J. Commun. Dis. 40 (2) 2008 : 111-120 Bacterial Meningitis: apid Diagnosis And Microbial Profile : A Multicentered tudy ultana hameem*, VinodKumar C**, Neelagund YF*** ABTACT Meningitis continues to be a formidable illness with high morbidity and mortality among children in India. The present study was undertaken to prospectively look for the prevalence of pyogenic meningitis at Gulbarga and to find out the utility of gram stain, Latex Agglutination Test and (LTA) and C-reactive protein in the rapid diagnosis of pyogenic meningitis from children. Over a 48-months period, 535 children with a presumptive clinical diagnosis of acute bacterial meningitis were investigated by direct microscopy, CP, bacterial culture, latex agglutination test (L TA), cell count and cell type and biochemical tests. Latex Agglutination Test (LA T) was done for detection of the antigens of treptococcus pneumoniae, Group B treptococci, E. coli, Neisseria meningitidis and Haemophilus influenzae type b. Among 535 suspected meningitis cases, 291 cases were diagnosed as pyogenic meningitis cases based on biochemical tests, cell count and cell type. Out of 291 cases, 55 cases have already received antibiotic treatment. Among 236 cases of untreated pyogenic meningitis cases, 199 CF samples were culture positive. treptococcus pneumoniae (44.7%) was the predominant organism identified, followed by H influenzae (25.6%) and Gp. B. treptococci (9.5%). 208 of 236 cases were gram-stain positive, 129 cases had elevated CF-CP and 214 cases were diagnosed as pyogenic meningitis by the detection of bacterial antigens by latex agglutination test. * Division of esearch in Medical ciences, Department of Microbiology, Gulbarga University, Gulbarga ** Department of Microbiology,.. Institute of Medical ciences & esearch Centre, Davanagere Correspondence to : Dr. Vinod Kumar C, Assistant Professor Department of Microbiology,.. Institute of Medical ciences & esearch Centre, Vidhyanagar post box -1, NH-4, Bypass Davanagere-5770. Email : vinodmicro@yahoo.com
112 ultana hameem et al Among 55 pretreated cases, only (9.1%) CF samples were culture positive, bacteria was observed in 36 gram stain smear, CP was elevated in 16 CF samples and 52 pretreated cases of suspected meningitis were diagnosed as pyogenic meningitis by latex agglutination test for detection of bacterial antigens. Many of the bacterial isolates were sensitive to gentamicin, cefotaxime and ceftriaxone and least sensitive to tetracycline and gentamicin. 13.1% of gram-negative bacilli were EBL producers. To conclude, inclusion of latex agglutination test for detection of bacterial antigen in the routine diagnosis adds a valuable adjunct in the rapid and accurate diagnosis of pyogenic meningitis. Key words : pyogenic meningitis, bacterial CF culture, gram stain and LAT. IODUCTION Pyogenic meningitis is a common medical emergency among the pediatric age group in 1 India. It contributes to 0.5 to 2.6% of the hospital admissions. Bacterial meningitis is a serious and sometimes fatal infection 2, 3 affecting the central nervous system.. Traditional laboratory diagnostic methods of culture for the identification of bacterial meningitis pathogens take up to 36 h or 4, 5 more. Furthermore, it has been observed that following an increase in the practice of starting antimicrobial therapy prior to clinical sample collection, the ability to confirm the pathogenic microorganisms of bacterial meningitis and septicemia has 4 decreased by approximately 30%. This has been noted in Public Health Laboratory ervice (PHL), Communicable Disease urveillance Centre (CDC) data which show a growing discrepancy between the numbers of clinically suspected and culture-confirmed cases of bacterial meningitis and septicemia, with particular reference to meningococcal 6 infection in India And also in recent years due to selection pressure of antibiotics in the hospital environment multiple resistance to antibiotics has been encountered among gram-negative bacteria. This is more so with 5-9 Klebsiella sps. Hence an accurate and rapid diagnosis is an essential pre- requisite to administer appropriate antibiotics for successful therapy. The conventional culture methods are time consuming and often give negative 5 results in treated cases. The simple rapid method like Gram stain is relatively non- 10 specific. Hence, there is a need