animals is not well studied. Hence, the present study was conducted to detect and characterize AstVs and AdVs in calves, piglets and puppies from Western Maharashtra, India. Out of the processed porcine (48), canine (80), and bovine (65) faecal samples, the porcine AstV (PAstV), bovine AstV (BAstV), canine AstV (CAstV), and porcine AdV (PAdV) were detected in 12.5%, 7.69%, 3.75% and 4.1% of samples, respectively. In the RNA-dependent RNA polymerase region-based phylogenetic analysis, the detected BAstV strains grouped with MAstV-28, MAstV-33, and MAstV-35, CAstV strains belonged to MAstV-5; PAstV strains belonged to MAstV-24, MAstV-26, and MAstV-31. However, in hexon gene-based phylogeny, both the detected PAdV were of genotype 3, exhibiting 91.9–92.5% nucleotide identity with Ivoirian and Chinese strains. The study reports first-time BAstVs from calves and PAdV-3 from piglets in India. The study revealed diversity in the circulation of AstVs in tested animals and AdVs in pigs, and suggested that they alone might be associated with other diarrhoea or in combination with other enteric pathogens, thus highlighting the necessity of extensive epidemiological investigations to develop diagnostic tools and control measures

VDR polymorphism, gene expression and vitamin D levels in leprosy patients from North Indian population.

BackgroundLeprosy is a chronic infectious disease caused by Mycobacterium leprae and mainly affects skin, peripheral nerves. Vitamin D receptor (VDR) gene polymorphism has been found to be associated with leprosy. Vitamin D has been shown to control several host immunomodulating properties through VDR gene. Vitamin D deficiency was also found to be linked to an increased risk for several infections and metabolic diseases.ObjectiveIn the present study, we investigated the association of VDR gene polymorphism, mRNA gene expression of VDR and the vitamin D levels with leprosy and its reactional states.MethodologyA total of 305 leprosy patients consisting of tuberculoid (TT), borderline tuberculoid (BT), borderline lepromatous (BL), lepromatous leprosy (LL), as well as 200 healthy controls were enrolled in the study. We identified single nucleotide polymorphisms (SNPs) of VDR Taq1, Fok1 and Apa1, as well as the expression of VDR mRNA gene using PCR-based restriction fragment length polymorphism (RFLP) analysis and real-time PCR respectively. We also performed ELISA to measure vitamin D levels.ResultWe observed that SNP of VDR gene (Fok1 and Taq1) are associated with the leprosy disease. The allelic frequency distribution of T and t allele (p = 0.0037), F and f allele (p = 0.0024) was significantly higher in leprosy patients and healthy controls. ff genotype of Fok1 was found to be associated with leprosy patients [p = 0.0004; OR (95% CI) 3.148 (1.662-5.965)]. The recessive model of Fok1 genotype was also found to be significantly associated in leprosy patients in comparison to healthy controls [p = 0.00004; OR (95% CI) 2.85 (1.56-5.22)]. Leprosy patients are significantly associated with t-F-a haplotype. Further, VDR gene expression was found to be lower in non-reaction group compared to that of reaction group of leprosy and healthy controls. Paradoxically, we noted no difference in the levels of vitamin D between leprosy patients and healthy controls.ConclusionBlood levels of vitamin D do not play any role in clinical manifestations of any forms of leprosy. ff genotype of Fok1 and tt genotype of Taq1 was found to be associated with leprosy per se. Association of t-F-a haplotype with leprosy was found to be significant and could be used as a genetic marker to identify individuals at high risk for developing leprosy. VDR gene expression was lower in TT/BT and BL/LL groups of leprosy in comparison to that of healthy controls

Comparative evaluation of PCR amplification of RLEP, 16S rRNA, rpoT and Sod A gene targets for detection of M. leprae DNA from clinical and environmental samples

