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Biomedical subjects

K N Brahmadathan

Publications and source records attributed to K N Brahmadathan.

At least 19 recordsLinked to original sources

Microbiological diagnosis of streptococcal pharyngitis: lacunae and their implications.

Post-streptococcal sequelae, especially acute rheumatic fever/rheumatic heart disease continue to occur in significant proportions in many parts of the world. Despite several attempts with various intervention strategies, little success has been achieved in the control of acute rheumatic fever/rheumatic heart disease in India. The success of the control programmes depends upon timely primary prophylaxis with benzathine penicillin for which a microbiological confirmation of group A streptococcal pharyngitis is essential. Isolation of beta hemolytic streptococci from throat cultures and their identification as GAS in the laboratory, clinches the microbiological diagnosis while demonstration of a 'significant rise' in antibody titers such as Anti-streptolysin O and Anti-deoxyribonuclease B differentiates it from a group A streptococcal carrier state or pharyngitis of a viral etiology. Despite the easiness with which these can be achieved, many laboratories in India are not equipped to do so. Enhancing bacteriological and serological facilities in laboratories across the country will drastically improve the clinician's ability to diagnose bona fide GAS pharyngitis and help to institute penicillin prophylaxis at the appropriate time. This will go a long way in enhancing the compliance to penicillin prophylaxis which is the cornerstone of any RF/RHD control program.

Culture Media↗

Evaluation of simplified DNA extraction methods for emm typing of group A streptococci.

Simplified methods of DNA extraction for amplification and sequencing for emm typing of group A streptococci (GAS) can save valuable time and cost in resource crunch situations. To evaluate this, we compared two methods of DNA extraction directly from colonies with the standard CDC cell lysate method for emm typing of 50 GAS strains isolated from children with pharyngitis and impetigo. For this, GAS colonies were transferred into two sets of PCR tubes. One set was preheated at 94 degrees C for two minutes in the thermal cycler and cooled while the other set was frozen overnight at -20 degrees C and then thawed before adding the PCR mix. For the cell lysate method, cells were treated with mutanolysin and hyaluronidase before heating at 100 degrees C for 10 minutes and cooling immediately as recommended in the CDC method. All 50 strains could be typed by sequencing the hyper variable region of the emm gene after amplification. The quality of sequences and the emm types identified were also identical. Our study shows that the two simplified DNA extraction methods directly from colonies can conveniently be used for typing a large number of GAS strains easily in relatively short time.

Antigens, Bacterial↗

Dynamics of anti-M antibody response in a mouse model following intranasal infection with group A Streptococcus M-18.

Dynamics of anti-M antibody response following intranasal infection with group A Streptococcus (GAS) M-18 were investigated in a Swiss albino mouse model. Mice arranged in three groups were inoculated intranasally with 2.0x10(7) c.f.u. ml-1 of GAS M-18 on 1, 2 alternate and 3 alternate days. Plasma collected from the retro-orbital plexus was tested for antibodies by an in-house indirect ELISA. The antibody titres of the plasma samples varied from 1 : 8 to 1 : 1024 in the 1 day dose, from 1 : 4 to 1 : 256 in the 2 day dose and from 1 : 4 to 1 : 128 in the 3 day dose. Peak titres were seen on day 42 or 56 and in all cases the titres had declined by day 84. Swiss albino mouse can thus serve as a useful animal model to study different aspects of type-specific anti-M immune responses against GAS disease when designing candidate streptococcal vaccines.

Animals↗

Antimicrobial susceptibility patterns of clinical isolates of Staphylococcus aureus in Nepal.

Isolates of Staphylococcus aureus (n = 117) from patients attending a tertiary care centre in western Nepal were tested for susceptibility to penicillin, oxacillin, gentamicin, erythromycin and ciprofloxacin. Eighteen (15.4%) were methicillin-resistant. Susceptibility among methicillin-resistant isolates varied from 0% (penicillin) to 16.6% (erythromycin and gentamicin), but varied among methicillin-susceptible isolates from 39.4% (penicillin) to 97.0% (ciprofloxacin). Fourteen (77.8%) of the methicillin-resistant isolates were resistant to all agents tested. Implementation of an appropriate antibiotic policy would reduce the risk of further development of antimicrobial resistance.