for rapid and reliable test for the diagnosis of acute pyogenic meningitis. Immunochemical demonstration of soluble bacterial antigen in CF speeds definitive diagnosis without the need of experienced bacteriological 11-14 laboratory person. The latex agglutination is one such test found to be useful for the diagnosis of pneumococcal, haemophilus 12-17 and meningococcal meningitis. Hence, this test was adopted in our laboratory for the diagnosis of acute pyogenic meningitis in children due to the common agents like treptococcus pneumoniae, Group B treptococci, E. coli, Neisseria meningitidis and Haemophilus influenzae type b in children. The objective of the present study is to prospectively look for microbial profile, antibiogram of bacterial meningitis and the utility of Latex agglutination tests in the rapid diagnosis of bacterial meningitis with special reference to treptococcus
apid diagnosis and microbial profile of bacterial meningitis 113 pneumoniae, Group B treptococci, E. coli, Neisseria meningitidis and Haemophilus influenzas type b in children. MATEIAL AND METHOD Five hundred thirty five (535) paediatric patients clinically suspected of having pyogenic meningitis were included in the study. They were admitted to Government Hospital, M.. medical college hospital, angameshwar pediatric hospital, and Basaveshwar hospital, Gulbarga, during the period from February 20 to January 2007. Information obtained at the time of admission included age, sex, race, period of illness, antibiotic intake before admission and clinical symptoms and signs at the time of admission and while the child was in hospital. After admission, 2-3 ml of cerebrospinal fluid (CF) was collected aseptically by lumbar puncture in two separate sterile test tubes. One of the two specimens from each patient was sent to the bacteriological laboratory for culture, Gram staining and latex agglutination tests. The second was used for cytology and for protein and sugar estimations. The CF was inoculated onto blood agar and chocolate agar and incubated aerobically in a candle jar at 37 C 18 immediately. Bacteriological studies on the CF were performed in the Department of P.G. studies in Microbiology, Division of research in Medical sciences, Gulbarga University, Gulbarga. On receipt, each specimen was centrifuged at 1000 rpm for 5 minutes. The supernatant was removed aseptically into a separate tube and used for latex agglutination tests for the detection of bacteriological antigens, using Wallcogen meningitis kit (Murex, England) & CP test (Tulip Diagnostic). The sediment was cultured using standard techniques and also used for Gram staining. All isolates were identified on the basis of their colony morphology and culture characteristics, and their biochemical 18 reactions according to standard procedures. Isolates were tested for their antibiotic sensitivity for commonly used antibiotics by 19 the Kirby & Bauer disk diffusion technique. taphylococcus aureus (ATCC no. 25923) and Escherichia coli (ATCC no. 25922) were used as control strains. EBL among gram-negative 20 was detected by double disk synergistic test. Latex Agglutination Test : The supernatant of the centrifuged CF samples were kept in boiling water bath for 5 minutes and used for LAT. For each specimen one drop of. pneumoniae Latex reagent (Wellcogen kit of Murex diagnostics Ltd. England) and one drop of control reagent was placed on a separate circle on the reagent 14, 17 card. One drop of processed CF was placed by the slide of test and control reagent. Contents of each circle were mixed and spread to cover the full area of circle. The card was rocked slowly and agglutination considered positive within three minutes by naked eye examination (as per the instruction manual of the kit). The test was repeated similarly with Group B treptococci, E. coli, Neisseria meningitidis and Haemophilus influenzae type b latex reagent. tatistical analysis was done using Chi square test. EULT Out of 535 suspected meningitis cases, cell count, cell type and biochemical results revealed that only 291 cases were pyogenic meningitis. Amongst 291 pyogenic meningitis cases, 55 cases were pre treated and 236 were untreated cases. Out of 291 cases male were affected more compared to female. The total male to female ratio was 1.2: 1. Most of the cases were between the age group 1 month to 3 years.
