Comparative evaluation of pan-fungal real-time PCR, galactomannan and (1-3)-ß-D-glucan assay for invasive fungal infection in paediatric cancer patients.

Limited specific data and investigations are available for the diagnosis of Invasive Fungal Infection (IFI) in paediatrics cancer patients. Three non-invasive tests; Platelia Aspergillus EIA for galactomannan (GM), ß-D-glucan (BDG) assay and pan-fungal real-time PCR for fungal DNA in blood were evaluated. One hundred twenty-five paediatrics cancer patients at the high risk of IFI were enrolled. Single blood and serum samples were evaluated by all the three methods. Patients were classified into 10 proven, 52 probable and 63 no IFI cases in accordance with EORTC MSG 2008 revised guidelines. The sensitivity, specificity, PPV and NPV of all the three tests in proven, probable and no IFIs cases were analysed singly and in combination. The sensitivity, specificity, PPV and NPV of GM, BDG and pan-fungal real-time PCR were: 87%, 61%, 81%, 69.5% for GM, 88%, 59.5%, 81%, 71.4% for BDG and 89%, 69.2%, 85%, 67.5% for PCR (95% CI). Among different combinations, best combination was found to be GM and PCR with sensitivity, specificity, PPV and NPV of 98.2%, 89.3%, 97.1% and 90% respectively. Single samples must be evaluated by combination of tests.

A study on pre-XDR & XDR tuberculosis & their prevalent genotypes in clinical isolates of Mycobacterium tuberculosis in north India.

BACKGROUND & OBJECTIVES: Pre-extensively drug resistant (pre-XDR) and extensively drug resistant tuberculosis (XDR-TB) have been areas of growing concern, and are posing threat to global efforts of TB control. The present study was planned to study the presence of pre-XDR and XDR Mycobacterium tuberculosis and their genotypes in clinical isolates obtained from previously treated cases of pulmonary TB. METHODS: A total of 219 isolates obtained from previously treated cases of pulmonary TB were subjected to first-line (streptomycin, isoniazid, rifampicin and ethambutol) and second-line (ofloxacin, kanamycin, capreomycin and amikacin) drug susceptibility testing on solid Lowenstein-Jensen medium by proportion method. Genotyping was done for pre-XDR and XDR-TB isolates using 12 loci Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats (MIRU-VNTR). RESULTS: Multi-drug resistance was observed in 39.7 per cent (87/219) isolates. pre-XDR and XDR M. tuberculosis isolates amongst 87 multi-drug resistant (MDR) TB isolates were 43 (49.4%) and 10 (11.4%), respectively. Two most dominant genotypes among pre-XDR and XDR M. tuberculosis isolates were Beijing and Delhi/CAS types. INTERPRETATION & CONCLUSIONS: Resistance to second-line anti-tubercular drugs should be routinely assessed in areas endemic for TB. Similar genotype patterns were seen in pre-XDR and XDR-TB isolates. Beijing and Delhi/CAS were predominant genotypes.

Molecular characterization of dengue viruses circulating during 2009-2012 in Uttar Pradesh, India.

Dengue is the most rapidly spreading mosquito-borne viral disease in the world; in India it has taken endemic proportion implicating all the four known dengue virus serotypes. Dengue infection is caused by a small, single stranded RNA virus comprising of four antigenically distinct virus serotypes designated as dengue virus type 1-4 (DENV-1-4). On the basis of genomic variations, each serotype is classified further into its genotypes. Epidemiological studies have shown that the emergence of a newer dengue serotype/genotype after an interval always leads to a major outbreak; therefore a continuous epidemiological surveillance is needed to monitor the epidemiology of dengue viruses. The present study was planned to identify the serotype/genotype of dengue viruses circulating in Uttar Pradesh, India. Of 433 dengue suspected patients, tested by reverse transcriptase PCR (RT-PCR), 136 were positive for dengue virus RNA. Of these, DENV-1, 2, and 3 were detected in 26 (19.1%), 77 (56.6%), and 33 (24.3%) patients, respectively. Of 136 RT-PCR positive samples, 24 samples were sequenced to identify their genotypes. For sequencing C-prM gene junction of dengue virus genome was chosen. Phylogenetic analysis of sequenced dengue strains revealed that all the 12 DENV-1 strains were genotype III, all the eight DENV-2 strains were genotype IV (Cosmopolitan genotype) and among four DENV-3 strains, three were genotype III and one was genotype I. In conclusion, the co-circulation of multiple dengue virus serotypes and genotypes is alarming in U.P., India.

