Extended spectrum -lactamases (ESBLs) are plasmid mediated, TEM and SHV derived enzymes, first isolated in Western Europe in mid 1980s, most commonly in Klebsiella spp., followed by Escherichia coli. These enzymes are capable of hydrolyzing broad spectrum cephalosporins and monobactams but inactive against cephamycins and imipenem. In addition, ESBL producing organisms exhibit coresistance to many other classes of antibiotics resulting in limitation of therapeutic option. Several risk factors have been suggested. A variety of classification schemes have been developed. Recently, Bush-Jacoby-Medeiros scheme integrated functional and molecular characteristics. ESBLs have serine at their active site and attack the amide bond in the lactam ring of antibiotics causing their hydrolysis. Because of inocolum effect and substrate specificity their detection is a major challenge. Two indicators of ESBLs are eight fold reduction in MIC and potentiation of the inhibitor zone of third generation cephalosporin in the presence of clavulanic acid. Incidence of these organisms is being continuously increasing through out the world with limited treatment alternatives. It becomes necessary to know the prevalence of these organisms and to formulate treatment policy. Moreover, restricted use of the third generation cephalosporins lead to withdrawal of selective pressure and use of lactam and -lactamase inhibitor combinations may exert reverse mutation on these enzymes.

Quality assurance (QA) is the total process whereby the quality of laboratory reports can be guaranteed. The term quality control covers that part of QA, which primarily concerns the control of errors in the performance of tests and verification of test results. All materials, equipment and procedures must be adequately controlled. Culture media must be tested for sterility and performance. Each laboratory must have standard operating procedures (SOPs). QA of pre-analytical, analytical and post-analytical stages of microbiological procedures should be incorporated in SOPs. The laboratory must be well lit with dust-free air-conditioned environment. Environmental conditions should be monitored. Supervisory and technical personnel should be well qualified. The laboratory should participate in external and internal quality assurance schemes.

PURPOSE: The purpose of this study was to know prevalence of extended spectrum -lactamase (ESBL) in multi drug resistant (MDR) strains of Klebsiella pneumoniae isolated from different clinical samples. METHODS: A total of 120 MDR strain of K. pneumoniae were selected for the study, 106 of which were resistant to atleast one of the third generation cephalosporins (3GC). They were studied for ESBL production by phenotypic confirmatory disc diffusion test (PCDDT) and by double disc synergy test (DDST). RESULTS: 88.3% (106) of the isolates were found to be resistant to atleast one of the 3GC tested (cefotaxime, ceftazidime and ceftriaxone) and 72% of the isolates were resistant to all the 3GC tested. ESBL was detected in 30.18% (32) of the K. pneumoniae by PCDDT and in 27.3% (29) by DDST. Among the ESBL producers 6 (18.75%) were sensitive to cefotaxime, 2 (6.25%) to ceftazidime and 3 (9.37%) to ceftriaxone by disc diffusion test. The minimum inhibitory concentrations (MICs) of 3GC for these strains ranged from 2-8 µg/mL while for non ESBL producer sensitive counterparts it ranged from 0.03-1 µg/mL. Resistance to cefotaxime was transferred to recipient E. coli K12 strains J62-1. All the K. pneumoniae isolates were sensitive to imipenem. Resistance against amoxicillin, gentamicin, ciprofloxacin and amikacin was found in 93.28, 70, 10.37 and 26.14% of the isolates respectively. CONCLUSIONS: Our study shows presence of ESBL producer K. pneumoniae in clinical isolates. The routine antimicrobial sensitivity test may fail to detect ESBL mediated resistance against 3GC and detection of ESBL production should be carried out as a routine in diagnostic laboratories by PCDDT as it is a simple and cost effective test.

