Incidence of Haemophilus influenzae type b meningitis in India.

BACKGROUND & OBJECTIVE: Vaccine policy depends on locally relevant disease burden estimates. The incidence of Haemophilus influenzae type b (Hib) disease is not well characterized in the South Asian region, home to 30 per cent of the world's children. There are limited data from prospective population incidence studies of Hib in Asia, and no data available from India. We therefore carried out this study to assess the burden of Hib meningitis in India. METHODS: A prospective surveillance study was carried out during 1997 and 1999 in hospitals for cases of Hib meningitis from 5 administrative areas of an Indian district (Vellore, Tamil Nadu) with 56,153 children under 5 yr of age, over a 24 month period RESULTS: Ninety seven cases of possible meningitis (> 10 WBC/microl in CSF) were reported, an annual incidence of 86 per 100,000 (95%CI 69 to 109) in 0-4 yr old children, and 357 per 100,000 in 0-11 month infants. Eighteen had proven bacterial meningitis, an annual incidence of 15.9 per 100,000. Eight CSF had Hib by culture or antigen testing, an annual incidence of 7.1 per 100,000 (95%CI 3.1 to 14.0) in children 0-59 months. In infants 0-11 months of age, the incidence of Hib meningitis was 32 per 100,000 (95%CI 16 to 67) and in the 0-23 month group it was 19 (95%CI 8 to 37). INTERPRETATION & CONCLUSION: Our data are the first minimal estimate of the incidence of Hib meningitis for Indian children. The observed incidence data are similar to European reports before Hib vaccine use, suggest substantial disease before 24 months of age, and provide data useful for policy regarding Hib immunization.

Comparison of phenotypic versus genotypic methods in the detection of methicillin resistance in Staphylococcus aureus.

BACKGROUND & OBJECTIVE: Conventional methods to detect methicillin resistance in Staphylococcus aureus are inadequate as expression of resistance is subject to environmental and conditional expression of PBP2a antigen. The objective of the present study was to determine methicillin resistance in S. aureus by conventional susceptibility (oxacillin disc diffusion and oxacillin MIC) and molecular methods (PCR) and to evaluate latex agglutination test for the detection of PBP 2a and to compare the results of these tests for its sensitivity, specificity and rapidity. METHODS: A total of 150 consecutive clinical isolates of Staphylococcus aureus received at the Department of Microbiology, Christian Medical College, Vellore, were included. Oxacillin (1 mg) disc diffusion and agar dilution method were used. The isolates were also subjected to latex agglutination test for detection of PBP2a and multiplex PCR to detect mecA and femB genes. RESULTS: Of the 150 isolates, 33 were found to be MRSA by oxacillin disc diffusion. By MIC method, 13 per cent of the isolates had values 32 microg/ml, 6 per cent between 16-8 mug/ml and 2.7 per cent had a value of 4 microg/ml; 100 per cent concordance was obtained between the oxacillin disc screening and MIC methods. The latex agglutination showed positive reaction for all MRSA with only one MSSA being falsely classified as MRSA. The specificity and sensitivity were 99 and 100 per cent respectively. Test results were obtained within 15 min. By multiplex PCR, all 22 per cent of MRSA were positive for mecA and femB genes and additionally one MSSA carried mecA gene. However, femB gene was not found in 6 MSSA isolates. Specificity and sensitivity of PCR for mecA detection was similar to latex agglutination test. PCR system required approximately five hours. INTERPRETATION & CONCLUSION: Our findings showed that the conventional methods for detection of methicillin resistance like disc screening, disc diffusion and MIC are cost-effective but time consuming. Latex agglutination though expensive is rapid and can be a good preliminary screen with high sensitivity and specificity. Multiplex PCR is a good confirmatory test though expensive.

An outbreak of cutaneous anthrax in a non-endemic district--Visakhapatnam in Andhra Pradesh.

BACKGROUND: Anthrax is a disease of herbivorous animals, and humans incidentally acquire the disease by handling infected dead animals and their products. Sporadic cases of human anthrax have been reported from Southern India. METHODS: Five tribal men presented with painless ulcers with vesiculation and edema of the surrounding skin on the extremities without any constitutional symptoms. There was a history of slaughtering and consumption of a dead goat ten days prior to the development of skin lesions. Clinically cutaneous anthrax was suspected and smears, swabs and punch biopsies were taken for culture and identification by polymerase chain reaction (PCR). All the cases were treated with intravenous followed by oral antibiotics. Appropriate health authorities were alerted and proper control measures were employed. RESULTS: Smears from the cutaneous lesions of all five patients were positive for Bacillus anthracis and this was confirmed by a positive culture and PCR of the smears in four of the five cases. All the cases responded to antibiotics. CONCLUSION: We report five cases of cutaneous anthrax in a non-endemic district, Visakhapatnam, Andhra Pradesh, for the first time.

