Early onset primary pulmonary cryptococcosis in a renal transplant patient.

We report a case of primary pulmonary cryptococcosis in a post-renal transplant patient. A 65-year-old male renal transplant patient was admitted to the hospital with a low grade fever of 1 month, radiologically mimicking tuberculosis (TB). Broncho-alveolar fluid (BAL) shows capsulated yeast, and Cryptococcus neoformans was grown on culture supported by cytology and histopathological examination. Cryptococcal antigen was positive (32-fold) in serum and was negative in cerebrospinal fluid (CSF). The patient was given amphotericin B and 5-flucytosine and clinical improvement was seen on a weekly follow up. The serum cryptococcal antigen test might contribute to the early detection and treatment of pulmonary cryptococcosis. The results of antifungal susceptibility were aid in selecting the drug of choice for treatment.

Antifungal susceptibility testing method for resource constrained laboratories.

PURPOSE: In resource-constrained laboratories of developing countries determination of antifungal susceptibility testing by NCCLS/CLSI method is not always feasible. We describe herein a simple yet comparable method for antifungal susceptibility testing. METHODS: Reference MICs of 72 fungal isolates including two quality control strains were determined by NCCLS/CLSI methods against fluconazole, itraconazole, voriconazole, amphotericin B and cancidas. Dermatophytes were also tested against terbinafine. Subsequently, on selection of optimum conditions, MIC was determined for all the fungal isolates by semisolid antifungal agar susceptibility method in Brain heart infusion broth supplemented with 0.5% agar (BHIA) without oil overlay and results were compared with those obtained by reference NCCLS/CLSI methods. RESULTS: Comparable results were obtained by NCCLS/CLSI and semisolid agar susceptibility (SAAS) methods against quality control strains. MICs for 72 isolates did not differ by more than one dilution for all drugs by SAAS. CONCLUSIONS: SAAS using BHIA without oil overlay provides a simple and reproducible method for obtaining MICs against yeast, filamentous fungi and dermatophytes in resource-constrained laboratories.

Detection of biofilm formation among the clinical isolates of Staphylococci: an evaluation of three different screening methods.

PURPOSE: The purpose of this study was to evaluate three methods for detection of biofilm formation in staphylococci. METHODS: For detection of biofilm formation, 152 clinical isolates of Staphylococcus spp. were screened by tissue culture plate (TCP), Tube method (TM) and Congo red agar (CRA) method. RESULTS: Of the 152 Staphylococcus spp. 88(57.8%) displayed a biofilm-positive phenotype under the optimized conditions in the TCP method and strains were further classified as high 22 (14.47 %) and moderate 60 (39.4 %) while in 70 (46.0 %) isolates weak or no biofilm was detected. Though TM correlated well with the TCP test for 18 (11.8 %) strongly biofilm producing strains, weak producers were difficult to discriminate from biofilm negative isolates. Screening on CRA does not correlate well with either of the two methods for detecting biofilm formation in staphylococci. CONCLUSION: The TCP method was found to be most sensitive, accurate and reproducible screening method for detection of biofilm formation by staphylococci and has the advantage of being a quantitative model to study the adherence of staphylococci on biomedical devices.

Evaluation of methods for AmpC beta-lactamase in gram negative clinical isolates from tertiary care hospitals.