Aetiology of childhood viral gastroenteritis in Lucknow, north India.

BACKGROUND & OBJECTIVES: Due to limited availability of data on viral aetiology of acute gastroenteritis in north India, the present study was planned to detect rotavirus, norovirus, sapovirus and astrovirus in stool samples of both in hospitalized and non-hospitalized children less than five years of age presenting with acute gastroenteritis. METHODS: A total of 278 stool samples from equal number of children were tested for rotavirus antigen using ELISA and for norovirus, sapovirus and astroviruses by reverse transcription (RT)-PCR. RESULTS: Of the 169 samples from hospitalized patients, rotavirus, norovirus, sapovirus and astrovirus were detected in 19.5, 2.3, 3.5 and 2.9 per cent samples, respectively. Of the 109 samples collected from the non-hospitalized patients, frequency of rotavirus and sapovirus detection was 9.1 and 1.8 per cent, respectively while norovirus and astrovirus were not detected. INTERPRETATION & CONCLUSIONS: Rotavirus was the most frequent cause of viral gastroenteritis in both hospitalized and non-hospitalized children. Maximum positivity of the viruses was seen in children less than two years of age.

In vitro validation of self designed "universal human Influenza A siRNA".

The genomic variability of Influenza A virus (IAV) makes it difficult for the existing vaccines or anti-influenza drugs to control. The siRNA targeting viral gene induces RNAi mechanism in the host and silent the gene by cleaving mRNA. In this study, we developed an universal siRNA and validated its efficiency in vitro. The siRNA was designed rationally, targeting the most conserved region (delineated with the help of multiple sequence alignment) of M gene of IAV strains. Three level screening method was adopted, and the most efficient one was selected on the basis of its unique position in the conserved region. The siRNA efficacy was confirmed in vitro with the Madin Darby Canine Kidney (MDCK) cell line for IAV propagation using two clinical isolates i.e., Influenza A/H3N2 and Influenza A/pdmH1N1. Of the total 168 strains worldwide and 33 strains from India, 97 bp long (position 137-233) conserved region was identified. The longest ORF of matrix gene was targeted by the selected siRNA, which showed 73.6% inhibition in replication of Influenza A/pdmH1N1 and 62.1% inhibition in replication of Influenza A/H3N2 at 48 h post infection on MDCK cell line. This study provides a basis for the development of siRNA which can be used as universal anti-IAV therapeutic agent.

Trend of dengue virus infection at Lucknow, north India (2008- 2010): a hospital based study.

BACKGROUND & OBJECTIVES: Dengue virus (DV) infection has emerged as a major health problem in north India. Here, we report the annual trend of dengue virus infection as seen in Lucknow, Uttar Pradesh, during 2008-2010. METHODS: Blood samples from clinically suspected cases of dengue virus infection were collected and history was taken on structured clinical data sheet. All samples were tested for dengue IgM by antibody capture ELISA. Selected samples were tested by conventional RT-PCR for dengue virus RNA. Weather information was continuously recorded from website of world weather information service . RESULTS: There was a gradual increase in number of dengue fever cases with increased occurrence in 2010. Cases referred in January - December 2008 were 398 (54.5% anti DV IgM positive), in January - December 2009 were 599 (51.9% anti DV IgM positive) and in January - December 2010 were 1602 (64.9% anti DV IgM positive). Serotypes circulating in years 2008, 2009 and 2010 were DV-2 & DV-3, DV -1, 2 & 3 and DV-1 and DV-2 respectively. There is no statistical significant correlation between weather data and increasing dengue positive cases. INTERPRETATION & CONCLUSIONS: Increased cases of dengue fever were seen in 2010, which was not correlated with any change in environmental factors. A change in circulating serotypes was noted.