Resistance of Mycobacterium tuberculosis to the first line anti tubercular drugs--a twenty year review.

Tuberculosis and more so the multi drug resistant variety has been thrust into the forefront as a serious and life threatening illness in recent years. The advent of AIDS contributes to this substantially, especially in the developed world where it had become practically non- existent. We reviewed our data over the past 20 years with a view to determine when drug resistance began to manifest in the strains.

Comparison of disc diffusion & E test methods with agar dilution for antimicrobial susceptibility testing of Haemophilus influenzae.

BACKGROUND & OBJECTIVES: Reliable methods of detection of antimicrobial resistance are of paramount importance in the treatment and management of infections caused by Haemophilus influenzae. The objective of the present study was to compare and evaluate the performance of disc diffusion and E test (Epsilometer test) with agar dilution method for antimicrobial susceptibility testing of H. influenzae. METHODS: A total of 46 isolates of H. influenzae from various invasive sites were included as test strains. Antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion method for ampicillin, chloramphenicol, trimethoprim-sulphamethoxazole (TMP-SMZ) and cefotaxime. Minimum Inhibitory Concentration (MIC) determination was performed by E test and agar dilution for the same set of antimicrobials. All tests were performed on Haemophilus test medium (HTM). RESULTS: Disc diffusion showed a very major (2%) and minor (4%) interpretative error with TMP-SMZ and minor interpretative errors to ampicillin (13%) and chloramphenicol (24%) when compared to agar dilution method. E test produced only minor interpretative errors to chloramphenicol (7%) and TMP-SMZ (2%) and no interpretative errors with ampicillin and cefotaxime as against agar dilution. E test showed good agreement with agar dilution for each of the antimicrobial tested. INTERPRETATION & CONCLUSION: Disc diffusion test may be used as a preliminary screen for susceptibility testing of H. influenzae. E test is simple, easy to perform and a reliable method for determination of resistance in H. influenzae. However its cost and limited availability in India may limit its use. The reference agar dilution method can be used reliably in routine susceptibility testing of H. influenzae.

Epidemiological typing of Streptococcus pneumoniae from various sources in India & Korea using Box A PCR fingerprinting.

BACKGROUND & OBJECTIVES: Epidemiological typing of Streptococcus pneumoniae is necessary to study genetic relatedness among strains. The Box A PCR assay, a good epidemiological tool for high resolution typing of pneumococcal isolates, was used for comparison of strains of S. pneumoniae from Korea, an area of high penicillin resistance and India with low resistance to penicillin. METHODS: The pneumococcal strains from Indian and Korean sources included in the study belonged to serotypes 19F, 23F, 6A & 6B. The penicillin susceptibility of the strains was confirmed by determination of minimum inhibitory concentration values. The strains were then fingerprinted using the Box A PCR protocol and the results were further analysed by Molecular Analyst Software. RESULTS: The Box fingerprinting technique produced sharp, clear and reproducible banding patterns for strains of S. pneumoniae included in the study. There was no similarity between Indian and Korean strains at the genetic level. Among the Korean strains with similar serotypes, the level of similarity varied. Except for one pair (serotype 23F), that showed a level of homology of about 90 per cent, almost all the others showed less than 80 per cent homology. It was also seen that there was a high percentage of homology (> 90% in nasopharyngeal isolates from India belonging to serotype 19F) between isolates from similar source. Comparison of the Indian and Korean isolates of similar serotype showed low homology, except for one cluster of two strains with serotype 6B which showed 84 per cent homology (blood isolates from India and Korea). INTERPRETATION & CONCLUSION: Box A PCR is a highly discriminatory and useful method for typing S. pneumoniae. The results of the study have shown low levels of homology between strains from within India and also between two geographically distinct areas.

Nasopharyngeal carriage of Haemophilus influenzae.

OBJECTIVE: Nasopharyngeal colonization of Haemophilus influenzae (H. influenzae) in young children may be important in developing countries. METHOD: In this study, we screened school going children for carriage of H. influenzae. A total of 44 H. influenzae isolates out of a collection of 162 were characterized for biotypes, capsular serotypes and antibiotic resistance. RESULTS: A significant proportion of H. influenzae (25/44) isolates were serotype b. High antibiotic resistance was observed against commonly administered antibiotics like ampicillin (79%), chloramphenicol (20%), trimethoprim sulfamethoxazole (84%) and erythromycin (95%). Comparison of antibiotic resistance profile of nasopharyngeal isolates was observed to be correlated with those of H. influenzae from disease. CONCLUSION: Multidrug resistant nasopharyngeal H. influenzae in young healthy children may act as reservoir. Monitoring of antibiotic resistance among nasopharyngeal H. influenzae as a surrogate for invasive H. influenzae seems an attractive option.

