Rapid detection of drug-resistant Mycobacterium tuberculosis directly from clinical specimens using allele-specific polymerase chain reaction assay

Background & objectives: Rapid detection of drug resistance in Mycobacterium tuberculosis (MTB) is essential for the efficient control of tuberculosis. Hence, in this study a nested-allele-specific (NAS) PCR, nested multiple allele-specific PCR (NMAS-PCR) and multiple allele-specific (MAS) PCR assays were evaluated that enabled detection of the most common mutations responsible for isoniazid (INH) and rifampicin (RIF) resistance in MTB isolates directly from clinical specimens. Methods: Six pairs of primers, mutated and wild type, were used for the six targets such as codon 516, 526 and 531 of rpoB, codon 315 of katG and C15-T substitution in the promoter region of mabA-inhA using allele-specific (AS) PCR assays (NAS-PCR, NMAS-PCR and MAS-PCR). The performance of AS PCR method was compared with phenotypic drug susceptibility testing (DST). Results: The usefulness of AS PCR assays was evaluated with 391 clinical specimens (251 Acid fast bacilli smear positive and MTB culture positive; 93 smear negative and MTB culture positive; 47 smear positive and MTB culture negative) and 344 MTB culture positive isolates. With culture-based phenotypic DST as a reference standard, the sensitivity and specificity of the NAS-PCR, NMAS-PCR and MAS-PCR assay for drug resistance-related genetic mutation detection were 98.6 and 97.8 per cent for INH, 97.5 and 97.9 per cent for RIF and 98.9 and 100 per cent for multidrug resistance (MDR). Interpretation & conclusions: The performance of AS PCR assays showed that those could be less expensive and technically executable methods for rapid detection of MDR-TB directly from clinical specimens.

Detection of rifampicin resistance in tuberculosis by molecular methods: A report from Eastern Uttar Pradesh, India.

Diagnosis of drug resistance tuberculosis (TB) by the gold standard method is labour intensive and time consuming. Hence, there is an urgent need for introduction of rapid diagnostic techniques. Line probe assay (LPA) and cartridge‑based nucleic acid amplification test (CBNAAT) have been introduced in India under Revised National Tuberculosis Control Program. Spot and morning sputum samples of previously treated patients by anti‑TB drugs were subjected to LPA or CBNAAT. Total 682/1253 (54.4%) were diagnosed as rifampicin‑resistant. The patients could be diagnosed early by molecular methods and put on second line treatment.

Mutations at embB306 codon and their association with multidrug resistant M. tuberculosis clinical isolates.

Purpose: The presence of embB306 mutation in ethambutol (EMB)‑susceptible (EMBs) clinical isolates questions the significance of these mutations in conferring resistance to EMB. The present study was carried out to determine the occurrence of embB306 mutation in EMB‑resistant (EMBr) and EMBs strains of M. tuberculosis. One hundred and four multidrug‑resistant tuberculosis (MDR‑TB) strains were also included to establish the relevance of excessive use of rifampicin (RIF) and isoniazid (INH) in occurrence of embB306 mutations in EMBs M. tuberculosis isolates. Materials and Methods: Deoxyribonucleic acid (DNA) from M. tuberculosis clinical strains was isolated by cetyltrimethylammonium bromide (CTAB) method. Phenotypic and genotypic drug susceptibility testing (DST) was performed on 354 M. tuberculosis isolates by using standard proportion method and multiplex‑allele‑specific polymerase chain reaction assay, respectively. Results: The overall frequency of embB306 mutations in EMBr isolates was found to be five times higher than its occurrence in EMB‑susceptible isolates (50% vs 10%). Further, the association between embB306 mutation and EMB‑resistance was observed to be statistically significant (P = 0.000). Conclusion: The embB306 is not only the main causative mutation of EMB resistance, but is a sensitive applicant marker for EMB‑resistance study.

Observation on integron carriage among clinical isolates of Klebsiella pneumoniae producing extended-spectrum β-lactamases.

