Detection of the classical G2576U mutation in linezolid resistant Staphylococcus aureus along with isolation of linezolid resistant Enterococcus faecium from a patient on short-term linezolid therapy: First report from India.

Purpose: Linezolid is an effective drug against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). We describe the emergence of linezolid resistance in MRSA and VRE from India. Material and Methods: One MRSA and two VRE strains were isolated from a patient on linezolid therapy of one week duration. All three isolates were resistant to linezolid with minimal inhibitory concentrations (MIC) ≥4 mg/L. The 746-bp region fl anking the possible G2576U mutation on the corresponding DNA from the 23S rRNA was amplifi ed by polymerase chain reaction (PCR) and amplicons were sequenced for all the three isolates. Conjugation experiments using the linezolid resistant MRSA (LRMRSA) and linezolid resistant VRE (LRVRE) isolates as donors and wild strains of corresponding genera as recipients were performed. Results: The MRSA isolate had the classical G2576U mutation. High quality value scores in the sequencing software validated the mutation. Conjugation studies did not indicate presence of transferable resistance for linezolid. Sequencing did not indicate presence of any mutation in the two LRVRE isolates. Conclusions: This is the fi rst report from India citing resistance in Staphylococcus and Enterococcus against Linezolid.

Multiple carbapenem hydrolyzing genes in clinical isolates of Acinetobacter baumannii.

Purpose: Carbapenem resistance in Acinetobacter baumannii has become highly rampant, which has been ascribed to the presence of multiple carbapenemases. The objective of the present study was to prospectively investigate the presence of multiple carbapenemase encoding genes in clinical isolates of A. baumannii. Materials and Methods: A total of 30 imipenem resistant, consecutive non-repeat clinical isolates A. baumannii from a Tertiary Care Centre of Delhi were subjected to antimicrobial susceptibility testing (AST), screening for carbapenemase production by modified Hodge test (MHT) and determination of minimum inhibitory concentration for imipenem by E-Test® . These were subjected to Real time PCR for blaIMP-1 and 2 , blaVIM-1 and 2 , blaOXA23, 24, 51 and 58 using SYBR green-I. These were grouped together on the basis of their genotype as each isolate harboured multiple carbapenemases and correlated with their AST profile. Detection of the novel carbapenemase blaNDM-1 was performed by real time PCR using TaqMan probes on 14 isolates. Results: Colistin appeared to be the most effective drug in vitro, followed by tetracycline and beta lactam/beta lactamase inhibitor combinations. All, but one isolate were positive for the MHT. All 30 isolates were positive for blaOXA-51 like gene as well as blaIMP-1 and blaVIM-1 genes. blaOXA 24 and 58 were not detected in any of the isolates. blaIMP-2 , blaVIM-2 , blaOXA-23 were present in 15, 6 and 14 isolates respectively. Grouping based on the genotypic profile did not correlate with susceptibility pattern. Nine among the 14 isolates also harboured the novel blaNDM-1 gene. Conclusions: This is the first study from North India, which comprehensively detected the presence of multiple carbapenemases as well the blaNDM-1 gene. The presence of the novel gene blaNDM-1 indicated ability of A. baumannii to acquire new carbapenemase genes despite the existence of multiple carbapenemase genes. The present study confirmed the presence of multiple genetic mechanisms for carbapenemases production among the clinical isolates of A. baumannii in north India.

Zinc-dependent carbapenemases in clinical isolates of family Enterobacteriaceae.