The purpose of this study was to simultaneously screen for Extended-spectrum beta-lactamases (ESBL) and AmpC beta-lactamases in gram negative clinical isolates from four tertiary care hospitals and further to compare two detection methods three-dimensional extraction method and AmpC disk test for AmpC beta-lactamases. A total of 272 isolates were screened for ESBL and AmpC beta-lactamase by modified double disk approximation method (MDDM). Synergy observed between disks of ceftazidime/cefotaxime and clavulanate were considered as ESBL producer. Isolates showing reduced susceptibility to either of the test drugs (ceftazidime or cefotaxime) and cefoxitin were considered as presumptive AmpC producers and further confirmed by three-dimensional extraction method and AmpC disk test. A total of 173 (64%) of the isolates were found to be ESBL positive and 61 (23%) showed resistant to cefoxitin. ESBL was detected in 80 (62%) isolates of E. coli and 71 (73%) of Klebsiella spp. The occurrence of AmpC beta-lactamases was found to be 8% (22) of the total isolates and the two detection methods for AmpC beta-lactamase showed concordant results. Screening for ESBL and AmpC can be simultaneously done by MDDM method and confirmation for AmpC beta-lactamase should be carried out routinely in tertiary care hospitals by AmpC disk test, as it is a simple and rapid procedure.

Detection of vancomycin resistant Staphylococcus aureus: a comparative study of three different phenotypic screening methods.

The objective of this study was to investigate screening methodologies, to detect Staphylococcus aureus strains with decreased susceptibility to vancomycin. Three methods were used to screen 160 Staphylococcus aureus clinical isolates along with ATCC quality control strains. Subsequently, MIC of all these 160 strains were determined by NCCLS methodology. The MIC of all the 160 clinical isolates was < or = 4 microg/mL and were classified as vancomycin susceptible by NCCLS criteria but 23 strains were positive by Hiramatshu method, two grew on MHA (5 microg/mL vancomycin) while CDC method correctly identified no vancomycin intermediate S.aureus (VISA) or vancomycin resistant S.aureus (VRSA) strains with reference to there MIC. CDC method was found to be the most appropriate screening methodology for detection of VISA or VRSA for diagnostic laboratories.

Mechanisms of resistance to fluoroquinolones.

Fluoroquinolones have some of the properties of an 'ideal' anti-microbial agent. Because of their potent broad spectrum activity and absence of transferable mechanism of resistance or inactivating enzymes, it was hoped that clinical resistance to this useful group of drugs would not occur. However, over the years, due to intense selective pressure and relative lack of potency of the available quinolones against some strains, bacteria have evolved at least two mechanisms of resistance: (i) alteration of molecular targets, and (ii) reduction of drug accumulation. DNA gyrase and topoisomerase IV are the two molecular targets of fluoroquinolones. Mutations in specified regions (quinolone resistance-determining region) in genes coding for the gyrase and/or topoisomerase leads to clinical resistance. An efflux pump effective in pumping out hydrophilic quinolones has been described. Newer fluoroquinolones which recognize both molecular targets and have improved pharmacokinetic properties offer hope of higher potency, thereby reducing the probability of development of resistance.

Antibodies to Cag A protein are not predictive of serious gastroduodenal disease in Indian patients.

OBJECTIVE: The present study was aimed at assessing the predictive utility of anti-Cag A antibodies in differentiating patients of duodenal ulcer (DU) and non ulcer dyspepsia (NUD) from asymptomatic controls in a developing country. METHODS: Sera from 120 subjects were tested for antibodies to Cag A using the immunodominant portion of a recombinant 37.5 kDa fusion protein by ELISA, in endoscopically proven cases of DU and NUD and healthy controls. RESULTS: The observed optical density (OD) in DU and NUD patients was 1,947 and 1,960 respectively, which was higher than that observed in controls (p < 0.01), but there was no difference in the anti-Cag A antibody titers between DU and NUD patients. CONCLUSION: Anti-Cag A antibodies do not seem to discriminate duodenal ulcer patients from non ulcer dyspepsia in the Indian population.

Study of Pseudomonas aeruginosa causing ventilator associated pneumonia.