Purpose: Klebsiella pneumoniae is considered an important pathogen causing nosocomial and community-acquired infections and is often associated with the production of extended-spectrum β-lactamases (ESBL) belonging to SHV and CTX-M families, which are frequently described as a part of complex integrons, facilitate their horizontal transfer to other related as well as unrelated microbes. The present study was undertaken to investigate the occurrence and characterization of integrons among K pneumoniae isolates producing ESBL in a tertiary referral hospital. Materials and Methods: A total of 136 clinical isolates of K pneumoniae were investigated for the presence of ESBL. Their ESBL genes were characterized by multiplex polymerase chain reaction (PCR). Integrase gene PCR was performed to detect the presence of integron. The isolates were further typed by random amplification of polymorphic DNA (RAPD). Result: Out of 136 K pneumoniae isolates, 63 (46%) were confirmed to be ESBL producers. SHV (68%) and CTX-M (67%) ESBL genes were the most common in our study. Of the 63 ESBL-positive isolates, 58 (92%) strains carried integrons; 52 strains (82%) carried only class 1 integron, whereas 6 (9%) isolates harboured both class 2 integrons and the class 1 gene. However, in ESBL negatives, only 29 (40%) strains were positive for class 1 integron and none for class 2 integron. Conclusion: The presence of class 2 integron amongst ESBL-producing K pneumoniae is being described for the first time in this part of the world. The findings of this study strongly suggest that integrons have a role in the dissemination of ESBL-mediated resistance among the nosocomial isolates of K pneumonia.

Increased prevalence of extended spectrum beta lactamase producers in neonatal septicaemic cases at a tertiary referral hospital.

Emergence of extended spectrum beta lactamases (ESBLs) producing strains of gram negative bacteria, as one of the leading cause of septicaemia often complicates the clinical and therapeutic outcome. The present study was undertaken to investigate the prevalence of ESBLs in bacteria isolated from neonatal septicaemic cases along with their antimicrobial sensitivity pattern. Blood samples were collected from 243 suspected cases of neonatal septicaemia. Apart from susceptibility testing, all the gram negative isolates were subjected to phenotypic tests for ESBL production. Amongst the positive test samples (n = 115), 84 were gram negative rods. ESBL was detected in 26 (32%) isolates. Results indicate that routine ESBL detection should be made imperative and empirical use of third generation cephalosporins must be discouraged.

Observations on carbapenem resistance by minimum inhibitory concentration in nosocomial isolates of Acinetobacter species: an experience at a tertiary care hospital in North India.

Acinetobacter species are emerging as an important nosocomial pathogen. Multidrug-resistant Acinetobacter spp. has limited the option for effective treatment. Although carbapenems are effective for the treatment of such infections, resistance to this drug has recently been reported. This study was undertaken to assess resistance to carbapenem in clinical isolates of Acinetobacter spp. from hospitalized patients by both disc-diffusion and minimum inhibitory concentration (MIC) methods. All clinical samples from suspected cases of nosocomial infections were processed, and 265 isolates were identified as Acinetobacter species. These isolates were tested for antibiotic resistance by the disc-diffusion method with 14 antimicrobials, including meropenem and imipenem. Thereafter, all Acinetobacter species were subjected to MIC for meropenem. More than 80% resistance to second- and third-generation cephalosporins, aminoglycosides, and quinolones was recorded. Thirty percent of the strains were resistant to cefoperazone/sulbactam. Resistance to meropenem was observed in 6.4% of Acinetobacter spp. while 8.3% of the isolates showed intermediate resistance detected by MIC. All carbapenem-resistant/intermediate strains were also resistant to other (>10) antibiotics tested by the disc-diffusion method. The rising trend of resistance to carbapenem poses an alarming threat to the treatment for such infections. Regular monitoring, judicious prescription, and early detection of resistance to carbapenem are necessary to check further dissemination of drug resistance in Acinetobacter spp.

Antimicrobial resistance profile of bacterial isolates from Intensive Care Unit: changing trends.

