“EVALUATION OF CONVENTIONAL CULTURE METHOD AND RAPID
ANTIGEN DETECTION METHOD FOR DIAGNOSIS OF BACTERIAL
MENINGITIS IN CHILDREN BELOW FIVE YEARS OF AGE.”
DISSERTATION
Submitted to the Rajiv Gandhi University of Health Sciences, Bangalore,
Karnataka in partial fulfillment of the regulations for the award of M.D degree
in Microbiology examination to be held in April 2011.
Guide
Dr. ASHA B PATIL,M.D.
Associate Professor ,
Department of Microbiology,
KIMS, Hubli.
Post Graduate
Dr. SYEDA FASIHA MOHAMMADI
DEPARTMENT OF MICROBIOLOGY
KARNATAKA INSTITUTE OF MEDICAL SCIENCES
HUBLI-22
2011
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA
DECLARATION BY THE CANDIDATE
I, Dr SYEDA FASIHA MOHAMMADI, declare that this dissertation titled
“EVALUATION OF CONVENTIONAL CULTURE METHOD AND RAPID
ANTIGEN DETECTION METHOD FOR DIAGNOSIS OF BACTERIAL
MENINGITIS IN CHILDREN BELOW FIVE YEARS OF AGE” is a bonafide
work done by me under the guidance of DR.ASHA B PATIL,M.D., ASSOCIATE
PROFESSOR, Department of Microbiology, Karnataka Institute of Medical Sciences,
Hubli. This is being submitted to the Rajiv Gandhi University of Health Sciences,
Bangalore, Karnataka, in partial fulfillment of regulations for the award of M.D.
Degree in Microbiology, examination to be held in April 2011.
This dissertation has not been submitted by me on any previous
occasion to any Universit y for the award of any degree.
Place: Hubli
Dr. Syeda Fasiha Mohammadi
Date:
P.G. in Microbiology,
Karnataka Institute of Medical
Sciences,Hubli.
KARNATAKA INSTITUTE OF MEDICAL SCIENCES
HUBLI
DEPARTMENT OF MICROBIOLOGY
CERTIFICATE BY THE GUIDE
This
is
to
certify
CONVENTIONAL
that
the
dissertation
CULTURE
METHOD
titled
“EVALUATION
AND
RAPID
OF
ANTIGEN
DETECTION METHOD FOR DIAGNOSIS OF BACTERIAL MENINGITIS
IN CHILDREN BELOW FIVE YEARS OF AGE” is a bonafide work done by
Dr.Syeda Fasiha Mohammadi, a post graduate student in Microbiology ,under my
guidance and supervision.
Place:Hubli
Date:
Dr.Asha B Patil, M . D .
Associate Professor,
Department of Microbiology,
Karnataka Institute of Medical
Sciences,Hubli.
KARNATAKA INSTITUTE OF MEDICAL SCIENCES,
(KIMS) HUBLI.
ENDORSEMENT BY THE HOD, PRINCIPAL/HEAD OF
THE INSTITUTION.
This
is
to
certify
CONVENTIONAL
that
the
dissertation
CULTURE
METHOD
titled
AND
“EVALUATION
RAPID
OF
ANTIGEN
DETECTION METHOD FOR DIAGNOSIS OF BACTERIAL MENINGITIS
IN CHILDREN BELOW FIVE YEARS OF AGE” is a bonafide research work
done by Dr. Syeda Fasiha Mohammadi, a post graduate student in Microbiology,
under the guidance and supervision of Dr.Asha B Patil,M.D. Associate Professor,
Department of Microbiology, KIMS, Hubli. This is being submitted to the Rajiv
Gandhi University of Health Sciences, Bangalore, Karnataka in partial fulfillment
of regulations for the award of M.D. Degree in Microbiology examination to be
held in April 2011.
Dr.Shobha D Nadag ir
Dr.U.S.Hangarga
Professor & HOD,
Principal,
Department of Microbiology,
Karnataka Institute of
Karnataka Institute of
Medical Sciences,Hubli.
Medical Sciences,Hubli
Date:
Date:
Place: Hubli
Place: Hubli
KARNATAKA INSTITUTE OF MEDICAL SCIENCES,
HUBLI.
DEPARTMENT OF MICROBIOLOGY
COPYRIGHT
DECLARATION BY THE CANDIDATE
I hereby declare that the Rajiv Gandhi University of Health Sciences, Karnataka shall
have the rights to preserve, use and disseminate this dissertation / thesis in print or
electronic format for academic / research purpose.