Identification, characterization and antibiotic susceptibility pattern of non fermenters from clinical specimens in Lucknow, Uttar Pradesh.

Present study was an attempt to study the prevalence of nonfermenter and its antibiotic susceptibility pattern at CSM Medical University, Lucknow. All the isolates and samples were selected from clinical specimens received in Bacteriology section, P.G. Depart of Microbiology, for culture. The observation were made on the nonfermenter isolates that can be isolated from clinical specimen using simple Laboratory media e.g. Blood Agar & Mac Conkey agar. All relevant history & information were recorded from the subjects. A total of 8340 specimen were screened for a period of one year. The prevalence of nonfermenters came to be 19.09% among all isolates. Most of spp. belongs to oxidase+ve group (77%). P. aeruginosa was found to be most common isolate (53%). Overall sensitivity profile for ciprofloxacin was 60%, P/T 58% & Amikacin 56%. Sensitivity of imepenem was 60% for multi-resistant isolates. The most resistant isolate was Sachrolytic Acinetobacter spp. The knowledge of synergism between drugs in context to different isolates may aid in effective therapy for these isolates.

High-level aminoglycoside resistance and beta-lactamase production in enterococci at a tertiary care hospital in India.

Enterococci, a family of important opportunistic pathogens, exhibits intrinsic resistance to a number of antimicrobial agents in addition to acquired multidrug resistance. The present study was conducted to determine whether enterococci at a tertiary care hospital in India exhibit high-level aminoglycoside resistance and beta-lactamase production. Enterococci were isolated from various clinical specimens and identified phenotypically. High-level resistance (HLR) to gentamicin, kanamycin, and streptomycin was determined by disc diffusion tests. Beta-lactamase production was detected using three methods: iodometric, acidometric, and chromogenic beta-lactamase assays. Among the 86 enterococci isolated, 34 were found to have HLR to one or more aminoglycosides; HLR to kanamycin was most common. Vancomycin resistance was present in four of the isolates. Only one enterococcus produced beta-lactamase, and it was sensitive to ampicillin on routine disc diffusion testing. Beta-lactamase production among enterococci, though not very common, may be missed on routine susceptibility testing. Frequent occurrence of HLR to kanamycin makes amikacin a poor choice for inclusion in combination therapy with cell wall-active agents.

Prevalence of reproductive tract infections in women attending a tertiary care center in Northern India with special focus on associated risk factors.

BACKGROUND: Reproductive tract infections (RTIs) continue to present major health, social, and economic problems worldwide, and their complications are the most important causes of morbidity and mortality for women, especially in developing countries. Interest in RTIs and their management has increased tremendously because the presence of a RTI in the sexual partner increases the risk of acquisition of HIV. AIMS: The aim of this study is to know the prevalence of RTIs, its correlation with clinical features and associated risk factors in women of reproductive age group attending a tertiary care center in Lucknow. MATERIALS AND METHODS: The present study was conducted on 318 women of the reproductive age group (18-45 years) attending the RTI/sexually transmitted infection clinic at our center; they were evaluated for the prevalence of following RTIs: Chlamydia, gonorrhea, syphilis, bacterial vaginosis, trichomoniasis, and candidiasis; their correlation with clinical features and associated risk factors. RESULTS: The prevalence of reproductive tract infections in women attending our centre reported 9.7%. The prevalence of candidiasis was maximum (11.5%) followed by chlamydia (4.1%), syphilis (4.1%), bacterial vaginosis (1.73%), and trichomoniasis (0.57%). None of the women were found positive for gonorrhea. The most common presentation was genital discharge (52.8%) followed by lower abdominal pain (45.2%). CONCLUSION: The factors found to be significantly associated with RTI were illiteracy (P < 0.05), unemployment (P < 0.05), history of RTI in patient (P = 0.001), and the presence of RTI in their partner (P < 0.05). The genital discharge was the most common presentation.

Etiological agents of diarrhea in hospitalized pediatric patients with special emphasis on diarrheagenic Escherichia coli in North India.

