Detection and characterization of metallo-β-lactamases in Pseudomonas aeruginosa by phenotypic and molecular methods from clinical samples in a tertiary care hospital / Detección y caracterización de la metallo-betalactamasa en la Pseudomonas aeruginosa mediante métodos fenotípicos y moleculares a partir de muestras clínicas en un hospital de atención terciaria

AIMS: The aim of this study was to detect and characterize the presence of metallo-β-lactamase (MBL) production in multidrug resistant (MDR) P aeruginosa collected from clinical samples in a tertiary care hospital. METHODS AND MATERIALS: A total of 67 non-repetitive isolates of MDR P aeruginosa recovered from various clinical specimens were screened for MBL production by IPM/MEM-EDTA combined disc test. Polymerase chain reaction was performed on all isolates using blaIMP and blaVIM consensus primers to characterize them genotypically. RESULTS: Among 67 P aeruginosa isolates, 62.7% (42/67) and 70.1% (47/67) were resistant to imipenem and meropenem respectively and 47 (70.1%) were found to be MBL producers. Among this 47 MBL-producing isolates, 41 (61.1%) strains carried the blaVIM gene and 2 (3%) strains carried the blaIMP gene. Three strains were phenotypically negative but positive genotypically for blaVIM gene. One strain was resistant to both imipenem and meropenem but did not show phenotypic positivity. CONCLUSION: This study confirms the dissemination of blaVIM genes among MDR Pseudomonas aeruginosa and hence it is indispensible to identify and aptly control the threat of horizontal and vertical transfer.

OBJETIVO: El objetivo de este estudio es descubrir y caracterizar la presencia de producción de metallo-betalactamasa (MBL) en P aeruginosa resistente a los multifármacos (RMF), recogida de muestras clínicas de un hospital de atención terciaria. MÉTODO: Un total de 67 aislados no repetitivos de P aeruginosa RMF obtenidos de varios specímenes clínicos, fueron tamizados en busca de producción de MBL, mediante una prueba de disco combinado IPM/MEM-EDTA. Se efectuó una reacción en cadena de la polimerasa sobre todos los aislados, usando iniciadores de consenso blaIMP y blaVIM para la caracterización genotípica. RESULTADOS: Entre los aislados de P aeruginosa, 62.7% (42/67) y 70.1% (47/67) fueron resistentes al Imipenem y al Meropenem respectivamente, mientras que se halló que 47 (70.1%) eran productores de MBL. De los 47 aislados productores de MBL, 41 (61.1%) cepas eran portadoras del gen blaVIM en tanto que 2 (3%) cepas eran portadoras del gen blaIMP. Tres cepas fueron fenotípicamente negativas, pero genotípicamente positivas con respecto al gen blaVIM. Una cepa fue resistente tanto al Imipenem como al Meropenem, pero no mostró positividad fenotípicamente. CONCLUSIÓN: El presente estudio confirma la diseminación de los genes blaVIM entre las Pseudomonas aeruginosa RMF. Es importante identificar así como controlar adecuadamente la amenaza de la transferencia horizontal y vertical.

Detection of Amp C genes encoding for beta-lactamases in Escherichia coli and Klebsiella pneumoniae.

Purpose : Amp C beta-lactamase are Ambler class C enzymes that confer resistance to extended spectrum cephalosporins and are not inhibited by beta-lactamase inhibitors. Their detection is crucial, since the phenotypic tests are not standardised leading to ambiguity in interpretation of results. This study was done to detect the types of Amp C prevalent in Escherichia coli and Klebsiella pneumoniae by multiplex polymerase chain reaction (PCR). Materials and Methods : Seventy-seven consecutive cefoxitin resistant clinical isolates of E. coli (n = 25) and K. pneumoniae (n = 52) were included in the study. Antibiotic susceptibility testing to various classes of antibiotics was performed by disc diffusion using Clinical Laboratory Standards Institute (CLSI) guidelines. Minimum inhibitory concentration (MIC) to cefoxitin, imipenem and meropenem were determined by broth microdilution method. Isolates were screened for production of Extended Spectrum Beta-Lactamase (ESBL). Multiplex PCR was performed for the detection of Amp C genes after phenotypic testing (Hodge test and inhibitor based test). Results : Cefoxitin Hodge test was positive in 40 isolates which included 20 E. coli and 20 K. pneumoniae. There was zone enhancement with boronic acid in 55 isolates, of which 36 were K. pneumoniae and 19 were E. coli. Multiplex PCR detected Amp C in 11/25 E. coli and 12/52 K. pneumoniae isolates. The Amp C genes detected were CIT (Amp C origin - Citrobacter freundii), DHA (Dhahran Hospital, Saudi Arabia), ACC (Ambler class C), EBC (Amp C origin - Enterobacter cloacae) groups. ESBL was co-produced in 54 isolates. Conclusions : Amp C was detected in 29.87% of the study isolates. Majority of them co-produced ESBL. The most common Amp C was the CIT family. Screen tests for cefoxitin resistance may be falsely positive due to production of carbapenamases.