Purpose: The emergence and spread of zinc-dependent carbapenem resistance has become a diagnostic challenge for clinical microbiologists. The objective of the present study was to screen zinc-dependent carbapenemase activity in clinical isolates of family Enterobacteriaceae. Materials and Methods: A total of 102 multidrug-resistant organisms (MDROs), non-repeat clinical isolates of family Enterobacteriaceae from two tertiary care centres in Delhi, were screened for carbapenemase production by a modified Hodge test (MHT) and additionally by a re-modified Hodge test, EDTA double disc synergy test, and combined disc test (or disc enhancement test) to determine zinc dependence of carbapenemases harbouring bacteria. Results: Of the total 102 clinical isolates (June through November 2010), 91 were from urine and 11 were from blood specimens. The isolates were obtained from patients visiting the outpatient department (18 isolates), admitted in non-ICU inpatient care units (74 isolates) and patients admitted in ICUs (4 isolates). MHT identified 92 (90.2%) isolates as carbapenemases producers. Among those found negative for MHT (n=10), metallo-beta-lactamases (MBLs) activity was demonstrated through the EDTA disc diffusion synergy test and the combined disc test in 8 and 9 isolates respectively. A total of 63 (61.7%) isolates demonstrated MBL activity despite in vitro sensitivity to Imipenem. Conclusions: The study demonstrated that supplementing the MHT with at least one of the screening methods increases the likelihood of picking up such isolates that may be missed by the MHT. The study also demonstrates the wide-spread presence of MBLs in Enterobacteriaceae members from patients visiting hospitals in east Delhi.

Zinc-dependent carbapenemases in clinical isolates of family Enterobacteriaceae.

Purpose: The emergence and spread of zinc-dependent carbapenem resistance has become a diagnostic challenge for clinical microbiologists. The objective of the present study was to screen zinc-dependent carbapenemase activity in clinical isolates of family Enterobacteriaceae. Materials and Methods: A total of 102 multidrug-resistant organisms (MDROs), non-repeat clinical isolates of family Enterobacteriaceae from two tertiary care centres in Delhi, were screened for carbapenemase production by a modified Hodge test (MHT) and additionally by a re-modified Hodge test, EDTA double disc synergy test, and combined disc test (or disc enhancement test) to determine zinc dependence of carbapenemases harbouring bacteria. Results: Of the total 102 clinical isolates (June through November 2010), 91 were from urine and 11 were from blood specimens. The isolates were obtained from patients visiting the outpatient department (18 isolates), admitted in non-ICU inpatient care units (74 isolates) and patients admitted in ICUs (4 isolates). MHT identified 92 (90.2%) isolates as carbapenemases producers. Among those found negative for MHT (n=10), metallo-beta-lactamases (MBLs) activity was demonstrated through the EDTA disc diffusion synergy test and the combined disc test in 8 and 9 isolates respectively. A total of 63 (61.7%) isolates demonstrated MBL activity despite in vitro sensitivity to Imipenem. Conclusions: The study demonstrated that supplementing the MHT with at least one of the screening methods increases the likelihood of picking up such isolates that may be missed by the MHT. The study also demonstrates the wide-spread presence of MBLs in Enterobacteriaceae members from patients visiting hospitals in east Delhi.

Development of TaqMan real-time polymerase chain reaction for the detection of the newly emerging form of carbapenem resistance gene in clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii.

Purpose: The newly emerging form of the so-called New Delhi Metallo-beta-lactamases (NDM-1) has been reported recently from patients worldwide and broadly thought as a potential source for the major global health problem. Thus, it is important to study the epidemiology of the so-called NDM-1 harbouring bacteria to prevent its further spread and to place effective control measures. The present study describes the use of the real-time polymerase chain reaction (PCR) assay for the detection of the bla NDM-1 gene using TaqMan probes among clinical isolates. Materials and Methods: Clinical isolates of Escherichia coli (11 strains), Klebsiella pneumoniae (17 strains) and Acinetobacter baumannii (six strains) that were resistant to either of the carbapenems (meropenem or imipenem) were included in the study. The presence of carbapenemases in such strains was confirmed using the modified Hodge test. A real-time PCR assay was optimized for the detection of NDM-1 using a cloned synthetic gene fragment followed by testing of the clinical isolates. The findings were further confirmed using PCR and gene sequencing. Results: TaqMan probe assay displayed a good detection limit with analytical sensitivity of the assay up to 10 copies of bla NDM-1 gene per reaction. The isolates of E. coli and K. pneumoniae revealed narrow range crossing point values (Cp values) between (12-17) cycles (mean Cp value 14), indicating number of bla NDM-1 gene copies of 106-108. The wider range of Cp values (15-34) cycles with a higher mean Cp value (23.6) was observed in A. baumannii with number of bla NDM-1 gene copies of 103-108. Conclusions: The study demonstrates that real-time PCR assay based on TaqMan chemistry is a useful technique for the detection of bla NDM-1 harbouring clinical isolates of E. coli, K. pneumoniae and A. baumannii. The assay has great precision in measuring the number of bla NDM-1 gene copies per specimen of DNA.

