Comparison of Manual and Automated Nucleic Acid (RNA) Extraction Methods for the Detection of SARS-CoV-2 by qRT-PCR.

Objectives During the COVID-19 pandemic, several laboratories used different RNA extraction methods based on the resources available. Hence this study was done to compare the Ct values in qRT-PCR, time taken (sample processing-loading to PCR), manpower requirement, and cost of consumables between manual and automated methods. Materials and methods A cross-sectional study was done on 120 nasopharyngeal/oropharyngeal swabs received in VRDL for RT-PCR testing. Based on the results of automated RNA extraction (Genetix, HT 96 Purifier) and RT-PCR (Trivitron PCR Kit) detecting E gene (screening) and ORF gene (confirmatory), the division into Group- I (Ct 15-22), Group- II (Ct 23-29), Group-III (Ct 30-36) and Group-IV (Ct >36) was done. Manual RNA extraction was done using magnetic beads (Lab system, Trivitron). Statistical analysis Data were analyzed by SPSS 19.0 version software. Ct values obtained in the two methods were compared by paired t-test, GroupWise. Z test was used to compare the other parameters. Results The difference in Ct values for target genes was statistically significant (p<0.05) in Group-I to III; however, no variation in result interpretation. The difference in time, manpower, and cost were statistically significant (p<0.05). The manual method required twice more manpower; 40 minutes more time & automated method cost 3.5 times more for consumables. Conclusion The study showed that RNA yield was better with automated extraction in comparison to manual extraction. The samples extracted by the automated method detected the virus at a lower Ct range by PCR than the manual method. Automated method processed samples in less time and with less manpower. Considering the cost factor, manual extraction can be preferred in resource-limited settings as there was no difference in the results of the test. The manual method requires more hands-on time with potential chances of cross-contamination and technical errors.

In vitro effect of fosfomycin on multi-drug resistant gram-negative bacteria causing urinary tract infections.

BACKGROUND: Rising rates of resistance to antimicrobial drugs among Enterobacteriaceae limit the choice of therapeutic agents to treat urinary tract infections. In this context we assessed the in-vitro effect of fosfomycin against extended-spectrum beta-lactamases, AmpC beta-lactamases and carbapenemase-producing strains of Escherichia coli, Klebsiella pneumoniae, Enterobacter spp, and P seudomonas aeruginosa isolated from the patients with urinary tract infection (UTI) and also studied the effect of fosfomycin on their biofilm formation. MATERIALS AND METHODS: A total of 326 multidrug-resistant (MDR) isolates comprising of Escherichia coli, Klebsiella pneumoniae, Enterobacter spp, and P seudomonas aeruginosa from the urine samples of the patients with a diagnosis of UTI were included in the study. MIC 50 and MIC 90 were detected by agar dilution method and the capacity to form biofilm in the presence of fosfomycin by these MDR isolates was assessed by the tissue culture plate method. RESULTS: The MIC50 for meropenem (0.5 µgm/mL) and nitrofurantoin (32 µgm/mL) was within the susceptible range only for E. coli. Fosfomycin was the only antibiotic that inhibited 100% E.coli, 70% Klebsiella spp, and 50% Pseudomonas spp and 40% Enterobacter spp which included the extended-spectrum beta-lactamases producers. It showed a similar effect on carbapenemase producers and AmpC producers. Fosfomycin disrupted biofilm in 67% (n=141) E.coli, 74% (n=50) Klebsiella spp, 88% (n=27) Pseudomonas spp and 36% (n=23) Enterobacter spp at 24 hrs of incubation with a concentration of 2 fold dilution lower than that of the MIC. CONCLUSION: Fosfomycin showed a good inhibitory effect on the biofilms produced by the MDR organisms studied here.

A study of virulence and antimicrobial resistance pattern in diarrhoeagenic Escherichia coli isolated from diarrhoeal stool specimens from children and adults in a tertiary hospital, Puducherry, India.