Date:
Name: Dr.Syeda Fasiha Mohammadi
Place: Hubli
Post Graduate student ,
Department of Microbiology,
Karnataka Institute of Medical
Sciences,, Hubli.
© Rajiv Gandhi University of Health Sciences, Karnataka
ACKNOWLEDGEMENT
Firstly I thank God for guiding me and giving me the knowledge and wisdom to do
this work.
I take this opportunity to express my sincere thanks and gratitude to my guide Dr. Asha B
Patil,
M.D.,
Associate Professor, department of Microbiology for her guidance, advice,
suggestions and enduring patience with my inexperience saw me through the completion
of this dissertation work.
With deep sense of gratitude I express my sincere thanks to my teacher Dr. Shobha D
Nadagir,M.D., Professor & Head, department of Microbiology, KIMS, Hubli for her
inspiring guidance, suggestions and advice during the course of my study.
I sincerely thank, Dr Mahesh kumar S, Dr Namratha Nandihal , Dr Mythri B A & Dr
Pramod N S for their unconditional support and help during my dissertation work.
I sincerely thank Dr. T. A. Shepur , Professor and Head Department of Pediatrics ,
KIMS, Hubli and Dr Devaraj V. Raichur Professor of Pediatrics, Department of
Pediatrics, KIMS, Hubli for their support.
I also thank Dr Kanchana H, Dr Chennabasappa M and Dr Venkatesh D for their
help.
It is my immense pleasure to thank my colleagues Dr Abdul Kaleem, Dr Madhusudhan,
Dr Mamata K, Dr. Manjunath A Hosamani , Dr Lakshminarayana, Dr Shyamala , Dr
Saroja, Dr Asma, Dr Thipperudraswamy and Dr Santhosh for their unconditional
support.
I am equally grateful to Dr M S Ravindra and Dr Umesh Dixit for all the possible help.
I express my sincere thanks to all the technicians who have helped me in doing the study.
I express my sincere thanks to Dr M.G. Hiremath, Director, KIMS, Hubli and
Dr. U.S.Hangarga, Principal, KIMS, Hubli for permitting me to do this study and to use
the institute facilities for the purpose.
Above all my whole hearted thanks to my parents and my husband for being the source of
constant inspiration and support.
Last but not the least, I express my deep sense of gratitude to all my patients and subjects
who have co-operated and without whom this study would not have been possible.
Place: Hubli
Date:
Dr. Syeda Fasiha Mohammadi
PG in Dept. of Microbiology
Karnataka Institute of Medical
Sciences Hubli.
LIST OF ABBREVIATIONS
A.baumannii
Acinetobacter baumannii
ABM
Acute Bacterial Meningitis
AST
Antibiotic susceptibility testing
A
Ampicillin
Ak
Amikacin
BBB
Blood Brain Barrier
Cpm
Cefepime
Ce
Cefotaxime
Ca
Ceftazidime
Ci
Ceftriaxone
CSF
Cerebrospinal fluid
C
Chloramphenicol
C.koseri
Citrobacter koseri
DS
Direct smear
E.coli
Escherichia coli
GBS
Group B Streptococcus
H.influenzae
Haemophilus influenzae
K.pneumoniae
Klebsiella pneumoniae
LAT
Latex agglutination test
N.meningitidis
Neisseria meningitidis
Pt
Piperacillin-tazobactam
P.aeruginosa
Pseudomonas aeruginosa
S.pneumoniae
Streptococcus pneumoniae
ABSTRACT
Objectives
To know the incidence of bacterial meningitis in children below five years of age.
To identify the specific etiological agents causing bacterial meningitis in children
below five years of age by CSF culture. To detect bacterial antigens in CSF by latex
particle agglutination test (LAT) and to compare conventional culture and antigen
detection methods.
Methods
Present study undertaken for a period of one year included 100 CSF samples of
clinically suspected meningitis cases in children below 5 years of age. The samples
were subjected to cell count, Gram stain, culture and LAT. The organisms isolated in
the study were characterized according to standard procedures.
Results.