Evaluation of nitrate reductase assay for direct detection of drug resistance in Mycobacterium tuberculosis: Rapid and inexpensive method for low-resource settings.

The aim of this study was to evaluate a nitrate reductase assay (NRA) for the direct detection of multidrug resistance (MDR) in Mycobacterium tuberculosis from 100 smear-positive sputum samples. The NRA results were compared with the reference proportion method for 100 sputum specimens for which comparable results were available. NRA results were obtained at day 7 for 61 specimens, results for 26 specimens were obtained at day 10, and the results for 13 specimens were obtained at day 14. Thus, 87% of NRA results were obtained in 10 days. NRA is a rapid, accurate, and cost-effective method for the detection of MDR in M. tuberculosis isolates as compared to the proportion method, which is time consuming. Therefore, NRA constitutes a useful tool for detection of tuberculosis drug resistance in low-resource countries with limited laboratory facilities due to its low-cost, ease of performance and lack of requirement of sophisticated equipment.

Higher Bifurcation of Brachial Artery with Superficial Course of Radial Artery in Forearm.

An unusual case of bilaterally symmetrical higher bifurcation of brachial artery into radial and ulnar arteries with superficial course of radial artery in right forearm is reported. Accurate information regarding these variations is important during vascular and re-constructive surgery and also in evaluation of angiographic images.

Seasonal variation in prevalence of hypertension: Implications for interpretation.

Objectives: To study seasonal variation in prevalence of hypertension. Materials and Methods: The study was carried out in the year 2006, in Gokulpuri, an urban slum located in eastern part of Delhi. 275 females 18-40 years of age were examined in summer. Blood pressure was measured in two seasons, summer and winter. Nutritional status of each individual was assessed by BMI. Results: The prevalence of hypertension based on SBP was 12.72% in summer which increased to 22.22% in winter. The prevalence of hypertension, using DBP criteria increased to more than double (summer vs. winter, 11.27% vs. 26.59%, P< 0.001). Overall prevalence of hypertension (SBP≥140 or DBP≥90 mm of Hg) was 1.9 times during winter compared to summer (P<0.001). Greater increase in prevalence of hypertension during winter among older females and underweight as well as normal females was observed. Conclusion: Significant increase in prevalence of hypertension during winter compared to summer indicates need for considering this factor while comparing prevalence reported in different studies as well as interpreting the surveillance data based on repeat surveys.

Hygeine Status and Food Handling Practices among Street Food Vendors in East Delhi An Observational Study.

Practices regarding personal and food hygiene of 100 street food vendors in East Delhi were studied by dose observations. A general physical examination of all vendors and bacteriological examination of nail scrapings of every alternate street food vendor was done. Majority of vendors (77%) were less than 40 years of age and 58% were illiterate. Only 13% of the vendors were found to wash fruits and vegetables before use. Most of the vendors used dirty utensils for storage of food (82%), 73% were storing food in uncovered utensils and 78% did not wash utensils before reuse. With regard to personal hygiene, majority (85%) did not wash hands before preparation, 93% did not follow this practice before serving. Most vending sites (61%) were observed to have dust and 49% had files in the environment. Majority of vendors had unkempt nails (87%), 22% had boils and 9% open wounds on hands. Microbiological examination of nail scrapings revealed K. pneumoniae and E. coli as the commonest pathogens isolated. There is II felt need for generating awareness regarding personal and food hygiene among the vendors, and a system for regular health examination of the vendors.

Antimicrobial resistance in invasive and colonising Streptococcus pneumoniae in North India.