INTRODUCTION: Infectious diarrhea is leading infectious cause of childhood morbidity, hospitalizations, and mortality particularly in children living in developing countries like India. The etiological agents differ depending on geographical area, and recent data suggest increase in drug resistance to various enteropathogens. AIMS AND OBJECTIVES: The aim of the study was to investigate emerging diarrheal agents and antimicrobial resistance profile of bacterial pathogens from children (<12 years of age) hospitalized with acute diarrhea. MATERIALS AND METHODS: A cross-sectional, hospital-based observational study was conducted over 1 year in which 100 children <12 years who were hospitalized due to diarrhea were recruited. Diarrhea was defined as the passage of three or more liquid stools in a 24-h period using the World Health Organization guidelines. Samples were processed for detection of various bacterial, viral, and parasitic agents by standard microbiological, serological, and molecular tests. Antimicrobial resistance testing was performed with the Kirby-Bauer disk diffusion method. ELISA was performed for Rotavirus and Escherichia coli O157. Multiplex polymerase chain reaction test was performed to detect diarrheagenic E. coli (DEC). RESULTS: Pathogenic diarrheal agents were found in 63% patients. Rotavirus was identified in 52.5%, DEC in 29%, Vibrio cholerae in 4%, Shigella flexneri in 3%, Aeromonas sp. in 1%, Giardia lamblia in 4%, and Entamoeba histolytica in 1% cases. Enteropathogenic E. coli (EPEC) in 19 (65.5%) cases was the most common agent followed by Enteroaggregative E. coli (EAEC) in 5 (17.2%), Enterotoxigenic E. coli (ETEC) in 2 (6%), and Enteroinvasive E. coli (EIEC) in 3 (10.3%) cases. Resistance rates of DEC to first-line therapeutic drugs were high, 97.3% to ampicillin and 95.95% to co-trimoxazole. DEC was susceptible to chloramphenicol in 58.11%, gentamicin in 48.19%, and amikacin in 58.11% cases. Shigella sp. and V. cholerae isolates were 100% sensitive to gentamicin and ofloxacin. CONCLUSION: EPEC is the most common DEC pathotype and EAEC, ETEC, and EIEC are also emerging as dominant diarrheal agents. Rotavirus was the most common causative agents of diarrhea especially in children <5 years. Most of the bacterial isolates showed high level of drug resistance to first-line empirical drugs and were multidrug resistant making them unsuitable for empiric treatment. Laboratory monitoring of drug susceptibility of stool isolates appears necessary to formulate antibiotic policy for treating diarrheal illness at the local level. There is an urgent need to strengthen diarrheal surveillance to monitor susceptibility to commonly prescribed antibiotics.

Synergy of clavine alkaloid 'chanoclavine' with tetracycline against multi-drug-resistant E. coli.

The emergence of multi drug resistance (MDR) in Gram-negative bacteria (GNB) and lack of novel classes of antibacterial agents have raised an immediate need to identify antibacterial agents, which can reverse the phenomenon of MDR. The purpose of present study was to evaluate synergy potential and understanding the drug resistance reversal mechanism of chanoclavine isolated from Ipomoea muricata against the multi-drug-resistant clinical isolate of Escherichia coli (MDREC). Although chanoclavine did not show antibacterial activity of its own, but in combination, it could reduce the minimum inhibitory concentration (MIC) of tetracycline (TET) up to 16-folds. Chanoclavine was found to inhibit the efflux pumps which seem to be ATPase-dependent. In real-time expression analysis, chanoclavine showed down-regulation of different efflux pump genes and decreased the mutation prevention concentration of tetracycline. Further, in silico docking studies revealed significant binding affinity of chanoclavine with different proteins known to be involved in drug resistance. In in silico ADME/toxicity studies, chanoclavine was found safe with good intestinal absorption, aqueous solubility, medium blood-brain barrier (BBB), no CYP 2D6 inhibition, no hepatotoxicity, no skin irritancy, and non-mutagenic indicating towards drug likeliness of this molecule. Based on these observations, it is hypothesized that chanoclavine might be inhibiting the efflux of tetracycline from MDREC and thus enabling the more availability of tetracycline inside the cell for its action.

Seroepidemiology of Spotted Fever Rickettsiosis in Uttar Pradesh: A Prospective Study.