Purpose: PCR assay is a highly sensitive, specific and reliable diagnostic tool for the identification of pathogens in many infectious diseases. Genome sequencing Mycobacterium leprae revealed several gene targets that could be used for the detection of DNA from clinical and environmental samples. The PCR sensitivity of particular gene targets for specific clinical and environmental isolates has not yet been established. The present study was conducted to compare the sensitivity of RLEP, rpoT, Sod A and 16S rRNA gene targets in the detection of M. leprae in slit skin smear (SSS), blood, soil samples of leprosy patients and their surroundings. Method: Leprosy patients were classified into Paucibacillary (PB) and Multibacillary (MB) types. Ziehl–Neelsen (ZN) staining method for all the SSS samples and Bacteriological Index (BI) was calculated for all patients. Standard laboratory protocol was used for DNA extraction from SSS, blood and soil samples. PCR technique was performed for the detection of M. leprae DNA from all the above-mentioned samples. Results: RLEP gene target was able to detect the presence of M. leprae in 83% of SSS, 100% of blood samples and in 36% of soil samples and was noted to be the best out of all other gene targets (rpoT, Sod A and 16S rRNA). It was noted that the RLEP gene target was able to detect the highest number (53%) of BI-negative leprosy patients amongst all the gene targets used in this study. Conclusion: Amongst all the gene targets used in this study, PCR positivity using RLEP gene target was the highest in all the clinical and environmental samples. Further, the RLEP gene target was able to detect 53% of blood samples as positive in BI-negative leprosy cases indicating its future standardization and use for diagnostic purposes

Molecular typing of Mycobacterium tuberculosis isolates from a rural area of Kanpur by spoligotyping and mycobacterial interspersed repetitive units (MIRUs) typing

Molecular typing of Mycobacterium tuberculosis isolates has greatly facilitated the understanding of epidemiology of tuberculosis (TB). This study was done to characterize prevalent genotypes of M. tuberculosis on a collection of 97 isolates based on spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing in rural area of Kanpur, North India. In this area different types of interventions are being undertaken and follow-up studies are progressing. Predominant spoligotypes prevalent in this region belonged to Central Asian-Delhi family (CAS1_Del) (37%), East African-Indian family (11%), T1 family (8%) and Beijing (4%) family. Highly distinct MIRU-VNTR genotypes were obtained. Significant spoligotypes such as Beijing and CAS1_Del type were further divided into subtypes with MIRU-VNTR. This preliminary study reveals that CAS is the most predominant family in this rural area of Kanpur. If confirmed in other areas, this combined approach of molecular typing can be preferably be used as first line tool for studying linkage and transmission dynamics of TB in India

A report of rifampin-resistant leprosy from northern and eastern India: identification and in silico analysis of molecular interactions

Presence of point mutations within the drug resistance determining regions of Mycobacterium leprae (M. leprae) genome confers molecular basis of drug resistance to dapsone, rifampin and ofloxacin in leprosy. This study is focused on the identification of mutations within the rpoB gene region of M. leprae that are specific for rifampin interaction, and further in silico analysis was carried out to determine the variations in the interactions. DNA and RNA were isolated from slit skin scrapings of 60 relapsed leprosy patients. PCR targeting rpoB gene region and amplicon sequencing was performed to determine point mutations. mRNA expression levels of rpoB and high-resolution melt analysis of mutants were performed using Rotor Gene Q Realtime PCR. Molecular docking was performed using LigandFit Software. Ten cases having point mutations within the rpoB gene region were identified and were clinically confirmed to be resistant to rifampin. A new mutation at codon position Gln442His has been identified. There is a 9.44-fold upregulation in the mRNA expression of rpoB gene in mutant/resistant samples when compared with the wild/sensitive samples. In silico docking analysis of rifampin with wild-type and Gln442His mutant RpoB proteins revealed a variation in the hydrogen-bonding pattern leading to a difference in the total interaction energy and conformational change at position Asp441. These preliminary downstream functional observations revealed that the presence of point mutations within the rifampin resistance determining regions of rpoB gene plays a vital role in conferring genetic and molecular basis of resistance to rifampin in leprosy