Pradyot Prakash

Evaluation of Nested PCR in Diagnosis of Typhoid Fever

ABSTRACT In this study, nested PCR using H1-d primers, which is specific for Salmonella enterica serovar Typhi, was compared to blood culture and the single-tube Widal test. Results indicate that nested PCR can be used as a gold standard to determine the cutoff titer of the Widal test for diagnosis of typhoid fever.

Rapid detection of dermatophytes from skin and hair

BackgroundDermatophytes 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.FindingsConventional 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).ConclusionResults 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

ABSTRACT 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.

INCREASED PREVALENCE OF EXTENDED SPECTRUM β 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.

Role of spoligotyping and IS6110-RFLP in assessing genetic diversity of Mycobacterium tuberculosis in India.

In the present study, genetic diversity analysis of Mycobacterium tuberculosis isolated from patients attending a tertiary care hospital, North India, has been attempted. Eighty three isolates of M. tuberculosis were subjected to DNA fingerprinting using spoligotyping and IS6110-RFLP techniques. Spoligotype patterns showed that central Asian (32.5%), ill defined T (13.2%) and Beijing (10.8%) families were predominant in ongoing transmission of the bacterium. Two STs; ST26 (CAS_Delhi) and ST1 (Beijing) represented 36.1% of the total M. tuberculosis population in eastern Uttar Pradesh, North India. IS6110 RFLP analysis showed that isolates having low and zero copy number of the IS element were 15.6% and 19.2%, respectively. Out of the 47 isolates clustered by spoligotyping, 40 could be further differentiated as unique strains by IS6110-RFLP. Therefore, this study recommends that both the techniques be used simultaneously for DNA fingerprinting of M. tuberculosis in India.

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 beta-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.

Role of β-lactamase inhibitors in enterobacterial isolates producing extended-spectrum β-lactamases

OBJECTIVES To determine the in vitro activity of beta-lactamase inhibitors (clavulanic acid and sulbactam) in combination with third-generation cephalosporins and monobactam against extended-spectrum beta-lactamase (ESBL)-producing members of the Enterobacteriaceae family. METHODS A total of 361 ESBL-producing enterobacterial isolates obtained from patients of a university hospital were screened for the status of co-production of AmpC beta-lactamase. These strains were further subjected to an MIC study using third-generation cephalosporins and monobactam, and reductions were observed after combining with beta-lactamase inhibitors at a fixed concentration of 4 mg/L. RESULTS Most of the isolates showed 8-fold reduction with sulbactam when combined with ceftriaxone, cefpodoxime and cefotaxime but not with ceftazidime and aztreonam, whereas clavulanic acid showed the same result with all the cephalosporins tested. Further, both the inhibitors showed greater reduced MIC when combined with aztreonam. CONCLUSIONS As the ability of clavulanic acid to induce AmpC production may interfere with ESBL detection, sulbactam is likely to be preferred over clavulanic acid after standardization of an appropriate concentration for ESBL detection in the scenario of increased prevalence of AmpC producers. Greater in vitro activity of these inhibitors when combined with aztreonam further indicates the need of studies to evaluate these combination antimicrobials in clinical settings as they can play a significant role for clinicians as viable alternatives to treat infections caused by such organisms.

Rapid Genotypic Detection of rpoB and katG Gene Mutations in Mycobacterium tuberculosis Clinical Isolates from Northern India as Determined by MAS-PCR

There is a growing need to develop rapid laboratory research methods to counter the menace of drug resistant tuberculosis (MDR‐TB) cases worldwide especially in developing countries. The present study was undertaken to investigate the type and frequency of rpoB and katG mutations in rifampicin (RIF) and isoniazid (INH) resistant strains respectively of Mycobacterium tuberculosis (MTB) circulating in Northern India and to explore the utility of multiplex‐allele‐specific (MAS)‐PCR assay for detection of drug‐resistant MTB isolates in low resource set up. J. Clin. Lab. Anal. 27:31–37, 2013.

Possible Role of Curcumin as an Efflux Pump Inhibitor in Multi Drug Resistant Clinical Isolates of Pseudomonas aeruginosa.

INTRODUCTION Multidrug resistant non-fermenters are continuously increasing in hospital and ICU settings. One of the mechanisms of developing drug resistance is possession of efflux pump through which bacteria extrude antimicrobial agents and other toxic substances. If these efflux channels are blocked or inhibited, increased drug concentration can be achieved in a bacterial cell with optimal drug dose. Present study was aimed to investigate role of curcumin as efflux pump inhibitor (EPI) and to compare its activity with a known EPI like phe-arg-beta-naphthylamide (PAβN). MATERIALS AND METHODS A total of 170 clinical isolates of Pseudomonas aeruginosa were taken, antimicrobial susceptibility was performed by disc diffusion test and minimum inhibitory concentration (MIC) against selected drugs before and after adding known synthetic EPI, PAβN (20mg/L). Out of these, 30 multidrug resistant strains were taken and MIC was performed with curcumin (50mg/L) with and without selected drugs. Results : Significant reduction in MIC was observed after adding curcumin (50mg/L) with selected antimicrobial agents in 9/30 (30%) of multi drug resistant (MDR) isolates of Pseudomonas aeruginosa, while no change in MIC was observed when curcumin (50mg/L) was used alone, indicating its efflux pump inhibitor activity. CONCLUSION This study suggests role of efflux pump in development of drug resistance which can be overcome by use of an efflux pump inhibitor, with more emphasis on compound like curcumin which will have less or no adverse effects if used in vivo.

Cutaneous zygomycosis: a possible postoperative complication in immunocompetent individuals.

Fungi in the class of zygomycetes usually produce serious infections in diabetics and immunocompromised hosts. Cutaneous zygomycosis is a less common form, with an unpredictable extent of anatomical involvement and clinical course. Here, we report two cases of primary cutaneous zygomycosis as postoperative complications in otherwise healthy females. Zygomycosis was suspected and specimens from the surgical debridement were examined by microbiological and histopathological studies for confirming the clinical diagnosis. Rapid diagnosis, liposomal amphotericin B, and proper debridement of affected tissue are necessary to avoid a fatal outcome.