The incidence of antimicrobial resistance has increased over the years resulting in limitation of therapeutic options. Strategies such as appropriate infection control measures and surveillance of resistance pattern are necessary to address the problem of resistance. Knowledge of the pattern of resistance in Intensive Care Units (ICUs) can help to determine antibiotic prescribing policy. A retrospective study has been carried out to determine the bacterial spectrum and the antibiotic resistance pattern of clinical isolates collected from patients admitted to the ICU. The data was compared with a similar study conducted during 1996-97. Amongst the gram-positive organisms Staphylococcus aureus (23%) was the predominant isolate, while Pseudomonas (23%), Acinetobacter (20.8%), Citrobacter (11.7%) accounted for the majority of the gram-negative organisms. Both gram positive and gram-negative organisms exhibited high resistance to most antimicrobial agents used for testing susceptibility. The frequency of resistance has markedly increased as compared to the previous study.

Prevalence of methicillin resistant staphylococcus aureus in a tertiary referral hospital in eastern Uttar Pradesh.

We report the prevalence of methicillin resistant Staphylococcus aureus (MRSA) infections and their antibiotic susceptibility pattern in our hospital located in eastern Uttar Pradesh. Out of total 549 strains of Staphylococcus aureus isolated from different clinical specimens 301 (54.85%) were found to be methicillin resistant. More than 80% of MRSA were found to be resistant to penicillin, cotrimoxazole, ciprofloxacin, gentamicin, erythromycin, tetracycline, 60.5% to amikacin and 47.5% to netilmicin. However, no strains were resistant to vancomycin. Many MRSA strains (32.0%) were multi-drug resistant. To reduce the prevalence of MRSA, the regular surveillance of hospital associated infection, monitoring of antibiotic sensitivity pattern and formulation of definite antibiotic policy may be helpful.

Antibiotic sensitivity pattern of the bacteria isolated from nosocomial infections in ICU.

The incidence of nosocomial infections in ICU is 4-5 times greater than in general ward. Critically ill patients are always at higher risk of developing nosocomial infections with resistant strains. This study is an attempt to know the antibiotic sensitivity pattern of the common isolates in ICU. Samples comprising urine, blood, endotracheal secretions and throat swabs were collected from 102 ICU patients of them, 56 patients showed evidence of nosocomial infection (54.9%), from whom 120 different organisms were isolated. Antibiotic sensitivity test was done according to Kirby Bauer method. Klebsiella pneumoniae were the most prevalent isolates from respiratory tract infections followed by Proteus spp, Escherichia coli, Staphylococci spp. and Acinetobacter spp. The gram negative enteric bacilli were uniformly resistant to betalactam antibiotics as well as betalactam-betalactamase inhibitors. Resistance to Ciprofloxacin and Ceftriaxone ranged from 50-100% and 25-83.3% respectively. Staphylococci were 100% resistant to penicillin and tetracycline, 80% to cotrimoxazole, 60% to erythromycin and gentamicin and 40% to amikacin. Acinetobacter spp. were highly resistant to most of the antibacterial agents except gentamicin while Pseudomonas spp. showed 75% resistance to it. The increased prevalence of resistant organisms in ICU probably reflects lack of proper antibiotic policy resulting in prolonged and indiscriminate use of antimicrobial agent.

Enterotoxigenic Campylobacter jejuni & C. coli in the etiology of diarrhoea in northern India.

Faecal specimens from subjects with (320) and without (450) diarrhoea were screened for Campylobacter jejuni and C. coli. C. jejuni and C. coli were detected in 5 per cent of subjects with diarrhoea and 0.7 per cent of those without diarrhoea and the difference was significant (P less than or equal to 0.01). The isolation rate was much higher in under five diarrhoeal children (8.3%), in comparison to the older group (3.0%). Eleven diarrhoeal isolates of C. jejuni and C. coli were tested in rat ileal loops for enterotoxigenicity. All the strains caused fluid accumulation in the loop model. However, 6 strains required up to 3 consecutive passage(s) to do so. Therefore, rat ileal loops were found to be sensitive and reproducible animal model for the demonstration of enterotoxin produced by C. jejuni and C. coli. The culture filtrates of 3 strains of C. jejuni and C. coli were subjected to neutralisation with cholera antitoxin. The fluid accumulation was completely neutralised up to 1 in 80 dilution showing immunobiological relationship between C. jejuni and C. coli enterotoxin and cholera toxin.