Of the 100 cases studied, 31 cases were diagnosed as ABM by Gram stain, culture
and latex agglutination test as per WHO criteria. The hospital frequency of ABM was
1.7%, Male to Female ratio was 1.63:1 showing male preponderance .15 (48.38) cases
were culture positive. Gram stain was positive in 22(70.96) cases and LAT in
17(54.83) cases. Haemophilus influenzae was the most common causative agent of
acute bacterial meningitis followed by S.pneumoniae. Case fatality rate was 45.16%.
The sensitivity and specificity of Gram stain was 53.33% and 83.52% , LAT was
66.66% and 87.91% respectively.
Interpretation and Conclusion
Bacterial meningitis is a medical emergency and early diagnosis and treatment is life
saving and reduces chronic morbidity. The simple Gram stain smear of CSF was the
most useful single test for identifying bacterial meningitis. LAT was more sensitive
compared to conventional Gram stain and Culture technique in identifying the
fastidious organisms like H.influenzae, S.pneumoniae and Group B Streptococcus.
However, the combination of Gram stain, Culture and LAT proved to be more
productive than any of the single tests alone.
Keywords: Acute Bacterial Meningitis; Gram stain; Culture; LAT.
TABLE OF CONTENTS
PAGE NO
1. INTRODUCTION
1
2. AIMS AND OBJECTIVES
3
3. REVIEW OF LITERATURE
4
4. MATERIALS AND METHODS
29
5. OBSERVATION AND RESULTS
32
6. DISCUSSION
51
7. CONCLUSION
57
8. SUMMARY
58
9. BIBLIOGRAPHY
60
10. ANNEXURE
66
ANNEXURE-I : PROFORMA
66
ANNEXURE-II: KEY TO THE MASTER CHART
71
ANNEXURE-III: MASTER CHART
72
LIST OF TABLES
Sl.No
1.
TABLES
Page No.
Relationship between Common Bacterial Pathogens and
Factors Predisposing to Meningitis.
2. Pathogenic sequence of Bacterial Neurotropism.
3. Empiric therapy for bacterial meningitis in
developed and developing countries.
4. Recommendations for Pathogen-specific antimicrobial
therapy of children with Bacterial Meningitis.
5. Age and sex distribution of cases.
6. Clinical presentation of cases.
7. Laboratory confirmed cases of ABM as per WHO criteria.
8. CSF Cell count among the laboratory confirmed
cases of ABM.
9. Distribution of cases according to age and causative
agent among the laboratory confirmed cases of ABM.
10. Etiological agents identified in CSF by various methods.
11. CSF LAT results compared with administration of antibiotics prior
to CSF collection among the laboratory confirmed cases of ABM.
12. CSF culture results compared with administration of antibiotics prior
to CSF collection among the laboratory confirmed cases of ABM.
13. CSF Gram stain smear results compared with administration of antibiotics
prior to CSF collection among the laboratory confirmed cases of ABM.
14. Comparison of CSF Gram stain, Culture and LAT.
15. Accuracy indices of CSF Gram stain against CSF culture
as gold standard.
16. Accuracy indices of CSF LAT against CSF Culture
as gold standard.
17. Table showing comparison of sensitivity , specificity, PPV& NPV
of CSF Gram stain and CSF LAT with culture as gold standard.
18. Resistance pattern of the isolates.
19. Mortality rate among the laboratory confirmed cases of ABM.
20. Comparative studies of male to female ratio.
21. Comparative studies of Cell count in ABM.
22. Comparison of etiological agents isolated/identified
in CSF with other studies.
23. Comparative studies of Gram stain in ABM.
24. Comparative studies of culture isolation in ABM.
25. Comparative studies of LAT in ABM
26. .Comparative studies of case fatality rate in ABM.
LIST OF FIGURES
Sl.No.
FIGURE
1. Pathophysiologic cascade in bacterial meningitis.
2. Cerebrospinal fluid
3. Working station tray of latex agglutination test
4. Test card of latex agglutination test
5. Latex agglutinatin test positive for S.pneumoniae
antigen.
6. Direct Gram stain smear showing pleomorphic
Gram negative bacilli.
7. Chocolate agar with growth of Haemophilus.
8. Culture smear of Haemophilus.
9. Satellitism.
10. AST of Haemophilus.
11. Brain heart infusion broth.
12. Direct Gram stain smear showing
Gram positive cocci in pairs.