INTRODUCTION: Spotted Fever Rickettsiosis (SFR), an acute febrile illness caused by Rickettsia rickettsii, R. conorii and R. akari which is associated with considerable morbidity and mortality. SFR is one of the most covert emerging infections of the present time which is prevalent in various parts of India as shown by the increase in the number of clinically diagnosed patients in various states except Uttar Pradesh. AIM: To diagnose SFR in clinically suspected patients using serological tests and recognition of common epidemiologic situations and clinical manifestations of SFR in the state of Uttar Pradesh. MATERIALS AND METHODS: Patients of all age groups presented with a diagnosis of Pyrexia of Unknown Origin (PUO) from May 2013 to February 2015 were evaluated. Testing was done using a nonspecific Weil felix test followed by more specific Enzyme Linked Immunosorbent Assay (ELISA) and a gold standard Immunofluorescence Assay (IFA) test for specific IgM antibodies against Rickettsia conorii. The data was statistically analysed on Graph Pad Prism (5.0) software by using Chi-square test. RESULTS: Of the 432 patient samples tested by non specific Weil felix test, 200 (46.29 %) samples showed titre 1:80 or more and were taken as positive. Similarly out of the 432 blood samples tested by both ELISA and IFA based test against Rickettsiaconorii IgM antibody, only 115 (26.62%) samples were found to be positive and these samples were also positive by Weil felix. The common symptoms noted were fever, hepatomegaly, thrombocytopenia, lymphadenopathy and rashes, nausea followed by icterus, cyanosis, headache, oedema and abdominal pain. Eschar was found in only four (3.4%) patients. We also found that 31 patients with SFR also had associated co-infections like typhoid, malaria, dengue and hepatitis. CONCLUSION: Our findings demonstrated that Weil Felix test can fill in as an underlying yet not sole strategy to perceive and analyse rickettsial ailments, as it needs specificity. So, it may be used to assess the burden in the area and later on other tests like ELISA or IFA can be added, as these are more specific diagnostic tests. Further, our results also showed that if a patient tests positive for the more common endemic infections, we must test for rickettsiosis so that appropriate treatment could be administered.

Effect of efflux pump inhibitors on the susceptibility of Mycobacterium tuberculosis to isoniazid.

BACKGROUND: Mycobacterium can develop drug resistance (DR) by mutation of its existing gene. However, the existence of DR without mutation shows the need to look for an alternative mechanism such as the role of efflux pumps. In this study, we examined the effect of efflux pump inhibitors on isoniazid (INH) susceptibility in clinical isolates of Mycobacterium tuberculosis (Mtb). MATERIALS AND METHODS: Resazurin microtiter assay was used to examine the effect of efflux pump inhibitors on minimum inhibitory concentration (MIC) levels of INH in eighteen Mtb clinical isolates. RESULTS: The observed reduction in INH-MIC was 2-16-fold in INH-resistant isolates with katG and inhA gene mutations, 2-8-fold in INH-resistant isolates without mutation and 2-4-fold in INH-sensitive isolates. The MIC reduction by verapamil (VER) was observed in 83% isolates, by carbonyl cyanide m-chlorophenylhydrazone (CCCP) 61% isolates, by chloropromazine (CPZ) 61% isolates, by reserpine (RES) in 61% isolates and by 2,4-dinitro phenol (DNP) in 55% isolates. INTERPRETATION AND CONCLUSIONS: The results obtained in this study confirm that MIC of INH decreased in the presence of efflux pump inhibitors (VER, CCCP, CPZ, DNP, RES) in clinical isolates of Mtb and that the inhibition of efflux pumps by the efflux pump inhibitors can enhance the clinical effect of a drug. The results showed that these efflux pump inhibitors are active against both drug susceptible and drug resistant isolates, indicating that the effect of efflux pump inhibitors is not dependent on the mutational profile of the isolate. We observed in this study that VER was the most effective efflux pump inhibitor.

Characterization of Acinetobacter from clinical isolates at Gandhi memorial and associated hospitals, Lucknow.

