Molecular & phenotypic characterization of Staphylococcus epidermidis in implant related infections.

Background & objectives: The discrimination between the Staphylococcus epidermidis colonizing the deep seated indwelling devices and those which are mere commensals has always been a challenge for the clinical microbiologist. This study was aimed to characterize the S. epidermidis isolates obtained from device related infection for their phenotypic and molecular markers of virulence and to see whether these markers can be used to differentiate the pathogenic S. epidermidis from the commensals. Methods: Fifty five S. epidermidis isolates from various device related infections such as endophthalmitis following intra-ocular lens (IOL) implantation, intravascular (IV) catheter related sepsis and orthopaedic implant infections, were studied for slime production, biotyping, antibiotic sensitivity; and mec A and ica positivity by the recommended procedures. Results: Twenty three (41.8%) isolates were multi-drug resistant, 26 (65.2%) were slime producers, 30 (54.5%) were adherent, 23 (41.8%) possessed the intercellular adhesin (ica) gene, and 28 (50.9%) harboured the mec A gene. Biotypes I and III were the commonest, most members of which were multi- drug resistant. Twenty two (73.3%) of the 30 adherent bacteria were slime producers as opposed to only 4 (16%) of the 25 non-adherent bacteria (P<0.001). A vast majority i.e. 21 (91.3%) of the 23 ica positive organisms were adherent to artificial surfaces in contrast to only 9 (28.1%) of the 32 non-ica positive organisms (P<0.001). Twenty (86.9%) of the 23 ica positive bacteria were slime producers, as opposed to only 6 (18.7%) of the 32 ica negative bacteria (P<0.001). Of the 23 multi-drug resistant isolates, 19 (82.6%) carried the mec A gene. Interpretation & conclusions: The present findings showed that ica AB and mec A were the two important virulence markers of S. epidermidis in implant infections and slime was responsible for the sessile mode of attachment on the devices.

The role of a commercial enzyme immuno assay antigen detection system for diagnosis of C. trachomatis in genital swab samples.

In the present pilot study, endocervical and urethral swabs collected from 100 patients attending sexually transmitted disease (STD) clinics and regional centre for STD in two referral hospitals in New Delhi were analyzed by enzyme immune assay (EIA), polymerase chain reaction (PCR) and direct fluorescent antibody (DFA) for detection of C. trachomatis. It was found that EIA could detect a very low number of cases (3/100) as against DFA (11/100) and PCR (9/100). Thus, in spite of the widespread availability, lower cost and ease of performance of the enzyme-linked-immunosorbent serologic assay, the present study highlights the need to employ sophisticated diagnostic tools like DFA and PCR for detection of Chlamydia trachomatis in STD patients.

Ultrastructural analysis of slime positive & slime negative Staphylococcus epidermidis isolates in infectious keratitis.

BACKGROUND & OBJECTIVE: Slime is a major determinant of Staphylococcus epidermidis adherence.The established methods of laboratory detection of slime production by this organism i.e., Christensen's tube method and congo red agar plate method, can both yield inconclusive and/or intermediate results. We, therefore tried to find out electronmicroscopically the localization of slime in relation to the bacterial cell wall and look for the effect, if any of the slime location on the staphylococcal adherence as well as on the quantum of slime production. METHODS: A total of 132 coagulase negative staphylococci from cases of infectious keratitis were identified as S. epidermidis following the recommended protocol. Slime was detected both by Christensen's tube method and congo red agar plate method. Antibiotic sensitivity testing was performed by standardized disc diffusion method. Adherence of the organisms to artificial surfaces was determined by a quantitative method and transmission electron microscopy was carried out by the conventional techniques. RESULTS: Of the total 132 isolates, 57 (43.2%) were slime positive and 75 (56.8%) were slime negative.Twenty seven (47.4%) of the 57 slime producing organisms were multi drug resistant as compared to only 12 (16%) of 75 nonslime-producing organisms (P<0.001). A majority i.e., 45 (78.9%) of 57 adherent organisms were slime producers as against 12 (16%) of 75 nonadherent organisms. Electron microscopic study revealed a thick viscid layer of slime anchoring to the bacterial cell wall, especially in adherent organisms and those yielding positive slime test. Some of the organisms showed loose nonadherent slime and those were mostly nonadherent to artificial surfaces. INTERPRETATION & CONCLUSION: Slime and multi drug resistance were the important virulence factors of S. epidermidis in bacterial keratitis. It was the adherent slime (i.e., slime in intimate association with the bacterial cell wall as shown by electron microscopy) only, which was responsible for resistance to multiple antibiotics and for the adhesion phenomenon observed in the quantitative slime test.