The present study was done to detect the antibiotic resistance in S. pneumoniae. One hundred twenty S. pneumoniae isolates from clinical specimens and 50 from nasopharyngeal sites were subjected to antimicrobial susceptibility testing by Kirby Bauer disk diffusion method and minimum inhibitory concentration (MIC) determination for penicillin and cefotaxime non-susceptible isolates. A total of 22 isolates (18.3%) from clinical sites and eight (16%) from nasopharyngeal sites showed decreased susceptibility to penicillin by oxacillin disk diffusion test. MICs of 26 of these resistant strains ranged from 0.12-1 microg/mL (intermediate resistance) by broth dilution and E test. Only four isolates, two from sputum and two from nasopharyngeal swabs, showed MIC of 2 microg/mL (complete resistance). However, MIC of two cefotaxime resistant isolates (by disk diffusion) was in the susceptible range (0.5 microg/mL). Highest antimicrobial resistance was seen to cotrimoxazole (55.2%) and tetracycline (61.2%). Antimicrobial resistance to cotrimoxazole and tetracycline was much more in clinical isolates than colonizing isolates. Multi-drug resistant phenotype was detected in 76.9% (20 of 26) of isolates that were intermediately sensitive to penicillin and 50% (2 of 4) of penicillin resistant isolates (co-resistant to tetracycline and cotrimoxazole). Routine screening for antibiotic susceptibility is recommended for clinical isolates of pneumococci. Strains with reduced susceptibility to penicillin should be subjected to MIC determination to detect relative resistance or true resistance as such strains are associated with increased virulence.The choice of antibiotics should be guided by the prevalence of local resistance patterns of pneumococci.

Ataxia and deafness in a young male: an unusual aetiology.

We report here a case of 18 year old male with tremors of hands, deafness, tendency to fall while walking, drowsiness and double vision of total duration 1(1/2) years. He had internuclear ophthalmoplegia, broken saccades, hypertonia and hyperreflexia of all four limbs, intention tremors, signs of gait and limb ataxia. Pupillary reactions and fundus examination were normal and signs of meningeal irritation or sensory neurological deficit were absent. MRI head and cervical spine with gadolinium enhancement revealed demyelination as evident from multiple oblong foci isointense on T1-weighted images and hyperintense on T2-weighted and fluid attenuated inversion recovery sequences in corpus callosum, sub-cortical white matter, right thalamus, pons and periaqueductal region of midbrain. Ill-defined linear hyperintense signals were observed in cervical spinal cord. No skeletal abnormality was noted in the skull or cervical spine. Oligoclonal bands were present in the cerebrospinal fluid. Brainstem auditory evoked potentials were abnormal, although visual evoked potentials were in normal range. A diagnosis of primary progressive multiple sclerosis (PPMS) was made fulfilling the revised criteria as laid down. In view of its presentation, it is a unique case of PPMS from India.

Visceral leishmaniasis masquerading as chronic liver disease.

We report a patient with fever, progressive jaundice and abdominal distension, having marked pallor, icterus, ascites and hepatosplenomegaly. Investigations revealed pancytopenia and deranged liver functions. Doppler study revealed portal hypertension and endoscopy showed grade II oesophageal varices. Liver biopsy suggested leishmanial hepatitis and bone marrow demonstrated multiple LD bodies. Diagnosis of "visceral leishmaniasis with leishmanial hepatitis with portal hypertension" was made. The case is being reported because of its rarity apart from it being an unusual presentation of kala-azar.

Simple and economical method for speciation and resistotyping of clinically significant coagulase negative staphylococci.

An attempt was made to speciate 102 clinically significant isolates of coagulase negative staphylococci (CoNS) by a practical scheme adapted from various references. This scheme utilizes slide and tube coagulase test, urease test ornithine decarboxylase, novobiocin susceptibility and aerobic acid from mannose for assigning species group. Inclusion of one or two additional tests in a species group could identify the isolates to species level. Ninety eight (97%) isolates were conveniently identified as S. epidermidis (41%), S. saprophyticus (16.6%), S. haemolyticus (14.7%), S. hominis (14.7%), S. lugdunensis (4.9%), S. schleiferi (1.9%) and S. capitis (1.9%). Only four isolates were not identified to the species level, two of which were probably S. capitis subspecies ureolyticus / S. warneri / S. simulans . Antibiotic susceptibility testing showed maximum resistance to ampicillin (89%) followed by cefotaxime (59%) with no resistance to vancomycin. The increasing recognition of pathogenic potential of CoNS and emergence of drug resistance amongst them denotes the need to adopt simple laboratory procedures to identify and understand the diversity of staphylococci isolated from clinical material.