114 ultana hameem et al Untreated cases:- In untreated cases, gram stained smear examination of the 236 CF samples showed organism in 208 (88.1 %) samples. Gram positive diplococci were the predominant organisms seen in the gram smears (Table-1). 199 (84.3%) CF samples were culture positive (table-2).. pneumoniae was the common isolate (44.7%) grown followed by Haemophilus influenzas type b (25.6%). Neisseria meningitides was isolated in 6.5%, taphylococcus aureus in 5.5%, Klebsiella sps in 4.0%, E.coli in 2.5% and Pseudomonas sps 1.5%. Among 236 untreated cases, 130 CF samples were positive for CP of which 1 Table 1: esults of Gram stained smears of CF in Untreated Pyogenic Meningitis (n=236) Bacteria No (%) Gram positive diplococci 80 33.9 Gram negative bacilli 70 29.7 Gram positive cocci in small chain 26 11.0 Gram negative diplococci 22 9.3 Gram positive cocci in clusters 10 4.2 No bacteria seen in the smear 24 10.2 Total Table 2: Culture results of CF specimens in Untreated pyogenic Meningitis patients (n=236) Organisms No. Positive (%). pneumoniae 89 44.7 H. Influenzae 51 25.6 Group B treptococci 19 9.5 Niesseria meningitides 12 6.0 taphylococcus aureus 11 5.5 Klebsiella spp. 08 40. Esch.Coli 3.0 Pseudomonas aeruginosa 1.5 Total 199 samples were positive for culture and CP, 28 were positive only for CP and Culture negative. 1 CP negative cases, 97 CF samples were culture positive 9 samples were culture negative (Table-3). Latex agglutination test for detection of bacterial antigens were done for all 236 untreated cases, of which 214 (90.7%) of CF samples were positive of one or other bacterial antigens. 199 CF samples were positive for both culture and LAT, 09 CF samples were positive for LAT, but negative for culture. But all these nine patients were clinically proven meningitis cases. The most Table 3. Comparison of Gram stain Latex Agglutination test and CP with culture in untreated cases (236) Test Culture tatistics Positive Negative Gram stain LAT CP Positive 199 09 Negative -- 28 Positive 199 15 Negative 22 Positive 1 28 Negative 97 09 P=0.0 P=0.0 P=0.0
apid diagnosis and microbial profile of bacterial meningitis 115 common bacterial antigens identified by LAT are 94 Pneumococcal meningitis, 61 Hemophilus influenza meningitis, 29 Group B treptococci meningitis 18, Neisseria meningitis, and. 12 E. coli meningitis. Treated cases:- A total of 55 patients received medication prior to hospitalization. In this treated group, the conventional bacteriological technique identified the causative organism in 5 cases, whereas 36 cases of pyogenic meningitis cases were presumptively identified by gram stain, 16 cases by CP and 52 cases were identified by LAT. All the above 55 treated cases were clinically diagnosed as meningitis cases (Table-4). Antibiotic sensitivity test Antibiotic susceptibility pattern of the organisms isolated from pyogenic meningitis is dipected in table-5. 33.3% of the organisms were resistant to two or more antibiotics. Amongst the grampositive bacteria, 23.6% of. pneumoniae were resistant to penicillin and all the isolates were sensitive to ampicillin. In taphylococcus aureus 63.6% of the isolates were resistant to tetracycline, 54.5% to amoxicillin, 72.7% to amikacin and 54.5% to ceftriaxone. Among gramnegative bacilli 100% of Pseudomonas sps were resistant to ampicillin, gentamicin, chloramphenicol, ceftazidime and ceftriaxone. Among Klebsiella sps, the largest percent of resistance was seen to tetracycline (87.5%), ampicillin (75.0%) and 62.5% for gentamicin and 50.0% to amikacin respectively. 13.1 % of the gram-negative bacteria were EBL producers. EBL production was observed in 27% of Klebsiella sps, 40% % of E.coli and 60.7%% of Pseudomonas sps. DICUION The majority of our pyogenic meningitis cases were found in children of age less then 2 year (64.9%) (P < 0.), which concurs with 17 the findings of other authors. In this age group, there were more male children affected than females (male to female ratio of 1.2:1). Other authors have made similar 1,6,7 observations. Laboratory investigations of CF specimens in suspected acute meningitis are extremely important for prompt Gram stain LAT CP *Chi-square test #Fisher's extract test Table 4. Comparison of Gram stain Latex Agglutination test and CP with culture in treated cases (55) Test Culture tatistics Positive Negative Positive 32 Negative -- 19 Positive 47 Negative -- Positive 13 Negative 37 P*=0.2 # P =0.6 P=0.2