BACKGROUND: Emergence of atypical enteropathogenic Escherichia coli (EPEC) and hybrid E. coli (harboring genes of more than one DEC pathotypes) strains have complicated the issue of growing antibiotic resistance in diarrhoeagenic Escherichia coli (DEC). This ongoing evolution occurs in nature predominantly via horizontal gene transfers involving the mobile genetic elements like integrons notably class 1 integron. This study was undertaken to determine the virulence pattern and antibiotic resistance among the circulating DEC strains in a tertiary care center in south of India. METHODS: Diarrhoeal stool specimens were obtained from 120 children (< 5 years) and 100 adults (> 18 years), subjected to culture and isolation of diarrhoeal pathogens. Conventional PCR was performed to detect 10 virulence and 27 antimicrobial resistance (AMR) genes among the E. coli isolated. RESULTS: DEC infection was observed in 45 (37.5%) children and 18 (18%) adults, among which [18 (40%), 10 (10%)] atypical EPEC was most commonly detected followed by [6 (13.3%), 4 (4%)] ETEC, [5 (11.1%) 2 (2%)] EAEC, [(3 (6.6%), 0 (0%)] EIEC, [3 (6.6%), 0 (0%] typical EPEC, and [4 (8.8%), 1 (1%)] STEC, and no NTEC and CDEC was detected. DEC co-infection in 3 (6.6%) children, and 1(1%) adult and sole hybrid DEC infection in 3 (6.6%) children was detected. The distribution of sulphonamide resistance genes (sulI, sulII, and sulIII were 83.3 and 21%, 60.41 and 42.1%, and 12.5 and 26.3%, respectively) and class 1 integron (int1) genes (41.6 and 26.31%) was higher in DEC strains isolated from children and adults, respectively. Other AMR genes detected were qnrS, qnrB, aac(6')Ib-cr, dhfr1, aadB, aac(3)-IV, tetA, tetB, tetD, catI, blaCTX, blaSHV, and blaTEM. None harbored qnrA, qnrC, qepA, tetE, tetC, tetY, ermA, mcr1, int2, and int3 genes. CONCLUSIONS: Atypical EPEC was a primary etiological agent of diarrhea in children and adults among the DEC pathotypes. Detection of high numbers of AMR genes and class 1 integron genes indicate the importance of mobile genetic elements in spreading of multidrug resistance genes among these strains.

Evaluation of the utility of conventional polymerase chain reaction for detection and species differentiation in human hookworm infections.

BACKGROUND: Human hookworm infection is caused mainly by Necator americanus and Ancylostoma duodenale. Among the zoonotic hookworm species, only Ancylostoma ceylanicum causes potent human infections where dogs and cats act as reservoir of infection. Hence, species differentiation is imperative because the eradication of both anthroponotic and zoonotic hookworm depends on the concurrent human and animal health programs, hygienic practices, and mass drug administration for humans and dogs. OBJECTIVE: This study was performed to evaluate the utility of polymerase chain reaction (PCR) for detection of hookworm infections. MATERIALS AND METHODS: A total of 209 stool samples were collected and subjected to stool microscopy, Kato-Katz method to identify the intensity of the infection, coproculture for L3 larval identification and species differentiation and semi-nested PCR with sequencing. RESULTS: The prevalence of hookworm was estimated as 7.6%. Highest hookworm prevalence was seen in 20-30 years of age group. Majority of the infections were mild intensity infections. Sensitivity of stool microscopy was found to be 81.2% and the specificity was 100%. Sensitivity of Kato-Katz method was 87.5% and specificity was 100%. True positivity by agar plate culture was 83.3% and false positivity rate was 16.6%. CONCLUSION: Stool microscopy is the major mode of detection, but it has a higher false negative rate. Coproculture is time-consuming and needs the expertise to differentiate the species. On the other hand, PCR is known to be a sensitive, specific, and a reliable investigative tool which can help in diagnosis as well as in species differentiation.

The Emergence of Quinolone Resistant Shigella sonnei, Pondicherry, India.

Ciprofloxacin resistant Shigella sonnei across the globe have been increasing alarmingly. In order to understand the emergence of S.sonnei with respect to ciprofloxacin resistance in our patient population, the following study was carried out. Of the 184 Shigella sp. Isolated from 2012 to 2015, 34 S.sonnei which were confirmed by standard methods and subjected to antimicrobial susceptibility testing were selected. The minimum inhibitory concentrations (MICs) of 16/34 quinolone resistant isolates tested ranged from 4micrograms/ml to 16micrograms/ml for ciprofloxacin, from 16 micrograms/ml to 64 micrograms/ml for ofloxacin and from 16micrograms/ml to 64micrograms/ml for levofloxacin. Sequence determination of the quinolone resistance determining regions of gyrA, gyrB, parC, and parE genes showed mutations in GyrA at Gln69/Trp, Phe71/Ser, Ser72/Pro, Met75/Leu, Ser90/Cys, Met94/Leu, His106/Pro, Asn161/His, Thr163/Ala and in ParC at Ala64/Asp. Among the plasmid-mediated quinolone resistance (PMQRs) targets investigated,qnrB was the most (93.7%) prevalent followed by qnrC (18.7%). None hadqnrA, qnrS and qepA. Two (0.1%) of the isolates harboured theaac(6')-lb gene. Drug accumulation assay detected the presence of efflux pump activity in 9/15 (60%) among ciprofloxacin resistant isolates. All isolates harboured the ipaH gene followed by ial (17.6%), sen (11.7%), set1A&set1B (5.8%) genes. None had stx1 element. PCR for Enterobacterial repetitive intergenic consensus (ERIC) sequences resulted in 4 unique clusters, of which Type III was the most (44%) dominant but there was no correlation between the ERIC types and the antibiotic resistance pattern or the virulence profile. A documented increase in S.sonnei harbouring the qnrgenes and some unusual genes like set1Aand indicate an ongoing process of horizontal gene transfer. The accumulation of novel mutations in GyrA and ParC in the presence of efflux pump and PMQR genes contributed to the raised MIC to quinolones. These findings are crucial in our understanding of quinolone resistance in these isolates.