BACKGROUND: Iron deficiency has been shown to induce the expression of siderophores and their receptors, the iron-regulated membrane proteins in a number of bacterial systems. In this study, the response of Leptospira biflexa serovar Patoc strain Patoc I to conditions of iron deprivation was assessed and the expression of siderophores and iron-regulated proteins is reported. MATERIALS AND METHODS: Two methods were used for establishing conditions of iron deprivation. One method consisted of addition of the iron chelators ethylenediamine-N, N'-diacetic acid (EDDA) and ethylenediamine di-o-hydroxyphenylacetic acid (EDDHPA) and the second method involved the addition of iron at 0.02 µg Fe/mL. Alternatively, iron sufficient conditions were achieved by omitting the chelators in the former method and adding 4 µg Fe/mL of the medium in the latter protocol. Triton X-114 extraction of the cells was done to isolate the proteins in the outer membrane (detergent phase), periplasmic space (aqueous phase) and the protoplasmic cylinder (cell pellet). The proteins were subjected to SDS-PAGE for analysis. RESULTS: In the presence of the iron-chelators, four iron-regulated proteins (IRPs) of apparent molecular masses of 82, 64, 60 and 33 kDa were expressed. The 82-kDa protein was seen only in the aqueous phase, while the other three proteins were seen in both the aqueous and detergent fractions. These proteins were not identified in organisms grown in the absence of the iron chelators. The 64, 60 and the 33 kDa proteins were also demonstrated in organisms grown in media with 0.02 µg Fe/mL. In addition, a 24 kDa protein was found to be down-regulated at this concentration of iron as compared to the high level of expression in organisms grown with 4 µg Fe/mL. The blue CAS agar plates with top agar containing 0.02µg Fe/mL showed a colour change to orange-red. CONCLUSION: The expression of siderophores and iron-regulated proteins under conditions of iron deprivation was demonstrated in the non-pathogenic L.biflexa serovar Patoc.

PURPOSE: Acinetobacter spp. is an emerging important nosocomial pathogen. Clinical isolates of this genus are often resistant to many antibiotics. The in vitro susceptibility of Acinetobacter isolates obtained from patients were tested for currently used antibiotics. In addition, the study aimed at biotyping of Acinetobacter baumannii. METHODS: A total of 66 isolates were phenotypically characterised through a large panel of 25 carbon assimilation tests and susceptibility through disc diffusion method with 10 antimicrobial agents were tested. MICs were determined only for second line broad-spectrum drugs such as cefotaxime, ceftazidime, amikacin, ciprofloxacin, and ofloxacin using NCCLS guidelines. RESULTS: Multiple drug resistance (MDR) was only witnessed in A. baumannii and not in other Acinetobacter species. Aminoglycosides such as amikacin, netilmicin were most active against the MDR isolates tested (60% susceptibility). Ceftazidime was more active than cefotaxime. MDR A. baumannii strains were susceptible only to amikacin, netilmicin and ceftadizime. Ciprofloxacin had poor activity irrespective of isolates belonging to different DNA groups tested (58% resistance overall, 79% among A. baumannii). Strains of Biotypes 6 and 19 of A. baumannii showed broader resistance than those of biotype 10 and others. CONCLUSIONS: Strains of A. baumannii from patients in our hospital, were generally more resistant to quinolones, -lactam antibiotics, first and second generation cephalosporins and partially resistant to third generation cephalosporins and aminoglycosides. The strains belonging to other DNA groups of Acinetobacter were comparatively less resistant than A.baumannii, except ciprofloxacin. This study suggests that, a combination therapy, using a third generation cephalosporin and amikacin, would be best choice for treating Acinetobacter infections.

This study was done to compare the ability of a newly developed rapid malaria test OPtiMAL, an immunochromatographic antigen detection assay for the diagnosis of malaria using parasite lactate dehydrogenase, against standard microscopy. Blood samples were obtained from 232 patients suspected of having malaria. A total of 122 samples (52.5%) were positive by blood films while 118 (50.8%) were positive by OPtiMAL test. The blood film indicated that 21.4% (26 of 122) of the patients were positive for P. falciparum and 78.6% (96 of 122) were infected with P. vivax. OPtiMAL test showed that 21.2% (25 of 118) were positive for P.falciparum and 78.8% (93 of 118) were infected with P. vivax. This assay had sensitivities of 88.4% and 96.8% compared to traditional blood films for detection of P.falciparum and P.vivax malaria respectively. Thus OPtiMAL test can be used with or without traditional blood film examination for detection of both P. vivax and P. falciparum malaria and can be effectively used for the rapid diagnosis of malaria.