Comparative evaluation of expression of proliferating cell nuclear antigen in variants of ameloblastoma and ameloblastic carcinoma.

Aim: To evaluate the use of proliferating cell nuclear antigen index in the different histopathological variants of ameloblastoma, such as the follicular, plexiform, and unicystic types, and in ameloblastic carcinoma by immunohistochemical staining. The proliferating cell nuclear antigen index values of the variants of ameloblastomas and ameloblastic carcinomas are compared in order to determine the biological behavior of these tumors. Materials and Methods: For the present study, archival tissues that had been diagnosed as ameloblastoma and ameloblastic carcinoma were collected from the department of oral pathology. Specimens were embedded in paraffin wax and were sectioned at a thickness of 5 μm and stained with hematoxylin-eosin for reconfirming the histologic pattern. It was also stained immunohistochemically for anti-proliferating cell nuclear antigen antibody. Results: Positive proliferating cell nuclear antigen expression is seen as a light brown, granular stain. The proliferating cell nuclear antigen values of ameloblastic carcinoma were almost five times the value of ameloblastoma. Analysis of variance test, Fischer's exact test/variance ratio test, and Student's t-test were performed and the probability values were determined. Summary and Conclusion: This study showed that ameloblastic carcinoma had the maximum proliferative capacity. Among the variants of ameloblastoma, the plexiform variety had the maximum proliferative capacity, followed by the follicular and unicystic varieties. Altogether, these data indicate that proliferating cell nuclear antigen is related to the biological behavior and proliferation of tumor cells in the variants of ameloblastoma and ameloblastic carcinoma.

OXA beta-lactamase-mediated carbapenem resistance in Acinetobacter baumannii.

Objectives: Acinetobacter baumannii is a significant pathogen in health care settings. In recent years, an increase in carbapenem resistance among A. baumannii due to Ambler class B metallo-beta-lactamases or class D OXA carbapenamases has been reported. In this study we detected the presence of OXA carbapenamases and coproduction of metallo-beta-lactamases (blaVIM and blaIMP ) by phenotypic and genotypic methods in carbapenem resistant clinical isolates of Acinetobacter baumannii. Materials and Methods: A total of 116 consecutive, non-duplicate carbapenem resistant A. baumannii isolated from various clinical specimens were included in the study. The modified Hodge test and inhibitor potentiated disk diffusion tests were done for the screening of carbapenamase and metallo-beta-lactamase production, respectively. Polymerase chain reaction (PCR) was performed for the detection of OXA (blaOXA 23 like, blaOXA 24 like, blaOXA-51 like and blaOXA-58 like genes) and metallo-beta-lactamases (blaVIM and blaIMP ) genes. Gene sequencing was performed for representative isolates. Results: Among 116 A. baumannii, OXA genes were detected in 106 isolates. BlaOXA 51 like (n = 99) and blaOXA -23 like (n = 95) were the most common and they coexisted in 89 isolates. blaOXA-24 like gene was detected in two isolates of which one also carried blaOXA-51 like and blaOXA-58 like genes. The modified Hodge test was positive in 113 isolates. The metallo-beta-lactamase screening test was positive in 92 isolates. blavim was detected in 54 isolates of which 1 also carried the blaIMP gene. Conclusions: blaOXA-23 like and bla OXA 51 like genes are the most common types of OXA carbapenamases while the blaVIM type is the most common type of metallo-beta-lactamase contributing to carbapenem resistance in clinical isolates of A. baumannii. The coproduction of OXA and metallo-beta-lactamases is not an uncommon phenomenon in A. baumannii.

OXA beta-lactamase-mediated carbapenem resistance in Acinetobacter baumannii.