Rapid assay for detection of chloramphenicol acetyl transferase in Haemophilus influenzae.

BACKGROUND & OBJECTIVES: Haemophilus influenzae causes a variety of life threatening infections in humans. Early detection of antimicrobial resistance is of importance in the treatment and management of infection. The modified Slack's method, a simple assay, has been evaluated in this study for the early detection of chloramphenicol resistance. METHODS: Fifty isolates of H. influenzae from invasive and non-invasive sites were included. Antimicrobial susceptibility testing was done by disc diffusion method and minimum inhibitory concentration (MIC) determination was performed for chloramphenicol only. Modified Slack's method was used to test for the production of chloramphenicol acetyl transferase (CAT). RESULTS: Invasive isolates showed higher degree of resistance to chloramphenicol (72%) compared to non-invasive ones (28%). One hundred per cent association was found between results of disc diffusion, MIC and CAT production amongst strains resistant to chloramphenicol. INTERPRETATION & CONCLUSION: The findings suggested that chloramphenicol still remains the drug of choice for treatment for non-invasive infection caused by H. influenzae. Modified Slack's method is a simple, rapid, inexpensive and reliable method for the detection of chloramphenicol resistance amongst H. influenzae.

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.

Changing trend in susceptibility pattern of Streptococcus pneumoniae to penicillin in India.

Prior to 1995 all strains of Streptococcus pneumoniae isolated at a tertiary care hospital in south India were uniformly susceptible to penicillin. However, since late 1995 strains of S. pneumoniae with intermediate resistance to penicillin have been observed. Altogether there were 25 such isolates, 9 from invasive (5 from CSF as well as blood, 1 from pleural fluid and 3 from CSF alone) and 16 from noninvasive sites (6 from throat, 6 from sputum, 3 from eye and 1 from ear) respectively, thus 4.6 per cent of S. pneumoniae showed intermediate resistance of a total of 535 strains studied so far. The minimum inhibitory concentration (MIC) values of penicillin, erythromycin, chloramphenicol and cefotaxime were determined by agar dilution method and for confirmation, E test was carried out for penicillin alone. The MIC range obtained for penicillin was between 0.125-1.0 microgram/ml. Kirby-Bauer disc diffusion method was adopted for testing of erythromycin, chloramphenicol, co-trimoxazole, cefotaxime, tetracycline and vancomycin. We observed that none of the strains with intermediate resistance to penicillin were multidrug resistant. These strains belonged predominantly to serotype 14 (n = 10), 7B (n = 9), 19A (n = 3), 7F (n = 2) and 23F (n = 1). Clonality was not observed in the 5 representative strains subjected to Box A finger printing method.

Surgical presentation of melioidosis in India.

BACKGROUND: Melioidosis, the disease caused by Burkholderia pseudomallei, is common in Southeast Asia. It has also been reported from India, where some investigators feel it is under-diagnosed and under-reported. We report our experience with melioidosis presenting as abscesses at unusual sites. METHODS: All consecutive patients with culture proven B. pseudomallei, who presented to a single surgical unit between 1995 and 1998, were evaluated. RESULTS: Three patients presented with splenic abscesses and one with a soft tissue abscess in the neck. One patient developed septicaemia. All patients responded favourably to ceftazidime and/or co-trimoxazole which was started as soon as the diagnosis was confirmed. CONCLUSION: Melioidosis is under-diagnosed in India, probably due to a low index of suspicion of this disease among clinicians. It should be considered as a possibility when abscesses are encountered at unusual sites. The pus must then be cultured to identify the causative agent.

E test as an alternative to conventional MIC determination for surveillance of drug resistant Streptococcus pneumoniae.

A commercial E test was compared with the standard agar dilution method for the determination of minimum inhibitory concentration (MIC) of penicillin, erythromycin, chloramphenicol and cefotaxime for 36 strains of Streptococcus pneumoniae from patients with invasive diseases. Additional strains were tested for MIC values for penicillin (6), erythromycin (14) and cefotaxime (13) for a better statistical evaluation. Besides, 5 reference standards with predetermined MIC values obtained from WHO pneumococcal reference center at Copenhagen, Denmark were tested for penicillin and erythromycin, for quality assessment using both agar dilution as well as E test methods. An overall agreement within +/- 2 dilutions was noted for 97 per cent of the strains tested for all the antimicrobials. A high degree of correlation was noted for erythromycin (r = 1), penicillin (r = 0.99), chloramphenicol (r = 0.95) and cefotaxime (r = 0.9). In MIC determination of a single antimicrobial for diagnostic purpose, E test was found to be more cost effective than conventional agar dilution method. E test was simple to perform, easy to interpret and a valid method for MIC determination of antimicrobials for S. pneumoniae in our center.