13. Chocolate agar with growth of S.pneumoniae.
14. Culture smear of S.pneumoniae.
15. Bile solubility test.
16. Optochin sensitivity test.
17. AST of S.pneumoniae.
18. Mac Conkey agar with growth of K.pneumoniae.
19. AST of K.pneumoniae.
20. Mac Conkey agar with growth of E.coli.
Page. No.
21. AST of E.coli.
22. Mac Conkey agar with growth of C.koseri.
23. AST of C.koseri.
24. Mac Conkey agar with growth of A.baumannii.
25. AST of A.baumannii.
26. Mac Conkey agar with growth of P.aeruginosa.
27. AST of P.aeruginosa.
28. Age and sex distribution of cases.
29. Laboratory confirmed cases of ABM.
30. Results of CSF Gram stain among the
laboratory confirmed cases of ABM.
31. Results of CSF culture among the
laboratory confirmed cases of ABM .
32. Results of CSF LAT among the
laboratory confirmed cases.
33. Etiological agents identified in CSF by all methods.
INTRODUCTION
Bacterial meningitis is an acute purulent infection within the subarachnoid space.1 It
is an important cause of childhood death and neurological sequelae in countries with
limited resources.2 The incidence of this disease varies widely in different parts of the
world ranging approximately 3.0 per 1,00,000 population in the united states to as
much as 45.8 per 1,00,000 population in northeastern Brazil.3The community
incidence of acute bacterial meningitis in India varies from 0.5% to 2.6% .4,5The
mortality rate due to ABM remains significantly high in India and other developing
countries, ranging from 16-32%.6
Etiology varies in different parts of the world and in different age groups or
depending on host immune status. The three organisms commonly associated with
ABM in early childhood in western countries are Haemophilus influenzae type b,
S. Pneumoniae and Neisseria meningitidis. 4
Clinical diagnosis of meningitis is not easy, because its early clinical manifestations
are often not specific, especially in babies and small children. On the other hand even
if it is recognized early and prompt treatment is given ,the mortality rate still is high
~30% in children and 20-30% in neonates. In addition ~10% to 20% of cases suffer
from neurologic sequelae.7
As a result of emergence of antimicrobial resistance being reported, recommendations
for therapy are changing. Laboratory surveillance of isolates is crucial to identify
targets for immunization, chart preventive strategies and to help formulate rational
empirical treatment for potentially fatal bacterial meningitis.6
Meningitis is a serious emergency in which the microbiological laboratory plays a
critical role in the early identification of the causative bacterium and its antibiogram.8
Gram stain of the CSF can provide a rapid preliminary identification of the infective
organism, but is liable to misinterpretation particularly in inexperienced hands.9
Isolation of the bacteria is the definite proof of the etiologic role of the organism.
However, CSF culture can be negative in case of 1) induction of antimicrobial therapy
prior to collection of specimen ; 2)improper collection and transportation; 3) presence
of autolytic enzymes. Besides the culture results are available only after two or three
days.10
From the middle 70s, simple, fast and cheap methods to identify etiologic agents in
purulent meningitis were made available with the development of immunochemical
techniques such as latex particle agglutination test [LPAT].11
The LPAT is highly sensitive and specific, simple to perform, no special equipment
required, technically easy, results are available in 10 minutes10 and finally no
alteration of results by prior antibiotic therapy.12
The present study was undertaken to know the incidence of bacterial meningitis in
children below five years of age, to identify the specific etiological agent causing
bacterial meningitis in children by CSF culture, to detect bacterial antigen in CSF by
latex particle agglutination test and to compare conventional culture method and
antigen detection by latex particle agglutination test.
AIMS AND OBJECTIVES
1. To know the incidence of bacterial meningitis in children below five years of age.
2. To identify the specific etiological agent causing bacterial meningitis in children
below five years of age by CSF culture.
3. To detect bacterial antigens in CSF by latex particle agglutination test.
4. To compare conventional culture method and antigen detection by latex particle
agglutination test.