Molecular epidemiology of clinical isolates of ampc producing Klebsiella pneumoniae.

PURPOSE: AmpC producing K. pneumoniae have been increasingly reported from India but epidemiological studies are lacking. In the present study, molecular epidemiology of extended-spectrum AmpC beta-lactamases (ESACs) producing clinical isolates of K. pneumoniae prevalent in our hospital was studied. METHODS: Fifty-one non-repeat, consecutive, clinical isolates of K. pneumoniae producing AmpC enzymes, were subjected to whole cell protein profile analysis (SDS-PAGE) and ribotyping. The antimicrobial susceptibility was determined using standard disk diffusion technique. The isolates showing decreased susceptibility to cefoxitin (< 18 mm) or cefotetan (< 16 mm) were subjected to modified three- dimensional test for detection of AmpC enzyme. RESULTS: Six different types of protein profiles were observed. Ribotyping could further discriminate between the strains that were clustered by protein fingerprinting. Twelve different ribo-patterns were identified. Ribotyping was found to have a better Discriminatory Index (0.98) than that of SDS-PAGE (0.78). Of the 26 isolates that showed decreased susceptibility to cefoxitin and/or cefotetan 13 isolates were found to harbour AmpC enzyme. CONCLUSIONS: The study demonstrated the usefulness of SDS-PAGE whole cell protein profile analysis and ribotyping to identify the clonality of the ESACs isolates, the latter having a higher discriminatory power. The presence of ESACs isolates in the community as well as in hospital settings emphasizes the need for regular monitoring of antimicrobial resistance.

Liver abscess caused by Edwardsiella tarda biogroup 1 and identification of its epidemiological triad by ribotyping.

Two clinical isolates and an environmental isolate of Edwardsiella tarda biogroup 1 (ETB1), recovered from liver pus, the stool specimen and from the pond water of the village of the patient, diagnosed to have liver abscess, were found to be identical by protein fingerprinting and ribotyping. It can be construed that the pond water served as the source of infection. The epidemiological triad of the agent (ETB1), host (the patient) and environment (pond water) was thus established. This is the first report in which the triad for extraintestinal Edwardsiellosis caused by ETB1 has been identified. This also constitutes the first report of typing of ETB1 strains by SDS-PAGE and ribotyping.

Stem cells: A new paradigm.

Stem cell therapy is emerging as a potentially revolutionary new way to treat disease and injury, with wide-ranging medical benefits. It aims to repair damaged and diseased body-parts with healthy new cells provided by stem cell transplants. Disease and disorders with no therapies or at best, partially effective ones, are the lure of the pursuit of stem cell research. Recently a plethora of work has been done in this field in world around including India. However, Stem cell research presents many ethical and scientific questions as well as future challenges. Nevertheless, stem cell therapy, a prologue to an era of medical discovery of cell-based therapies that will one day restore function to those whose lives are now challenged every day, is still at the beginning of the road.

Diagnosis of neurocysticercosis by enzyme-linked immunosorbent assay.

Neurocysticercosis, a disease caused by larvae of T solium produces variable and non-specific symptoms. Computerised tomography, magnetic resonance imaging, immunological tests in the serum and cerebrospinal fluid are available options to diagnose the condition. Two hundred serum samples collected and stored frozen at -20 degrees C and were tested for cysticercosis by commercial Melotest cysticercosis kit. Diagnosis was confirmed by other investigations. After the confirmative diagnosis results of ELISA for cysticercosis were compared and the values of sensitivity, specificity, prevalence and precision were obtained along with positive and negative predictive values. Overall prevalence was found to be 10% in this study. Only 26.5% serologically positive cases had neurocysticercosis positively. The positive predictive value of the serological test was only 26.4% suggesting that the diagnosis of neurocysticercosis by ELISA is not reliable in an endemic area.

Phenotypic characteristics of clinical isolates of Klebsiella pneumoniae & evaluation of available phenotypic techniques for detection of extended spectrum beta-lactamases.