116 ultana hameem et al Table 5. Antimicrobial susceptibility pattern of the organisms isolated from pyogenic meningitis Organisms Penicillin Tetracycline Ampicillin Gentamicin Amoxicillin Amikacin Chloramphenicol Cefotaxime Ceftazidime Ceftriaxone treptococcus Pneumoniae (89) 21 (23.6) 68 (76.4) -- 89 -- -- -- -- -- -- -- -- H. Injluenzae(51) 34 17 (33.3) 42 (82.3) 09 (17.7) 34 17 (33.3) 43 (86.6) 08 (15.4) 08 (15.4) 43 (84.6) 17 (33.3) 34 -- -- -- 51 51 51 GpB treptococci (19) - 19 -- -- -- -- -- -- -- -- N. Meningitidis (13) 13 -- -- -- -- -- -- -- 13 -- 13 -- 13 13 13 13 13 13 taphylococcus aureus (11) 07 (63.6) 04 (36.4) (54.5) (45.5) (27.3) 08 (72.7) (54.5) (45.5) 08 (72.7) (27.3) (54.5) (45.5) (27.3) 08 (72.7) (45.5) (54.5) (54.5 (45.5) Klebsiella sps (08) 07 (87.5) (12.5) (75.0) (25.0) (62.5) (37.5) (62.5) (37.5) 04 (50.0) 04 (50.0) (62.5) (37.5) (25.0) (75.0) 07 (87.5) (12.5) 07 (87.5) (12.5) E. coli () 04 (80.0) (20.0) -- 04 (80.0) (20.0) Pseudomonas sps () (33.3) -- -- -- (33.3) -- (33.3) -- --
apid diagnosis and microbial profile of bacterial meningitis 117 recognition of the nature of the infecting organism as management and therapy of the patient depend on this information. The incidence of specific pathogens causing bacterial meningitis varies around 1,14,20 the world. The isolation rate of acute pyogenic meningitis ranges from 15.8-85%. The isolation rate in our study was 84.3%.. pneumoniae (33.8%) was the predominant organism isolated, followed by H. influenzae (25.6%), Gp. B. treptococci (9.5%), taphylococcus aureus (5.5%). imilar findings 19 have been reported from previous study and from other parts of the world, although in many other reports N. meningitides was more common. This is probably due to the endemicity of the organism and to the larger number of cases that may be involved in epidemic situations. The predominance of Gram-negative organisms reported as etiological agents of 16 bacterial meningitis was not seen in our study, where Gram- positive bacteria were isolated from 59.8%, and Gram- negative bacteria were found only in 40.2% of the cases. The simple Gram-stain smear of CF is reported to be the most useful single test for 3,7,9 identifying bacterial meningitis. In this study, Gram staining revealed the probable etiological organisms in 88.1 % of cases in untreated meningitis cases and 67.2% of cases in treated cases, while only 84.3% were positive by CF culture in untreated cases and 9.1% were positive in treated meningitis cases. Our findings corroborates with other 14 studies. The sensitivity of gram stain was 100%, which is more than that of Vishwanath 22 25 et al., (89%) and ajkumar et al., (44%). This confirms the value of the Gram stain as a rapid presumptive diagnostic aid in treated and untreated cases of meningitis. Bacterial antigens of the primary causative agents of childhood meningitis, namely those of N. meningitides,. pneumoniae Gp. B treptococci, E. coli and H. influenzae type b, can be used for the early diagnosis of pyogenic meningitis. In the present study, simple latex agglutination tests for these pathogens detected 90.7% untreated cases and 94.5% of pretreated meningitis. ensitivity of LAT was 100% in case of untreated cases and 94.5% in case of pretreated cases. Our finding correlates with 26 that of ao et al., 89% and Vishwanath 22 et al., (90%). The low culture positivity rate when compared to LAT may be probably due to viral meningitis, antibiotic therapy prior to the collection of CF or presence of autolytic 11,21 enzymes. The LAT positivity rate reported in the present study is slightly higher compared to the others workers because we have included LAT for N. meningitides, Gp. B. treptococci, and E.coli. In treated group LAT was able to identify the causative agent in 52(94.5%) cases whereas by the doing only conventional method, bacterial agent was identified in 5(9.1%) cases. This clearly indicates LA T is useful to determine the pretreated cases of pyogenic meningitis from CF specimen. Our findings are in 12,14 concurrence with other authors. The results obtained reassure the role of LAT for the antigen detection in CF samples even in pretreated cases and also signifies that LAT is superior to conventional tests for the etiological diagnosis of pyogenic 22 meningitis. LAT has many advantages over the conventional methods in being simple, rapid and more sensitive than culture besides also being useful in diagnosing treated cases.