The study was conducted in 4140 clinical samples sent to Microbiology department from different department of G.M. and associated hospitals. The samples included 2270 urine, 960 pus, 300 blood, 210 sputum, 180 CSF, 20 intercostal drainage tubes and 150 other swabs like vaginal and urethral, conjunctival smear 30, 10 ascitic fluids and 10 gastric aspirates. Apart from this, 30 specimens were collected from hospitals environment, like linen and trolley. From clinical samples, 43 acinetobacter strains (1.04%) were isolated. 17 strains (0.41%), were from pus, 12 (0.28%), from respiratory tract, 1, was (0.02%) from intercostal drainage secretions, urine 9 (0.22%), blood 1 (0.2%) and CSF 3 (.72%). From environmental samples, 7 strains (23.33%) were isolated. All the isolated strains were identified by routine biochemical tests. They were preserved in 1 % agar media for characterization. Characterization was done on the basis of growth at 37 degrees c, 41 degrees c and 44 degrees c, hemolysis, gelatin hydrolysis, acid from glucose, utilization of citrate, L-phenyl alanine, malonate, B-alanine, L-arginine, L-ornithine and L-aspartate. Among species identified Acinetobacter baumannii was 30 (69.67%), from clinical specimens and 5 (71.42%) from environment, Acinetobacter lwoffi was 10 (23.25%) from clinical specimen and 2 from environmental specimen, Acinetobacter hemolyticus was 3 (6.97%) and none from the environment. All the strains were resistant to penicillin. The sensitivity pattern showed gentamycin 64% sensitive, cotrimaxazole 42% cefotoxin 32% ciprofloxacine 26% and tetracycline 16%.

Comparative Analysis of Disc Diffusion and E-test with Broth Micro-dilution for Susceptibility Testing of Clinical Candida Isolates Against Amphotericin B, Fluconazole, Voriconazole and Caspofungin.

BACKGROUND: Antifungal susceptibility testing remains an area of intense interest because of the increasing number of clinical isolates resistant to antifungal therapy. Clinical and Laboratory Standards Institute has proposed reference broth micro dilution (BMD) method for susceptibility testing. The reference method is time-consuming and poorly suited for the routine clinical laboratory setting. Agar-based susceptibility testing methods, disk diffusion (DD) method and the E-test method can be an easier, reliable and less time consuming alternative for the BMD method. AIM: To compare the results of Amphotericin B, fluconazole, voriconazole, and Caspofungin susceptibility testing by DD, and the E-test method with the CLSI reference method for clinical Candida isolates. MATERIALS AND METHODS: Broth Microdilution (BMD), E-test and Disk diffusion testing of the various clinical Candida isolates was performed in accordance with CLSI documents. The results obtained were analysed and compared. RESULTS: The categorical agreement for Amphotericin B, fluconazole, voriconazole, and Caspofungin susceptibility results by E-test and DD method was 65.2%, 67.4%; 100%, 82.6%; 100%, 100%; 100%, 97.8% respectively. CONCLUSION: The agar-based E-test and disk diffusion methods are reliable alternatives to the BMD method for Candida isolates when test susceptible to fluconazole, voriconazole, and Caspofungin, however the susceptibility testing results must be interpreted with caution in case of Amphotericin B.

Comparative Evaluation of Disc Diffusion and E-test with Broth Micro-dilution in Susceptibility testing of Amphotericin B, Voriconazole and Caspofungin against Clinical Aspergillus isolates.