Anti-Bacterial Agents↗

Increasing erythromycin resistance among group A streptococci causing tonsillitis in a tertiary care hospital in southern India.

Group A streptococci (GAS) (n = 1313) isolated from patients with clinical symptoms of pharyngitis or tonsillitis attending a tertiary care hospital in southern India during 1986-2002 were tested for susceptibility to penicillin and erythromycin. The overall erythromycin resistance rate was 2.7% (n = 36). During 1986-1993, erythromycin resistance was observed in only one (2%) isolate in 1987, but reappeared in 1994 (2.7%), increased to 5.8% in 1999, and reached a maximum frequency of 13.8% in 2002. All isolates were susceptible to penicillin. The data indicate the need for continued surveillance of susceptibility patterns among GAS isolates in order to monitor the development of antibiotic resistance.

Drug Resistance, Bacterial↗

Outcome of bacterial culture from mastoid granulations: is it relevant in chronic ear disease?

OBJECTIVE: To detect the presence of bacteria in mastoid granulations and compare its prevalence in both types of chronic suppurative otitis media (CSOM). To find out if stage of disease activity, age, duration of disease, and aditus patency relate to obtaining positive cultures. STUDY DESIGN AND SETTING: A prospective, parallel group study done at a tertiary care referral centre. Mastoid granulations from 79 patients with CSOM undergoing mastoidectomy were processed for anaerobic and aerobic bacteria. RESULTS: Aerobes were isolated from 57.55 per cent of the tubotympanic and 74.4 per cent of atticoantral disease (p=0.18). Anaerobic cultures were positive in one case from each group. Monomicrobial growth was detected in 37.5 per cent of tubotympanic and 48.5 per cent of atticoantral disease. Polymicrobial growth occurred in 20 per cent and 25.6 per cent in the tubotympanic and atticoantral groups, respectively. The predominant aerobic isolate was coagulase negative Staphylococcus, followed by Pseudomonas aeruginosa, Staphylococcus aureus, non-fermenting Gram-negative bacteria, Enterobacter and Enterococcus, Proteus species, Citrobacter, non-pathogenic Neisseria, aerobic spore formers were grown only in atticoantral disease. A single isolate of Aspergillus was grown. Correlating the state of disease activity of the ears with positive mastoid granulation cultures, six out of the eight inactive ears were culture positive along with seven out of the nine active and 10 out of the 23 quiescent ears. Positive mastoid granulation cultures were obtained in 60 per cent of those with blocked aditus and 42.9 per cent with patent aditus. CONCLUSION AND SIGNIFICANCE: In this study, we found that mastoid granulations are not sterile but harbour polymicrobial pathogens. Positive cultures were obtained irrespective of stage of disease activity, age, duration of disease and aditus patency. The pattern of organisms cultured from safe and unsafe CSOM and also from ears in active, quiescent and inactive stages, were similar. These findings suggest that these organisms may be responsible for mastoid granulations. We also noted that positive cultures had no statistical correlation with aditus patency and duration of disease. We suggest further studies to evaluate the significance of asymptomatic mastoid granulations harbouring organisms and whether opening the mastoid antrum and achieving aditus patency, irrespective of the stage of disease activity, will help improve the long-term surgical outcome and also prevent recurrence of ear discharge.

Adult↗

Incidence of carbapenem resistant nonfermenting gram negative bacilli from patients with respiratory infections in the intensive care units.

Resistance to carbapenems is commonly seen in nonfermenting gram negative bacilli (NFGNB). We document herein the prevalence of carbapenem resistance in NFGNB isolated from patients with respiratory tract infections in the intensive care units (ICUs). A total of 460 NFGNB were isolated from 606 endotracheal aspirate specimens during January through December 2003, of which 56 (12.2%) were found to be resistant to imipenem and meropenem. Of these, 24 (42.8%) were Pseudomonas aeruginosa , 8 (14.2%) were Acinetobacter spp. and 24 (42.8%) were other NFGNB. Stringent protocols such as antibiotic policies and resistance surveillance programs are mandatory to curb these bacteria in ICU settings

Carbapenems↗

Detecting mycobacteraemia for diagnosing tuberculosis.