A simple alternative method for rapid detection of slime produced by Staphylococcus epidermidis isolates in bacterial keratitis.

BACKGROUND & OBJECTIVES: Slime is a known virulence factor of Staphylococcus epidermidis. The conventional Christensen's method for detection of slime in the laboratory takes at least 48 h. We, therefore, tried to evaluate the efficacy of the Congo red agar method as a routine procedure for detecting slime among isolates from corneal ulcers. METHODS: A total of 244 isolates from corneal ulcers were identified as S. epidermidis by the standard procedures. Slime was detected both by the conventional Christensen's method as well as by the Congo red agar method. RESULTS: Ninety two (37.7%) isolates were positive and 86 (35.2%) were negative for slime by both the techniques. Fifty four (22.1%) isolates were positive in Congo red agar, but negative by Christensen's method; whereas only 12 (4.9%) were negative by Congo red but positive by Christensen's method. Detection of slime by Congo red agar method was rapid i.e., all the 146 strains were positive within 24 h of incubation. On the other hand, Christensen's method had a delayed response; 42.3 per cent (44/104) strains being negative during the first 24 h of incubation. INTERPRETATION & CONCLUSION: Our results suggested that culture on Congo red agar was a sensitive and rapid test for detecting slime. This might help in the quick identification in a routine laboratory of slime positive isolates in bacterial keratitis.

Species specific chlamydiae antibodies in voluntary blood donors of Delhi.

BACKGROUND & OBJECTIVES: Different species of genus Chlamydia have been associated with ocular, genitourinary and respiratory infections, and coronary artery disease. Since the majority of these infections remain asymptomatic or subclinical, antibodies may be present in apparently healthy individuals, and the determination of species specific Chlamydia antibodies in a healthy population may reflect exposure. We therefore screened the sera of healthy blood donors for species specific Chlamydia antibodies by microimmunofluorescence assay. METHODS: Sera of 844 voluntary blood donors from Delhi were screened by microimmunofluorescence assay using specific antigens of C. trachomatis (18 serovars divided in 3 pools of serotypes A-C, D-K and L1-L3), C. psittaci and C. pneumoniae for Chlamydia antibodies. RESULTS: A total of 470 (55.69%) blood donors were found positive for Chlamydia antibodies. Of these, 361(42.77%) were positive for C. pneumoniae, 106 (12.5%) for C. trachomatis [of which 72 (8.5%) were against serotypes D-K and 34(4%) were against serotypes A-C]. There donors (0.3%) had antibodies to C. psittaci. INTERPRETATION & CONCLUSION: The results suggested that more than half of the study population (55.69%) is exposed to one or other species of chlamydiae. Majority of the donors (44.7%) had C. pneumoniae antibodies, suggesting the presence of widespread apparent or inapparent C. pneumoniae infection. The findings also suggest that Chlamydia antibody testing for the diagnosis of chlamydial infections may not be helpful due to the presence of antibodies in a large proportion of healthy individuals.

Slime production as a virulence factor in Staphylococcus epidermidis isolated from bacterial keratitis.