BACKGROUND: Clinical importance of Aspergillus has increased over the past few decades because of rise in immunosuppressive drugs and immune-modulating diseases. Antifungal susceptibility of Aspergillus is rarely performed by clinical laboratories because of lack of easier method. This study has investigated and compared susceptibility pattern of Aspergillus isolates by disc diffusion, E-test and broth micro-dilution for amphotericin B, voriconazole and caspofungin. MATERIALS AND METHODS: Disk diffusion (DD) method of antifungal susceptibility (AFS) was evaluated for three different classes of antifungals: amphotericin B (AMB), voriconazole (VCZ) and caspofungin (CAS). Forty four clinical isolates of Aspergillus were selected; these included 34 A.fumigatus, 8 A.flavus and 2 A. terreus. AFS by DD and E-test was done on non-supplemented Mueller Hinton Agar (MHA) and was compared to Clinical Laboratory Standard Institute(CLSI) broth micro-dilution (BMD) method of AFS. RESULTS: Disk diffusion method for amphotericin B showed 87.5% agreement while E-test showed 93.8% agreement with broth micro-dilution. The agreement with broth micro-dilution was similar for both disk diffusion and E-test in case of voriconazole (93.8%) and caspofungin (100%). 31.8% and 9.1% Aspergillus isolates were found to have amphotericin B and voriconazole MIC values above epidemiological cut off value (ECV) respectively. All isolates were within ECV for caspofungin. CONCLUSION: CLSI method of DD promises to be easier, reproducible and cost effective method of susceptibility testing, but this method must be interpreted with caution in case of amphotericin B susceptibility testing. E-test correlates better than DD with BMD.

Prevalence of gyrA and B gene mutations in fluoroquinolone-resistant and -sensitive clinical isolates of Mycobacterium tuberculosis and their relationship with MIC of ofloxacin.

The study was done to know the prevalent mutations of gyrA and gyrB genes, and their significance with drug resistance in clinical isolates of Mycobacterium tuberculosis. A total of 100 ofloxacin- (OFX) resistant and 100 OFX-sensitive isolates of M. tuberculosis were consecutively selected from routine Tuberculosis laboratory. Drug resistance pattern of these isolates was recorded. MIC of OFX was tested in all these isolates by absolute concentration method. Quinolone resistance determining region (QRDR) of gyrA and gyrB genes of 320 and 428 bp, respectively, were amplified and sequenced. Sequencing data were analyzed by BLAST on NCBI with reference strain H37Rv. Of 100 OFX-sensitive isolates, 30 were pansusceptible, 28 were monoresistant, 10 were polyresistant and 32 were multidrug resistant (MDR). Among 100 OFX-resistant isolates, 19 were OFX monoresistant, 16 were polyresistant and 65 were MDR. Mutations in gyrA and gyrB genes were observed in 79% and 5% of OFX-resistant isolates, respectively. Most prevalent mutation was found at codon 94 in QRDR of gyrA gene. Double mutations found in gyrA gene and in both gyrA and gyrB genes signifies higher levels of OFX resistance. In one isolate, a substitution at codon 592 (Pro592Ser) was found as a novel mutation outside the QRDR of gyrB gene. Our findings support previous studies that the OFX resistance to M. tuberculosis is associated with mutations in the QRDR of gyrA gene; however, the level of OFX resistance may not be predicted based on the mutation patterns in the gyrA gene.

Observations on Citrobacter species from a tertiary care health center with special reference to multi-drug resistance and presence of CTX-M gene.

BACKGROUND: Citrobacter is an important nosocomial pathogen and its multi-drug resistant (MDR) isolates are increasingly being reported across the globe. They are known to produce extended spectrum beta lactamase (ESBL) and harbor CTX-M gene. OBJECTIVE: The aim was to isolate Citrobacter sp. from clinical specimens, analyze their MDR status and look for the presence of CTX-M gene. MATERIALS AND METHODS: In this prospective study, Citrobacter isolates positive for ESBL on screening, were confirmed by combined disc method along with minimum inhibitory concentration (MIC) for cefotaxime. In selected cefotaxime resistant isolates, multiplex polymerase chain reaction was done for blaCTX-M gene. RESULTS: Of 146 Citrobacter sp. isolated, most (73%) were from admitted patients and hospital stay of >72 h and prior antibiotic intake were the most common associated factors. Maximum isolates were from pus (41.1%). Citrobacter freundii was the commonest species (49%) followed by Citrobacter koseri (28%); 79 were ESBL producers. Seventy were cefotaxime resistant as shown by MIC. blaCTX-M gene was detected in 15/40 of these isolates, all belonged to CTX-M group 1. CONCLUSION: Overall incidence of Citrobacter in our setup is low, but they were mostly MDR, and ESBL production was high, which is a cause of concern. blaCTX-M gene detection is important because of its rapid transmission to other bacterial species.