Objectives: Acinetobacter baumannii is a significant pathogen in health care settings. In recent years, an increase in carbapenem resistance among A. baumannii due to Ambler class B metallo-beta-lactamases or class D OXA carbapenamases has been reported. In this study we detected the presence of OXA carbapenamases and coproduction of metallo-beta-lactamases (blaVIM and blaIMP ) by phenotypic and genotypic methods in carbapenem resistant clinical isolates of Acinetobacter baumannii. Materials and Methods: A total of 116 consecutive, non-duplicate carbapenem resistant A. baumannii isolated from various clinical specimens were included in the study. The modified Hodge test and inhibitor potentiated disk diffusion tests were done for the screening of carbapenamase and metallo-beta-lactamase production, respectively. Polymerase chain reaction (PCR) was performed for the detection of OXA (blaOXA 23 like, blaOXA 24 like, blaOXA-51 like and blaOXA-58 like genes) and metallo-beta-lactamases (blaVIM and blaIMP ) genes. Gene sequencing was performed for representative isolates. Results: Among 116 A. baumannii, OXA genes were detected in 106 isolates. BlaOXA 51 like (n = 99) and blaOXA -23 like (n = 95) were the most common and they coexisted in 89 isolates. blaOXA-24 like gene was detected in two isolates of which one also carried blaOXA-51 like and blaOXA-58 like genes. The modified Hodge test was positive in 113 isolates. The metallo-beta-lactamase screening test was positive in 92 isolates. blavim was detected in 54 isolates of which 1 also carried the blaIMP gene. Conclusions: blaOXA-23 like and bla OXA 51 like genes are the most common types of OXA carbapenamases while the blaVIM type is the most common type of metallo-beta-lactamase contributing to carbapenem resistance in clinical isolates of A. baumannii. The coproduction of OXA and metallo-beta-lactamases is not an uncommon phenomenon in A. baumannii.

The utility of IS6110 sequence based polymerase chain reaction in comparison to conventional methods in the diagnosis of extra-pulmonary tuberculosis.

IS6110 sequence based polymerase chain reaction (PCR) was compared with conventional bacteriological techniques in the laboratory diagnosis of extra-pulmonary tuberculosis (EPTB). One hundred and ninety one non-repeated clinical samples of EPTB and 17 samples from non-tuberculous cases as controls were included. All the samples were processed for Ziehl-Neelsen staining for acid fast bacilli (AFB) and 143 samples were processed by culture for M. tuberculosis . All the samples were processed for PCR amplification with primers targeting 123 bp fragment of insertion element IS6110 of M. tuberculosis complex. Of the total 191 samples processed, 34 (18%) were positive by smear for AFB. Culture for AFB was positive in 31(22%) samples among the 143 samples processed. Either smear or culture for AFB was found positive in 51(27%) samples. Of the total 191 samples processed 120 (63%) were positive by PCR. In 140 samples, wherein both the conventional techniques were found negative, 74 (53%) samples were positive by PCR alone. Among 51 samples positive by conventional techniques, 46 (90%) were found positive by PCR. PCR assay targeting IS6110 is useful in establishing the diagnosis of EPTB, where there is strong clinical suspicion, especially when the conventional techniques are negative.

Recent advances in immunodiagnosis of leprosy.

Although prevalence of leprosy is considerably reduced, the unabated emergence of about 300,000 cases worldwide indicates that the source of infection and transmission are not being addressed. Early diagnosis and treatment still remain the cornerstone of leprosy control. Many diagnostic issues hinder the correct and timely diagnosis and classification of leprosy. Delayed and missed diagnosis of infectious leprosy patients and the lack of tests to measure asymptomatic M. leprae infection in contacts also hamper the assessment of transmission of M. leprae infection. An important goal would be the development of improved diagnostic tools to diagnose difficult cases and to detect M. leprae infection before clinical manifestation. The search for an ideal immunodiagnostic tool for leprosy had gone through various phases and development over the years, with inherent limitations in the sensitivity and specificity of the immunodiagnostic tests for leprosy. With improvement in technology many modifications of previously used PGL-1 assay in the form of rapid and less expensive techniques, such as dipstick, ELISA, ML flow test, have been introduced. Many new skin test antigens with potential for improving their efficiency, such as MLSA LAM, MLCwA and their fractionates, have been studied. After the completion of genome sequencing of M. leprae in 2000, many genes that were studied in M. tuberculosis and found potential for the immunodiagnosis of tuberculosis, such as CFP-10 and ESAT-6 proteins, have been investigated in M. leprae also. Genes that are unique to M. leprae with no homologous in M. tuberculosis have been explored for novel M. leprae-specific antigens. In order to overcome the problem of cross-reactivity, a number of workers have synthesized overlapping short peptides of different M. leprae recombinant proteins and studied their sequence divergence and attempted to identify M. leprae-specific B- and T-cell epitopes. This review makes an effort to present an overview of all these developments in the field of immunodiagnosis of leprosy.