REVIEW OF LITERATURE
Meningitis is as old as 460 B.C. when Hippocratus described it and recognized
the relationship between meningitis and aural infections.13
 Pyogenic meningitis has been known from the time of
Celsus and
Galen(22 B.C.) who described it as one of the entities of phrenitis, the
brain disorders.13
 Thomas Willis in 1661 described a case of meningitis.13
 Vieusseux in 1805 described an outbreak at Geneva and gave the 1st
definite knowledge of cerebrospinal fever.14
 Rose and Back in 1884 described streptococcal meningitis.13
 Weichselbaum of Vienna (1887) published his paper on Gram negative
diplococci in cases of acute cerebrospinal meningitis and named it
diplococcus cellularis meningitides.14
 Weichselbaum (1888) reported 1st case of meningitis due to Klebsiella
pneumoniae previously discovered and described by
Friedlander in
1882.15
 Schere in 1895 recorded 3 cases of Escherichia coli meningitis for the
1st time in infants under 3 months of age.15
 Slawyk in 1899 described the 1st case of meningitis due to H. influenza.15
 Flexner Simon (1913) an American bacteriologist from Kentucky
prepared the anti serum for treatment of cerebrospinal meningitis.16
 Whittle H C and Coworkers (1974) were the first to report the use of
LAT in the diagnosis of bacterial meningitis in a developing country.
They showed that the test was quick, cheap and easy to carry out and
commented that LAT was more sensitive than gram stain and culture in
diagnosing Hib, Pneumococcal and meningococcal meningitis. 17
 Gibson in 1935 isolated Listeria monocytogenes from a fatal case
of meningitis.15
Chinchankar N, Mane M, Bhave S, Bavdekar A, Bapat S, Dutta A, et al in their study
conducted at KEM hospital Pune (2002)4 found that 1.5% of all hospital admission
cases were ABM in less than 5 yrs of age (54 cases). 78% percent were below one
year and 52% were under the age of six months. Chief presentation was high fever,
refusal of feeds, altered sensorium and seizures. Meningeal signs were present in only
26%. The CSF C-reactive protein was positive in 41%, Gram stain was positive in
67%, LAT was positive in 78% and culture grew causative organisms in 50% of
cases. The final etiological diagnoses (as per LAT and /or cultures) were
Streptococcus pneumoniae in 39%, Haemophilus influenzae type b in 26% and others
in 35%. The others included one case of Neisseria meningitidis and 10 were LAT
negative and culture sterile. Blood cultures were positive in 4 (H.influenzae, alphahaemolytic streptococcus, Pseudomonas and S.aureus). Positive blood culture, CSF
culture and positive LAT was present only in one patient with H.influenzae infection.
Of the 54 patients with ABM 10 (19%) died in hospital within 1-7 days of admission.
The study concluded that the etiological diagnosis can be enhanced by LAT and good
culture. H. Influenza type b and S. pneumoniae account for more than 60% of ABM in
early childhood.
Deivanayagam N, Ashok TP, Nedunchelian K, Ahamed SS, and Mala N, in
their study at Institute of child health Chennai (1993)
10
observed that of the 114
consecutive CSF samples tested definitive diagnosis was evident in only 55 based on
CSF culture and/or LAT. Culture was positive in 12 (22%) cases whereas LAT was
able to identify the etiological agents in 46 (84%). Among the organisms identified by
LAT, only 3 out of 46 were culture positive, all Streptococcus pneumoniae. The
organisms identified by culture were Staphylococcus aureus in 4, Escherichia coli in
1, Klebsiella in 1, Pseudomonas in 1, and Proteus in 1. Ninety percent of H.influenzae
b and 67% of S.pneumoniae infections occurred in children below one year of age.
Eighty percent of ABM occurred in children below one year of age. Twenty six
percent of cases had received antibiotics prior to admission. They found LAT test
useful in arriving at etiological diagnosis of ABM rapidly and recommended the use
of LAT if Gram stain is negative
Of 50 CSF samples tested by Mirdha BR, Gupta U and Bhujwala RA, in their
study conducted at AIIMS hospital (1991)18,Gram stain was positive in 13 (26%)
cases and 18 (36%) cases were culture positive (N.meningitidis in 4, Streptococcus
pneumoniae in 3, H.influenzae in 2 and Staph aureus and Staph albus isolated in 2
each, Klebsiella pneumoniae 1, and E.coli 1, and others 3). In this study LAT was
positive in all culture positive cases for pneumococcus, meningococcus and
H.influenzae b. LAT detected 12 additional cases when cultures were negative. Using
all the three techniques, an etiological diagnosis was made in 30(60%). LAT is an
adjunct to conventional techniques in the etiological diagnosis of ABM when other
tests fail to identify the agent. It has a high utility in many hospital and health centers
without requiring highly trained staff and expensive laboratory.