Ps. aeruginosa is a frequent and prominent cause of nosocomial pneumonia especially in persons on assisted ventilation in the intensive care units. In a year long surveillance of ventilator associated pneumonia (VAP) we isolated 42 strains from broncho alveolar lavage samples collected and processed from 102 patients. By pyocin typing 40 of the 42 strains could be typed into 39 types but this designation changed each time the test was repeated. SDS-PAGE analysis of the whole cell proteins grouped the 42 strains of Ps. aeruginosa into 20 groups. After ribotyping, using an 18 mer DIG labelled oligonucleotide to the conserved region of 16S rRNA gene, the strains were designated into 18 types. The major type contained 8 isolates, but there was no clustering of isolates, indicating that each infecting strain was acquired separately and not from a common source. It would, therefore, appear that cross infection with a single clone was not the predominant mode of Ps. aeruginosa infection causing VAP in our ICU.

Role of circulating immune complexes in prognostic evaluation and management of genitourinary cancer patients.

Circulating immune complexes (CIC) were estimated in 48 Patients of genitourinary cancer by polyethylene glycol precipitation (PEG pptn.) test and latex agglutination inhibition (LAI). The results were compared with 25 healthy control volunteers. Pathological levels of CIC were observed in 79.18 percent patients of genitourinary cancer by combination of PEG pptn. and LAI tests, while no seropositivity for CIC was observed in control group (p < 0.001). Sequential increase in seropositivity for CIC was observed with advancing stage of genitourinary cancer i.e. number of seropositive patients in cancer stage I were 60 percent, stage II-71.42 percent, stage III-85.71 percent and stage IV-100 percent. Variation IN CIC levels in different patients within the same stage are compared. Circulating antigen antibody complexes have a significant role as prognostic monitors in management of genitourinary cancer patients. Statistical evaluation of data on intra- and inter-assay variation has been given. CIC levels rise with increases in tumor burden in vivo hence variation in CIC levels within the same stage in different patient have a significant role as prognostic monitors in management of individual patients.

Comparative evaluation of two indigenously developed tests for rapid detection of group-A streptococci directly from throat swabs.

The efficacy of two indigenously developed rapid tests, latex agglutination and antibody capture assay (a colour immunochromatographic assay) to detect group A Streptococcus pyogenes (GAS) was evaluated in comparison with the traditional culture method ('Gold Standard') in both asymptomatic children (1500) and symptomatic patients (233). Throat swabs were taken in duplicate and rapid tests performed on one swab and culture on the other. Both latex agglutination and antibody capture assays showed a sensitivity of 100 per cent and specificity of > 98 per cent as compared to culture and isolation in symptomatic patients. However, among asymptomatic carriers sensitivity of 100 per cent and 87.5 per cent and specificity > 95 per cent were observed for latex agglutination and antibody capture assays respectively. Latex agglutination showed no false negative results and sensitivity was not affected by low beta-haemolytic counts in asymptomatic children. The rapid tests described here will help in the detection and thereby the management of GAS infection.

Legionella as a lower respiratory pathogen in north India.

One hundred patients of lower respiratory tract infection (LRTI) were prospectively studied over 2 years to find out if Legionella is a causative agent in these patients. In addition, 50 environmental samples and 50 age and sex matched controls were studied. Culture and direct fluorescent antibody testing (DFA) of respiratory tract secretions, and serodiagnosis by indirect immunofluorescence (IIF) and ELISA, were employed to detect Legionella. Respiratory tract secretions from all patients were negative for Legionella on culture and DFA. Low antibody titters to Legionella were observed in 21 patients and these could be attributed to cross reaction with other gram-negative bacteria. All environmental samples and controls tested negative for Legionella. Legionella does not seem to be an important lower respiratory tract pathogen in this part of the country and empirical addition of erythromycin to treatment regimens for pneumonia is not warranted in our setting.

A preliminary study of fingerprinting of Pseudomonas aeruginosa by whole cell protein analysis by SDS-PAGE.