Evaluation of the modified leprosy elimination campaign in a high leprosy endemic district of Jharkhand.

An evaluation of the third Modified Leprosy Eradication Campaign (MLEC) was carried out in Potka block in the high endemic district of East Singhbhum, Jharkhand State, India, by our external evaluation team, from 29 October to 8 November 2001. The searchers in this block detected 389 suspects during the MLEC; of these, 181 (46%) were examined, and 69 (38%) of them were confirmed as cases by the Programme staff. The evaluators examined 189 (48.5%) of the total 389 suspects detected by the searchers, including 31 of the 69 cases confirmed by the Programme staff. Concordance of diagnosis of leprosy cases by the Programme staff and the evaluators was found to be high (90%). However, concordance of the type of leprosy was found to be variable (PB 38%, MB 72%, SSL 100%). Specificity and sensitivity of diagnosis by the Programme staff (as against those by the evaluators) were found to be 85.7% and 79.2% respectively. There was no case of re-registration. The evaluators examined 108 of the suspects detected by the Search Team, but not screened by the Programme staff, and diagnosed 47 cases (44%; PB 20, MB 9, SSL 18) from among them. The evaluators also diagnosed additional 30 new cases (PB 18, MB 5, SSL 7), during their visit. An assessment of knowledge about the disease and treatment among confirmed cases revealed that most of the patients did not know correctly about their disease. All the cases were referred by the searchers. About 45% of cases were aware of the duration for which they needed to take the treatment, 97% of cases showed the blister calendar packs and had taken the supervised dose. Availability of MDT to the patients and drug compliance were found to be adequate. Assessment of the impact of IEC activities on the awareness of leprosy among the community showed that about 50% of those interviewed were aware of the campaign. Most of them had information about the availability of leprosy drugs and knew that treatment was free. A majority of those aware of the disease said that they would refer suspects, if they come across any, to PHC centres for treatment.

Isolation and characterisation of nocardia from clinical specimens.

Sixteen isolations of nocardia of which 12 were from pulmonary infections, one from wound infection, one from mycetoma and 2 from eye infections were studied from June, 1989 to May, 1990. The importance of Gram's stain findings of primary smear is being highlighted. The nocardia species were identified utilising the morphological characters including acid fastness and cultural and biochemical characters. Notable among the isolates were Nocardia brasiliensis, one each from mycetoma and pulmonary infection, which are rare in South India and Nocardia asteroides from a case of endophthalmitis probably of endogenous origin.

HIV infection amongst leprosy patients in south India.

In a pilot study, 463 leprosy patients (374 males and 89 females) were investigated for HIV-1 and HIV-2 antibodies by screening tests. Sera positive by the screening tests were subjected to confirmatory tests. Three cases were confirmed to be positive for HIV, two for HIV-1 and one for HIV-2. All the three positive cases were young males, who had visited commercial sex workers. No correlation was found between the type of leprosy and HIV infection. This is the first report of HIV infection amongst leprosy patients from South India.

Non-organ specific & organ-specific auto-antibodies in hepatitis-B virus associated glomerulonephropathies.

Eighty eight patients of glomerulonephropathies (HBsAg positive 67; HBsAg negative 21) and 88 matched and healthy controls were screened for non-organ specific autoantibodies-ANA, AMA, ASMA and APCA by indirect immunofluorescent technique. The 2.3 per cent positivity in the test group and the 8 per cent positivity in the control group did not suggest the involvement of hepatitis-B virus (HBV), as an influencing or associated agent. When 48 patients with glomerulonephropathies and 23 controls were screened for liver cell membrane (LMA) and renal cell membrane antibodies (RMA) by indirect immunofluorescent technique using isolated rat hepatocytes and renal cells, 79.2 per cent LMA positivity was seen in the HBsAg positive group and 41.7 per cent in the negative group and RMA positivity was 58 per cent in the positive group and 25 per cent in the negative group. Simultaneous positivity for both LMA and RMA was recorded in 50 per cent of the HBsAg positive patients and 15.7 per cent of the negative ones. The results suggest the possibility of an organ specific autoimmune trigger more frequently in HBV associated glomerulonephropathy.