Finlay FO, Witherow H and Rudd PT, in their study conducted at pediatric
ward Royal United Hospital, Bath (1995)
19
found that in 48 cases considered in this
study from 72 children admitted with bacterial meningitis etiology was confirmed in
28 cases (58%) by LAT, and in 30 cases (63%) by Gram staining and CSF culture
was positive in 39 cases (80%).Twenty six children received antibiotics before lumbar
puncture and the LAT was positive in 13 cases (50%). The LAT confirmed the
etiology in three out of five cases (60%) of S.pneumoniae, 14 out of 15 cases (93%) of
H. influenzae, 12 out of 18 cases (67%) of culture positive meningococci and 3 out of
9 cases (33%) where there was clinical diagnosis of meningococcal meningitis and
culture was negative. It was felt that LAT is not justified if Gram stain is positive and
concluded that LAT may be performed if the Gram stain does not identify an
organism.
Bhisitkul DH, Hogan AE and Tanz RR in their retrospective chart review
study of 146 consecutive patients with bacterial meningitis done at Children’s
Memorial Hospital Chicago (1994)20 observed that the CSF LAT was positive in 100
cases (76%) and CSF culture in 131 cases (90%). The CSF gram stain was positive in
93 cases (71%). The causative organisms were H.influenzae B in 89 cases (61%),
pneumococci in 28 cases (19%), meningococci in 16 cases (11%), Group B
streptococcus in 10 cases (7%), and E.coli in 3 cases (2%). In patients who had
received prior antibiotics, the CSF LAT was positive in 87%, the CSF Gram stain in
71% and CSF culture in 75%. Twenty five percent of patients who had prior antibiotic
were culture negative but LAT positive. The study indicated the need for CSF LAT is
performed whenever the patient has received antibiotic therapy before the CSF culture
is obtained.
Das BK, Gurubacharya RL, Mishra OP, Mohapatra TM, in their study
conducted at Department of Pediatrics, Institute of Medical Sciences, Banaras Hindu
University, Varanasi, India (2003)21
studied 30 cases of ABM. Using LAT, an
etiological diagnosis could be made in 30 cases (83%). In contrast CSF Gram stain
was positive in 13(36%) and culture was positive in 2 (6%) cases. The sensitivity and
specificity of LAT test were 83% and 100% respectively. The common etiological
organisms were S. pneumoniae, H. influenzae type b and N. meningitidis A. S.
pneumoniae was isolated in all age groups while H. influenzae type b was found only
in below one year of age. In the study it was observed that LAT was a rapid and
superior diagnostic tool as compared to Gram stain and culture. The study
recommends LAT as an adjunct laboratory test for rapid etiological diagnosis of
ABM.
Among the 65 cases included in the study by Surinder K, Bineeta K, Megha M
, at Department of Microbiology, Maulana Azad Medical College, New Delhi,
India(2007)12,CSF culture was positive in 15 (23.1%). N. meningitidis 4,
S.pneumoniae 3, Acinetobacter species 2, E.coli 2, and one each of Staphylococcus
aureus,
Klebsiella
pneumoniae,
Pseudomonas
aeruginosa,
and
Group
B
Streptococcus. Of these 15 cases Gram stain was positive in 11 (73.3%). LAT
detected bacterial antigens in 10 (15.4%) cases. This study showed that Gram stained
CSF examination is likely to suggest etiological agent and all the samples that were
positive on culture or by LAT showed more than 5 to 6 pus cells per high power field
on Gram stain smear. The study concluded that LAT can be an adjunct laboratory test
in the etiological diagnosis of ABM particularly in pretreated cases.
In a study conducted at Department of Neuromicrobiology, NIMHANS,
Bangalore, India (2007)6 by Mani R, Pradhan S, Nagarathna S, Wasiulla R,
Chandramukhi A, noted that among 385 patients diagnosed as ABM an etiological
agent was identified in 284 (73.8%) cases by culture and/or smear and/or LAT. Gram
stain was positive in 253 (65.7%) patients and culture yielded growth in 157 (40.8%).
S. pneumoniae was the predominant agent accounting for 238 (61.8%) cases, in which
116 were culture positive, 60 detected by Gram stain and 62 by LAT. H. influenzae
was detected in 7 (1.8%) both culture and Gram stain positive in 4 and LAT detected
additional 3 cases. Meningococcal meningitis was identified in 4 (1%) cases Gram
stain was positive in all 4, two were culture positive and other two were detected by
LAT. The high yield of pathogens on Gram stain was attributed to routine use of
cytospin to concentrate the smear. Smear negative CSF samples with neutrophilic
pleocytosis in suspected ABM warrants antigen testing by LAT. The conclusion of
the study was antigen testing for pneumococci should be performed first, since it is
the most common pathogen causing community acquired ABM.