Forty two strains of Pseudomonas aeruginosa isolated from bronchoalveolar lavage fluid from intubated patients admitted to the Intensive care unit in AIIMS between December 1993 to June 1994 were included in the study. After obtaining typical biochemical profile, antimicrobial susceptibility was performed against ceftazidime, amikacin, gentamicin, ampicillin, cefotaxime and ciprofloxacin. Pyocin typing of these 42 strains was performed by scrape and streak method using 22 indicator strains. Forty strains could be typed showing excellent discrimination but on repeated testing the group designation changed indicating that the system had low reproducibility. SDS-PAGE of whole cell protein profile indicated the presence of 45 protein bands of different molecular weights, individual isolates had 37 to 42 protein band ranging in molecular weight from 340 kDa to 14.3 kDa. On the basis of Dice index of similarity the strains could be grouped into 20 types. Since all strains could be typed, the system has adequate typability. Similar results were obtained on repeated testing indicating good reproducibility.

Risk factors for wound infection following elective cholecystectomy.

A study of risk factors for wound infection among patients undergoing elective cholecystectomy was undertaken. Over a 2-Year period 177 patients who underwent elective cholecystectomy for symptomatic gall stone disease were randomized into groups, one receiving antibiotics (96 patients) and the other not receiving antibiotics (81 patients). Gall bladder bile and wound swab were cultured to detect bacterial growth. Duration of preoperative hospital stay, type of skin incision and operating time were noted for each patient. Postoperatively wound infection developed in 22/177 (12%) patients. The infection rate was lower in the antibiotic group 3/96 (3%) as compared to the non-antibiotic group 19/81 (23.5%). Wound sepsis occurred in 11/37 (23%) of patients with bactibilia as compared to 11/140 (7.8%) patients with sterile bile. Stepwise logistic regression analysis showed that bactibilia and use of prophylactic antibiotics were the most significant predictors of wound infection in low risk patients undergoing elective cholecystectomy.

A pilot programme of MRSA surveillance in India. (MRSA Surveillance Study Group).

This surveillance study was conducted simultaneously at three centres across India. A total of 13,610 test samples from various sites were obtained. Microbiological methods employed were similar at the three centres. Identification of S aureus was based on the recognition of the production of coagulase with positive isolates being recorded as S aureus. Both tube coagulase tests and slide coagulase test were performed. Antimicrobial susceptibility testing of the isolated strains of staphylococcus aureus and staphylococcus epidermidis to various antimicrobial discs were carried out according to standardized disk diffusion method recommended by NCCLS. Of the total 739 cultures of S aureus, 235 (32%) were found to be multiply resistant with the individual figures for resistance being 27% (Bombay), 42.5% (Delhi) and 47% (Bangalore). MRSA is emerging to be a significant problem pathogen in the surgical setting with vancomycin probably the only reliable choice for these infections.

Prognostic significance of circulating immune complexes in malignant tumours of head and neck.

Circulating immune complexes (CIC) were estimated in 31 cases of head and neck malignancies by polyethylene glycol (PEG) precipitation and latex agglutination inhibition (LAI) techniques. Results were compared with 25 age and sex-matched control volunteers. Seropositivity for CIC by PEG precipitation test was 54.83% compared to 61.29% by LAI test. No positive case was detected in control group. Seropositivity for CIC by combination of both test results was 67.74%. In stage I cancer seropositivity for CIC by both techniques was 33.33%. In stage II it was 36.36% by PEG precipitation test and 45.45% by LAI test. In stage III it was 64.28% by PEG precipitation test and 71.42% by LAI test. In stage IV all cases were seropositive by both techniques. LAI test was more sensitive in CIC detection in cancer stages II and III. PEG precipitation test had the advantage of detecting quantitative CIC levels by PEG index. Follow-up in 10 postoperative cases revealed significant decline in CIC levels by both tests (p < 0.01) after 3 months of surgery; 90% patients became seronegative for CIC by PEG precipitation technique while 80% became seronegative by LAI technique. However, 15 patients were followed up after 3 1/2 years of surgery; 75% patients remained seronegative while 25% patients became seropositive by both tests and manifested clinical recurrences.