BACKGROUND & OBJECTIVES: In human immunodeficiency virus (HIV) infected persons with pulmonary tuberculosis (TB), sputum may not always show acid fast bacilli (AFB). Moreover, in most cases of suspected extrapulmonary TB (irrespective of HIV status) mycobacteria-containing material is not readily available for investigation. This study evaluated whether blood culture for Mycobacterium tuberculosis bacteraemia (mycobacteraemia) help in diagnosing TB in such cases. METHODS: A total of 93 consecutive subjects with a clinical diagnosis of tuberculosis with or without laboratory confirmation, 42 with and 38 without coexisting HIV infection, and 13 patients with HIV infection without clinical evidence of TB were enrolled. Mycobacterial blood cultures were done using lysis centrifugation technique followed by subculturing onto the modified Lowenstein-Jenson medium (LJ-1) and Selective Kirchner's medium followed by subculturing onto the modified Lowenstein-Jenson medium (LJ-2, LJ-3). RESULTS: Of the 15 (16.2%) subjects with evidence of mycobacteremia in 4 (26.7%) blood was the first/ only source of diagnosing TB. Among 80 patients with clinical diagnosis of TB whether supported by laboratory tests or not, 14 (17.5%) had mycobacteraemia. Among the 21 HIV infected patients with laboratory proven TB, 9 (43%) had mycobacteraemia. INTERPRETATION & CONCLUSION: Blood culture appears to be a useful additional test to diagnose TB in persons with HIV infection. In patients without HIV infection, but with clinical picture compatible with TB, blood culture for mycobacteraemia may occasionally help in the diagnosis. We recommend the use of the lysis centrifugation technique followed by direct smear of the sediment along with inoculation of the sediment into both modified Lowenstein-Jenson medium and the Selective Kirchner's medium with subsequent subculturing onto the modified Lowenstein-Jenson medium for mycobacterial blood culture for detecting mycobacteraemia.

Adolescent↗

Comparison of different culture media and storage temperatures for the long-term preservation of Streptococcus pneumoniae in the tropics.

OBJECTIVE: The preservation of Streptococcus pneumoniae by standard freezing methods for subsequent tests--such as serotyping and antibiotic susceptibility--is not possible or is difficult in many developing countries because of the high cost of equipment, inadequate equipment maintenance, and irregular power supply. We evaluated alternative low-cost methods, by comparing different culture media and storage temperatures. METHODS: Clinical isolates of five capsular types (1, 5, 7, 19, and 23) of S. pneumoniae were preserved in rabbit blood, sheep blood, skimmed milk, or glycerol-chocolate broth, and stored at -20 degrees C or -70 degrees C. The cultures were also preserved by lyophilization or sand desiccation, followed by storage at room temperature and 4 degrees C. The viability of the preserved cultures was determined by making serial colony counts on day 0 and after 1 week, 4 weeks, 4 months and 16 months. The viability of cultures preserved by sand desiccation and storage at 4 degrees C was also determined every 6 months for up to 68 months. FINDINGS: Irrespective of the media used, cultures maintained at -20 degrees C became nonviable by the fourth month, while those maintained at -70 degrees C were still viable at 16 months. Cultures preserved by lyophilization or sand desiccation lost their viability by the fourth month when maintained at local room temperature (30-42 degrees C), but remained viable when stored at 4 degrees C for up to 68 months. CONCLUSIONS: Our results confirm that freezing at -70 degrees C, or lyophilization and storage at 4 degrees C are the ideal methods for the preservation of S. pneumoniae. In laboratories where lyophilization is not feasible, sand desiccation and storage at 4 degrees C offers an alternative low-cost method for the long-term preservation of S. pneumoniae.

Cell Culture Techniques↗

Investigation of antimicrobial use pattern in the intensive treatment unit of a teaching hospital in western Nepal.