A total of 126 coagulase negative staphylococci (CONS) isolated from corneal scrapings of patients of bacterial keratitis and 50 isolates from healthy eyes (controls) were tested for slime production. Eighty eight (69.84%) of 126 isolates from patients and 11 (22%) of 50 isolates from controls were slime producing (P < 0.001). Of these 88 isolates, 42 were Staphylococcus epidermidis biotype II, 30 were S. epidermidis biotype I, 8 were S. epidermidis biotype III and the rest belonged to CONS other than S. epidermidis. Amongst the corneal ulcer isolates, multidrug resistance (resistance to 3 or more antibiotics) was observed in 82.9 per cent (73/88) slime producing organisms as against only 18.4 per cent (7/38) nonslime producing organisms (P < 0.001). Similarly, of the total 99 slime positive and 77 slime negative isolates, 79 (79.8%) and 22 (28.6%) respectively were multidrug resistant (P < 0.001). Although, slime production is known to be one of the major virulence factors of CONS in extraocular systemic staphylococcal infections, the present study detected slime in isolates from ocular infections. It was found that S. epidermidis I and II were the common biotypes associated with bacterial keratitis and, slime production and multidrug resistance were the two important virulence factors. These observations have clinical and therapeutic significance.

An epidemic of viral acute haemorrhagic conjunctivitis in Delhi in 1994.

An epidemic of acute haemorrhagic conjunctivitis affecting persons of all ages and both sexes occurred in Delhi and surrounding areas during the monsoon season of 1994. The symptoms lasted on an average for 4-5 days. In some of the patients corneal involvement was observed. Conjunctival swabs from the affected patients were processed for viral antigen detection, virus isolation and bacterial culture and sensitivity. Viral antigen was detected in 62% (31/50) of the smears tested by indirect immunofluorescence assay. In 22 (44%) of the specimens Coxackie A 24 (Cox A 24) virus antigen and in 9 (18%) of the specimens Entero Virus 70 (EV 70) antigen were detected. In confluent monolayers of Hep 2 cells cytopathic virus was isolated in 10 (30.30%) of the 33 specimens processed. The isolated viruses were identified as either Cox A 24 (7 isolates) or EV 70 (3 isolates) using indirect immunofluorescence assay. Super added bacterial infection was observed in 33% (89/270) of the cases, Staphylococcus albus being the predominant bacteria isolated.

Development of an antigen capture enzyme immuno assay using genus specific monoclonal antibodies for detection of Chlamydia in clinical specimens.

An antigen capture enzyme immuno assay (EIA) for Chlamydia antigen detection was developed using polyclonal rabbit immunoglobulins against C. trachomatis, a genus specific antichlamydial murine monoclonal antibody (IgG) against major outer membrane protein (MOMP) antigen of C. trachomatis and a commercial anti mouse IgG immunoglobulin conjugated with HRPO. The test was evaluated against a direct immunofluorescence assay (DFA). Conjunctival specimens from 178 patients with follicular conjunctivitis and cervical specimens from 82 patients with cervicitis were tested for Chlamydia antigen detection by both the tests. Chlamydia antigen was detected in 69/178 (38.76%) and 68/178 (38.20%) of the conjunctival specimen by using EIA and DFA tests respectively. It could also be detected in 24/82 (29.27%) and 22/82 (26.83%) of the cervical specimens by EIA and DFA tests respectively. The sensitivity of the EIA test was 92.64 per cent and 86.36 per cent for the conjunctival and cervical specimens respectively against the reference DFA test. The specificity of the EIA test was found to be 94.54 and 91.66 per cent respectively against the reference DFA test.

Development & use of antichlamydial monoclonal antibodies for rapid diagnosis of follicular conjunctivitis.

We developed four independent murine hybrid clones producing IgG class and genus specific monoclonal antibodies against the major outer membrane protein (MOMP) of Chlamydia trachomatis, L2 serovar. All antibodies reacted with a single epitope of MOMP. In indirect immunofluorescence assay using one of the antibodies chlamydiae could be detected in 12 of 23 conjunctival scrapings from patients of follicular conjunctivitis. Giemsa staining could detect classical inclusion bodies in only 5 of these 23 smears. Antigen detection using these monoclonal antibodies may be used as a rapid diagnostic test in clinical and epidemiological screening for chlamydial infection.