In vitro activity of netilmicin against clinical isolates of methicillin resistant and susceptible Staphylococcus aureus.

BACKGROUND: The emergence of methicillin-resistant Staphylococcus aureus and its multidrug-resistant property has led to the search for an effective antibiotic to combat staphylococcal sepsis. At present, vancomycin remains the most effective antibiotic. This study evaluated the in vitro efficacy of netilmicin (an aminoglycoside) and compared its activity with 4 other antibiotics, viz. vancomycin, amikacin, tobramycin and ofloxacin. METHODS: The minimum inhibitory concentration of the antibiotics was determined by the agar dilution method. Thirty strains each of methicillin-resistant and -susceptible S. aureus isolated from pus and blood cultures were included. RESULTS: The susceptibility to netilmicin was found to be 100% and was the same as that observed for vancomycin. CONCLUSION: All the methicillin-resistant S. aureus strains tested showed 100% susceptibility to netilmicin, suggesting its use in patients with such infections as an alternative to vancomycin. However, this finding needs to be verified in the clinical setting.

Prevalence of pneumococcal serotypes in invasive diseases in southern India.

Forty two pneumococcal strains obtained in Vellore, Tamil Nadu, from 42 patients with invasive diseases, namely pneumonia, meningitis, or peritonitis were typed. Over one-third of pneumococci in children and nearly half in adults were serotype followed by serogroups/types 5, 6 and 7 which were most common in children and adults taken together. These 4 serogroups/types accounted for 11 (79%) of 14 strains in children and 20 (71%) of 28 strains in adults. The remaining 11 strains belonged to 8 serogroups/types, namely 3, 4, 10, 11, 12, 13, 19 and 20.

The spectrum of antimicrobial resistance among methicillin resistant Staphylococcus aureus (MRSA) in a tertiary care centre in India.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen globally, including India. Staph. aureus strains isolated from pus or blood of patients during January 1993 to November 1994 were tested for antimicrobial susceptibility using Kirby-Bauer disc diffusion technique. Among 1382 isolates of Staph. aureus, 332 (24%) were MRSA. Among the latter, 97 per cent were resistant to trimethoprim-sulphamethoxazole; 85.5 per cent to gentamicin and 45 per cent to amikacin. While over 90 per cent were resistant to norfloxacin and ciprofloxacin, only 53 per cent were resistant to ofloxacin. Fifty seven per cent were susceptible to rifampicin and 87 per cent to netilmicin. All tested strains were susceptible to vancomycin. Therefore, when antimicrobials other than vancomycin are considered for therapy, their choice requires the results of in vitro susceptibility testing of every isolate of MRSA.

Melioidosis In India: the tip of the iceberg?

Diagnosis of melioidosis by the isolation of Burkholderia pseudomallei from one or more body fluid/tissue specimens of 6 Indian subjects, 5 of whom had not travelled outside India, is reported. The places of residence of these 6 and one patient previously reported, namely Tripura (2), Kerala (2), Orissa (1), Tamil Nadu (1) and Maharashtra (1) are therefore potentially endemic for melioidosis. B.pseudomallei closely resembles common contaminant Pseudomonas sp. and are easily mis-identified in microbiology laboratories. We surmise that melioidosis is underdiagnosed and underreported in India and we alert clinicians, microbiologists and public health professionals to the possibility of melioidosis being far more common than previously recognised.

Changing patterns of group B streptococcal serotypes associated with human infections.

One hundred strains of group B streptococci (GBS) isolated from diverse clinical specimens of patients seen in our hospital were subjected to serotyping by an indigenously prepared coagglutination system. Serotype NT/c was the most predominant (24%), followed by Ia (23%), II/c (12%) and Ib (11%). Type Ia was the most predominant in all specimens except genital samples where NT/c was the most predominant. Comparison of the distribution of serotypes identified during 1975-78 with that of the present study showed a great increase in the prevalence of types NT/c, II/c and Ia and a dramatic decline of types III, Ia/c and Ib. Despite the inclusion of reagents for newer serotypes, IV and V two strains were nontypable indicating the prevalence of hitherto unidentified GBS serotypes in our community.