BACKGROUND: Inappropriate use of antimicrobials is of special importance in the intensive treatment unit because of the large number of drugs prescribed, the chance for drug errors, and the likelihood of development of drug resistance. METHODS: A total of 297 records of patients admitted to the intensive treatment unit of the Manipal teaching hospital, a tertiary care hospital in Pokhara, western Nepal, were studied to determine the prescribing frequency and rationality of use of antimicrobials. Patient outcome, duration of stay in the intensive treatment unit, and the age and sex distribution of the patients were also studied. RESULTS: Mean+/-SD drugs per patient was 3.4+/-1.8. About half (50.2%) of the patients received an antimicrobial; 84.6% of the antimicrobials were used without obtaining bacteriologic evidence of infection. The commonest organisms isolated on culture were Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, and Staphylococcus aureus. A total of 28.9% of the antimicrobials were prescribed for lower respiratory tract infections on the basis of the putative site of infection; 61.9% of the antimicrobials were prescribed by the parenteral route and mainly the older generation of antimicrobials were used. In 39 of the 149 patients prescribed an antimicrobial, the use was irrational. CONCLUSIONS: Prescriber education to improve prescribing patterns and regular auditing of antimicrobial prescriptions to prevent their inappropriate use and unnecessary cost to the patients are required. The high percentage of inappropriate use of antimicrobials raises concerns about the development and spread of drug resistance, which must be addressed.

Adolescent↗

Evaluation of serological tests for the diagnosis of tuberculosis.

To evaluate the use of antibody detection kits in the diagnosis of pulmonary tuberculosis in an endemic area, serum samples from cases (sputum smear positive for AFB) and controls (healthy young adults) were collected and tested using five different kits. Sensitivity, specificity and predictive values were calculated using smear positivity as gold standard. Sensitivity of tests varied from 46% to 68% and the specificity from 68% to 100%. None of the kits evaluated can be used as a single screening test for tuberculosis. However kits with good specificity may be used in conjunction with conventional methods for diagnosis.

Antigens, Bacterial↗

Typing of Acinetobacter baumannii isolated from hospital-acquired respiratory infections in a tertiary care centre in southern India.

In an attempt to define the epidemiology of Acinetobacter baumannii infection, 27 isolates, obtained from hospital-acquired respiratory infections, were typed using random amplified polymorphic DNA (RAPD) profile and antimicrobial susceptibility patterns. Ten different patterns were obtained with ERIC2 primer: 14 isolates had a similar profile representing a single strain. Within RAPD types, isolates could be further classified based on their antibiogram; however, strains of different types had similar antibiograms. This study showed that many different genetic types of A. baumannii are prevalent in our hospital. While antibiograms alone are not sufficiently discriminatory, RAPD typing helps in identifying outbreaks and in assessing infection control procedures within a hospital.

Academic Medical Centers↗

Persistent rhinorrhoea in rural Indian children: prevalence and consequences.

To determine whether persistent rhinorrhoea constitutes a significant problem requiring intervention, 17 rural day care centres (Balwadis) in Tamilnadu, India, were visited. Among 414 children in the Balwadis 92 (22 per cent) children with persistent rhinorrhoea (15 days duration or longer) were identified. Demographic and clinical data and nasopharyngeal swabs for bacterial culture were obtained from 56 such children and 91 age-matched controls from the same Balwadi. Type of housing or nutritional status did not appear to be significant risk factors. There was a significantly higher number of children aged 5-15 years in the household of cases as compared to controls (1.23 +/- 1.08 vs. 0.83 +/- 0.95, p = 0.02). Other illnesses were noted in 25 (44.6 per cent) cases and seven (7.7 per cent) controls (OR 11.5; CI, 4.13-33.4; p < 0.00001). Notably, chronic ear discharge was noted in 6 (11.7 per cent) cases but in none of the controls (p = 0.007). Streptococcus pneumoniae was isolated from nasopharyngeal swabs in 42/49 (85.7 per cent) cases and 44/80 (55 per cent) controls (p < 0.001) and H. influenzae from seven cases and five controls; S. pneumoniae was isolated in all children with chronic ear discharge and H. influenzae from one child. Serotypes of pneumococci commonly associated with otitis media, i.e., types 6, 14, 19, and 23 were isolated from 25 (51 per cent) cases and 16 (20 per cent) controls (OR 4.17; 95% CI, 1.78-9.85; p < 0.001). Persistent rhinorrhoea, presumably due to pneumococcus, is a common condition among rural Indian children and appears to be associated with chronic otitis media.

Adolescent↗

Systemic bacterial infections in bone marrow transplant patients.