Sahai S, Mahadevan S, Srinivasan S, Kanungo R, in their study conducted at
Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry
India (2001)22 observed that among 100 cases of ABM, organisms were isolated in
35% cases only. Culture negative meningitis accounted for majority of cases. Almost
88% of children with H.influenzae meningitis were below 2 years of age.
S.pneumoniae was uniformly distributed in all the age groups. 42% cases had received
antibiotics prior to admission. Long duration of pre admission illness, moderate CSF
cellular response and good outcome with standard therapy characterized the group of
children with culture negative meningitis. Short prediagnostic history was associated
with higher mortality irrespective of the organisms.
Rao BN, Kashbur IM, Shembesh NM, El-Bargathy SM, in their study
conducted at Al-Fateh Children’s Hospital, Al-Arab Medical University, Benghazi
(1998)8 concluded that among 77 ABM cases Gram stained smears identified more
etiological agents than culture. Culture of CSF was positive in 48 (62.3%) cases while
Gram stain was positive in 66 (85.7%). LAT was positive in 41 (53.2%) cases.
Among the identified etiological agents, H.influenzae was predominant agent (33.8%)
followed by S.pneumoniae (26%) and Klebsiella species (6.5%). Higher proportion of
H. influenzae infection was observed in children < 1 year of age. Thus the simple
Gram stain combined with the LAT will help give a rapid diagnosis of bacterial
meningitis and may indicate the most probable causative organisms in more than 85%
(66/77) of cases before culture results are available.
Berkley JA, Mwangi I, Ngesta CJ, Mwarumba S, Lowe BS, Marsh K et al in
their study conducted at Kilifi District Hospital, Kenya (2001)2,examined 905 CSF
samples,among them 45 met the criteria for acute bacterial meningitis. CSF culture
was positive in 37 cases- 17 S. pneumoniae, 14 H.influenzae, three non-typhoidal
salmonellae, one Pseudomonas aeruginosa, one group A streptococcus and one group
B streptococcus. In the eight children with negative CSF culture, LAT identified six
organisms, 5 cases of H.influenzae and one case of S.pneumoniae. The other two were
seen in Gram stained smears. Overall Gram stain identified 22 (59.5%) of culture
positive cases and four culture negative cases (three gram-negative rods and one
gram-positive cocci). The sensitivity of LAT for a positive CSF culture of
H. influenzae type b or S. pneumoniae was 96.6% (28/29). The study concluded that
without reliable laboratory facilities third of ABM cases may be missed and CSF
leukocyte counting misses sixth of ABM cases even when done accurately. CSF
culture is clearly the ideal but practically not available.
Sippel JE, Hider PA, Controni G, Eisenach KD, Hill HR et al in their study
conducted at Naval Biosciences Laboratory, Oakland, California and Naval Medical
Research Unit No. 3, Cairo, Egypt (1984)23 got the following results: Directigen LAT
detected H. influenzae type b antigen in 83% (69 of 83) of the specimens obtained
from patients with H. influenzae disease, pneumococcal antigen in 77% (30 of 39) of
the specimens from patients with pneumococcal disease, and N. meningitidis antigen
in 93% (37 of 40 ) of the specimens from patients with disease caused by N.
meningitidis serogroups A and C . If only culture positive specimens are considered,
Directigen LAT detected antigen in 86% (57/66) of H.influenzae specimens, 80%
(24/30) of the pneumococcal specimens and 91% (20/22) of meningococcal CSF
specimen. No false positive reactions were reported with the Directigen reagents.
Nonspecific reactions (agglutination with more than one of the four Directigen Latex
reagents) were noted with five specimens. The nonspecific reactions were resolved in
four of the five specimens by heating (100°C for 3 min). The data in this study
demonstrated Directigen meningitis test is sensitive for detection of antigens in
cerebrospinal fluid.