The treatment guidelines are generally decided on the basis of either percent resistant (%R) or percent sensitive (%S) bacterial population tested with a given antimicrobial that vary geographically and represent only a part of total bacterial population existing in response to the antimicrobial used. The isolates with intermediate sensitivity (%I) are either not reported or clubbed with resistant isolates though the two may differ in clinical response. Sensitivity Index (SI) of an antimicrobial is sensitive to change in any of the three co-existing bacterial population and may be a better criterion for rational use of antimicrobial.

Fluoroquinolones are antibiotics that are very effective against many gram negative microorganisms, including P. aeruginosa. However, resistance to these antibiotics has been reported in recent years as well. In this study, the sensivity of 136 P. aeruginosa strains, isolated from various clinical materials, to fluoroquinolones has been investigated. The lowest resistance rate was in ciprofloxacin with 12.5%. The resistance rates of the others were as follows: norfloxacin 14.7%, levofloxacin 16.9%, ofloxacin 19.9% and pefloxacin 28.7%. The 88.2% of the resistant strains to all fluoroquinolones were originated from intensive care unit.

World over antimicrobial resistance is a major public health problem. The WHONET software program puts each laboratory data into a common code and file format, which can be merged for national or global collaboration of antimicrobial resistance surveillance. In this study, antimicrobial sensitivity of 4,289 bacterial isolates was studied by Kirby-Bauer disk diffusion method. -lactamase production was assessed by iodometric test method. Extended spectrum -lactamase (ESBLs) were screened by ceftazidime disk sensitivity. Drug resistance was high in most of the isolates. It was maximum (80-94%) for ampicillin, nalidixic acid and cotrimoxazole. It varied between 40-60% for gentamicin, clindamycin, fluoroquinolones and coamoxyclav. It ranged from 21 to 38% for amikacin and third generation cephalosporins. Constitutive -lactamase production was highest in S.aureus (28.9%) and ESBL production was maximum in Klebsiella spp. (53.6%). WHONET software has in-built analysis program which helps in forming hospital drug policy, identification of hospital outbreaks and recognition of quality control problems in the laboratory.

Microsporidia (Enterocytozoon bieneusi) and Cyclospora cayetanensis have been reported worldwide causing diarrhoea in AIDS patients. Stool samples from HIV infected patients were subjected to routine examination for parasites, followed by special staining techniques to detect microsporidia and Cyclospora cayetanensis. Confirmed positive cases of these parasites were further processed for electron microscopy identity of the parasites and characteristic details. Scanning and transmission electron microscopy showed better morphological and structural details of the parasites.

Ocular nocardiosis is an opportunistic infection and is believed to be a rare entity. We describe a rare case report of a patient with culture-positive Nocardia asteroides canaliculitis who presented with complaints of watering, purulent discharge and painful swelling of left lower eye lid. A purulent tenacious material was expressed from the punctum of lower eye lid and subjected to microbiological investigations such as smears and cultures. Smears and culture proved the presence of Nocardia asteroides in the sample collected from punctum.

We report here a case of keratomycosis by Exserohilum rostratum. An 18-year old female patient presented with pain and watery discharge from the left eye since 10 days. Clinically the case was diagnosed as keratomycosis. Gram stain and KOH preparation of corneal scrapings revealed fungal elements. Fungal isolate was identified as Exserohilum rostratum by standard techniques.

A case of Salmonella typhi isolated from L4-L5 spine is reported here. The causative organism was not suspected preoperatively. The patient responded favourably to surgical drainage and appropriate antibiotic therapy.