118 ultana hameem et al Hence, it may be recommended for routine diagnosis of acute pyogenic meningitis in peripheral laboratory and in clinical setups where a facility for culture is not available and also in the referral hospitals for the rapid diagnosis. The other sensitive assay CF CP latex agglutination tests are increasingly used for biological application. There is no uniform agreement about sensitivity of CF-CP level in predicting pyogenic meningitis. High sensitivity rates were found in some studies, whereas low levels were reported in neonates 22 using laser nephalometry techniques. In our study 129 (54.7%) out of236 untreated cases were positive for CF-CP, and 16 (29.1%) out of 55 treated cases were positive. ensitivity of CF- CP in untreated cases was 54.7% and 29% in pretreated cases. Culture of CF was positive in 84.3%. Thus, the simple Gram stain combined with the latex agglutination test will help in a rapid diagnosis of bacterial meningitis, and may also indicate the most probable causative organisms before culture results are available. This is extremely Important in the 7,12,21 rapid management of such cases. However, culture remains important in assessing the antibiotic susceptibility pattern of the causative organisms. It is worth noting that 15.7% of bacterial meningitis cases had an unidentified etiology, which has also been observed by other workers. This is not likely to be the result of prior antibiotic therapy as many think, since it was reported even before the 22 antibiotic era. ome of these cases may be caused by N. meningitides, which is known in some cases to show a lack of organisms In smears, CF and blood cultures, and even in 22 antigen detection tests. The development of new and very sensitive tests may throw light on this group. The antimicrobial sensitivity pattern of our isolates showed that all Gram-positive, and many of the Gram-negative organisms were resistant to two or more antibiotics tested in the present study. 23.8% of. pneumoniae were resistant to penicillin. Ampicillin and tetracycline were the least effective. These findings are in accordance 7,19,22 with the findings of other investigators. There is increasing resistance among the major pathogens which cause meningitis to most of the traditional antimicrobial agents used as initial therapy prior to the availability of bacteriological results. A high percentage of ampicillinresistant (54%) and chloramphenicolresistant (41%) H. influenzae were isolated in the present study. imilar findings have been 19,24 observed by other workers. Tests for the production of b-iactamase mediated resistance revealed that 13.1 % of the total gram negative bacilli were EBL producer. Klebsiella sps and the E.coli were the predominant organisms producing EBL. The standard antibiotic therapy, using ampicillin and chloramphenicol before the bacteriological report reaches the pediatrician, may need to be modified. In our study, we found that the majority of isolates were highly sensitive to cefotaxime ceftriaxone and amikacin. The case fatality rate among the 236 cases was 11.6% despite treatment. Although lower mortality rates have been reported in industrialized countries such as the United 24 tates of America (2.6%), higher rates have been reported in some developing countries and countries in the Middle East, such as 9 23 Turkey (38%), audi Arabia (14.7%), udan 11 8 (28.6%) and India (21.8%).
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