The clinical microbiology department at CMC&H, Vellore in conjunction with the haematology department carries out routine surveillance of patients admitted to the hematology department. Since 1994 in a sample population of 55 patients with various underlying clinical conditions who have had bone marrow transplant, sepsis was observed in 16 patients (29%). The predominant Gram negatives associated with sepsis were non-fermenting Gram negative bacilli and all the 5 Gram positives were coagulase negative staphylococci. These organisms were susceptible to most of the routinely used antimicrobial agents. Continued surveillance is needed to determine changing trends with respect to organisms causing systemic infections and their susceptibility to antimicrobials.

Adolescent↗

Nasopharyngeal colonization of infants in southern India with Streptococcus pneumoniae.

To investigate the dynamics of nasopharyngeal colonization with Streptococcus pneumoniae, and to determine the prevalent serogroups/types (SGT) and their antimicrobial susceptibility, we studied 100 infants attending our well-baby clinic. Nasopharyngeal swab specimens were obtained at 6, 10, 14, 18 and 22 weeks and at 9 and 18 months of age and submitted for culture, serotyping and antimicrobial susceptibility testing of S. pneumoniae. Colonization with pneumococcus was seen on at least one occasion in 81 infants. The median age of acquisition was 11 weeks and the median duration of carriage was 1 3 months. The common SGTs identified were 6, 19, 14 and 15. SGT 1, which was a common invasive isolate in children in our hospital during this period, was not isolated from these children. Sequential colonization by 2, 3 or 4 SGTs was observed in 18, 5 and 2 children, respectively. Resistance to penicillin, chloramphenicol, cotrimoxazole and erythromycin was observed in 0, 13 (6%) 11 (5 %) and 5 (3 %) isolates, respectively. There was a significant difference in susceptibility to cotrimoxazole between colonizing and invasive isolates (5 % vs. 40 %, P<0.0001).

Drug Resistance, Microbial↗

Effects of intranasal challenge with group A beta haemolytic streptococcus M type 49 in Swiss albino mice.

Mice are susceptible to natural infections with streptococci and therefore can serve as suitable animal models to study experimental streptococcal infections. In an earlier study, we had shown the development of pharyngeal colonization, antibody response and histopathological changes in the heart following intranasal (IN) challenge with a rheumatogenic serotype of group A beta haemolytic streptococcus, the M type 18. To determine if nonpharyngitis associated serotypes can also elicit similar responses, 30 Swiss albino mice were challenged intranasally with 2 x 10(7) colony forming units of a skin associated serotype of group A beta haemolytic streptococcus, the M type 49. Pharyngeal colonization varied from 64% (n = 30) in the first week to 69% (n = 16) during the fourth week after IN challenge. Eleven (36.7%) of the 30 animals studied showed antibody response to DNase B (ADNB) with peak titers varying from 150 to 1200 units. Wide variations were seen in ADNB titers in individual mice. Histopathological evidence for cardiac lesions were seen in three animals. The changes were mild and varied from mild to chronic endocardial inflammation to calcification. The study shows that Swiss albino mice are also susceptible to IN challenge with skin associated strains of GABHS and therefore can serve as useful models to study the effects of experimental infection with diverse serotypes of GABHS.

Administration, Intranasal↗

The Griess test: an inexpensive screening test for asymptomatic bacteriuria in pregnancy.

This study was designed to compare urine microscopy and the Griess test as screening tests for asymptomatic bacteriuria (AB) in pregnancy, using urine culture as a gold standard. Urine microscopy, Griess test and urine culture were carried out on urine samples from 483 antenatal women. AB was detected by urine culture in 30 (6.8%) of the patients. The Griess test and urine microscopy were found to have a sensitivity of 63.3% and 60%, specificity of 99.5% and 83.6%; and positive predictive value of 90.5% and 21.1% respectively. Most of the infections missed by the Griess test were either <100,000 colony count on culture or caused by gram positive cocci. The cost of the Griess test was only one tenth of that of urine microscopy. The Griess test is a simple and inexpensive test that when compared to urine microscopy has equal sensitivity, and better specificity and positive predictive value for the detection of AB in pregnancy than urine microscopy.

Bacteriological Techniques↗