BACKGROUND & OBJECTIVES: Identifying organisms that harbour extended spectrum beta lactamases (ESBLs) is a major challenge for a diagnostic clinical microbiology laboratory. Wide variety of ESBLs produced and lack of a sensitive phenotypic method for their detection make the detection of ESBLs difficult and is responsible for under-recognition. The present study was undertaken to evaluate phenotypic characteristics, initial screening tests and established confirmatory phenotypic methods for detection of ESBLs Klebsiella pneumoniae isolates prevalent in a hospital in north India. METHODS: One hundred, non-repeat clinical isolates of K. pneumoniae collected over a period of six months were included in the study. Susceptibilities of the isolates to 20 different antimicrobial agents were determined. Agar dilution and broth dilution methods were used to determine minimum inhibitory concentrations (MICs) of ceftazidime (CAZ) and cefotaxime (CTX). CAZ and CTX were used with and without clavulanic acid to detect ESBL harbouring isolates. Using agar dilution and broth dilution, MIC reduction of two and three doubling dilutions were evaluated as a criterion for ESBL harbouring isolates. Standard double disk synergy test (DDST) with disks placed at 30 mm and modified DDST with disks placed at 16 mm center-to-center distance, using at least two different third generation cephalosporins and combined disk method were also performed to detect ESBL harbouring isolates. RESULTS: Multi-drug resistance (resistance to three or more antimicrobials of different classes) was found among 94 per cent of the isolates. Pooling the results of all the three confirmatory techniques MIC reduction of >3 doubling dilutions using broth dilution method (using CTX and CAZ), combined disk method [(using CTX, ceftriaxone [(CRO), CAZ and aztreonam)] and standard DDST (using CTX, CRO, CAZ and aztreonam), revealed as many as 87 per cent of the isolates as ESBL producers. CTX had greater sensitivity in identifying isolates that harboured ESBLs. Modified DDST using CTX was as sensitive method as broth dilution method and combined disk method in detecting ESBL harbouring isolates. MIC reduction technique using agar dilution method and standard DDST had lowest overall sensitivity in detecting ESBLs. INTERPRETATION & CONCLUSION: Modified DDST using at least two different third generation cephalosporins was considered to be the best technique for detection of ESBL producing K. pneumoniae at our hospital. MIC reduction test with >2 doubling dilution reduction in MICs was found to be a better criterion than the presently recommended >3 doubling dilution reduction. For screening of potential ESBL producers, MIC determination using agar dilution was as good as that using broth dilution method. However, while performing MIC reduction test agar dilution method was found highly unreliable for detection of ESBL harbouring isolates.

The 2003 outbreak of Dengue fever in Delhi, India.

Dengue fever (DF) and Dengue hemorrhagic fever (DHF) are widespread in Southeast Asia. An outbreak of DF/DHF in Delhi in 2003 started during September, reached its peak in October-November, and lasted until early December. This study describes the clinical and laboratory data of the 185 cases of DF/DHF admitted to Lok Nayak Hospital, New Delhi. The mean age of the patients was 26 +/- 10 years. Fever was present in all the cases with an average duration of fever being 4.5 +/- 1.2 days with headache (61.6%), backache, (57.8%), vomiting (50.8%) and abdominal pain (21%) being the other presenting complaints. Hemorrhagic manifestations in the form of a positive tourniquet test (21%), gum bleeding and epistaxis (40%), hematemesis (22%), skin rashes (20%) and melena (14%) were also observed. Hepatomegaly and splenomegaly were observed in 10% and 5% of cases, respectively. Laboratory investigations revealed thrombocytopenia (with a platelet count of < 100,000/microl) in about 61.39% of cases, Leukopenia (WBC <3,000/mm2) and hemoconcentration (Hct >20% of expected for age and sex) were found in 68% and 52% of the cases, respectively. The mortality rate was 2.7%. Despite widespread measures taken to control outbreaks of DF, it caused major outbreaks. More stringent measures in the form of vector control, improved sanitation and health education are needed to decrease morbidity, mortality and health care costs caused by a preventable disease.