Hardiono P, Hanifah O, Dalima A, Veronica F in their study conducted at
Mangunkusumo hospital, Jakarta, Indonesia (1998)7 noted that among 100 subjects,
16 were enrolled as suspected bacterial meningitis. A final diagnosis of bacterial
meningitis was established in 11 patients. Six were culture positive ,2 with H.
influenzae and ,1 each with Neisseria meningitidis, Staphylococcus aureus, Klebsiella
ozaenae and Escherichia coli. Three of the six had LAT positive ,2 H. influenzae and,
1 N. meningitidis. LAT was negative in two (Klebsiella ozaenae and Staphylococcus
aureus) and cross reacted with N. meningitidis in one (E.coli). LAT was negative for
all 10 with negative CSF culture. Gram stained smear was positive in only one of the
six cases with positive CSF culture and was negative in all 10 cases with negative
culture. It was observed in this study that LAT correlated well with culture whereas
Gram stain has had low sensitivity, perhaps reflecting deficiencies in technique. The
number of children admitted with bacterial meningitis has been declining, perhaps
because of early treatment with antibiotics in the community.
Bercion R, Bobossi-Seregbe G, Godby JC, Beyam EN, Manirakiza A and Le
Faou A in their study conducted at Complexe Pediatrique of Bangui, Central African
Republic (2007)24 found that over one year period 417 patients were enrolled as
suspected bacterial meningitis who underwent lumbar puncture. One hundred thirty
were proven meningitis confirmed when gram stained smear identified bacteria, when
CSF culture yielded growth and when antigen was detected in the supernatant of
centrifuged CSF. Among these S. pneumoniae was the most common (62 cases 48%)
followed by H.influenzae (46 cases 35%) and by N.meningitidis and Salmonella
species (8 cases 6% each).
Ceyham M, Yildirim I, Balmer P, Borrow R, Dikici B, Turgut M et al in their
study conducted in 12 hospitals participating in the study in Turkey(2008)25 noted
that over one year period there were 408 children with clinical suspicion of
meningitis. Of the 408 cases bacterial meningitis was confirmed by culture, LAT and
Polymerase Chain Reaction (PCR) in 243 (59.6%). Culture was positive in 41/243
(17%) cases -23 N.meningitidis, 12 S.pneumoniae and Hib in six cases. LAT detected
antigens in 56/243 (23%) cases – 37 N.meningitidis, 10 H.influenzae, and in 9 S.
pneumoniae. Blood culture was positive in 12/243 (4.9%) 4 each of N.meningitidis,
Hib and S.pneumoniae. Seven of 41 cases with positive CSF culture and 111 of 202
with negative CSF culture had a history of use of antimicrobial agents before lumbar
puncture. PCR analysis was by far the most reliable method of confirming bacterial
meningitis, accounting for all confirmed cases with 243 positive results.
ACUTE BACTERIAL MENINGITIS
Meningitis is defined as the inflammation of the membranes that surround the
brain and spinal cord.26The overall attack rate for bacterial meningitis is
approximately 3.0 cases per 100000 population, although variability based on age
,race and sex is noted.27The three most common meningeal pathogens, Haemophilus
influenzae, Neisseria meningitidis, and Streptococcus pneumoniae account for more
than 80% of cases.28Approximately 10% of patients with bacterial meningitis die, and
40% have sequelae including hearing impairment and other neurologic sequelae.26
Classification: Meningitis is classified as follows:
1. According to presentation
a. Acute meningitis
b. Chronic meningitis
2. According to etiology
a. Bacterial meningitis
b. Viral meningitis
c. fungal meningitis
3.According to epidemiology
a.Sporadic
b. Epidemic1
EPIDEMIOLOGY
TABLE -1:Relationship between Common Bacterial Pathogens and Factors
Predisposing to Meningitis .28
Predisposing Factor
Bacterial Pathogens
Age
<1 mo
Streptococcus agalactiae, Escherichia coli,
Listeria monocytogenes, Klebsiella pneumoniae
1–23 mo
S. agalactiae, E. coli, Haemophilus influenzae,
Streptococcus
pneumoniae,
Neisseria
meningitidis
2–50 yr
S. pneumoniae, N. meningitidis
>50 yr
S.
pneumoniae,
N.
meningitidis,
L.
monocytogenes, aerobic gram-negative bacilli
Immunocompromised state
S.
pneumoniae,
N.
meningitidis,
L.
monocytogenes, aerobic gram-negative bacilli
(including Pseudomonas aeruginosa)
Basilar skull fracture
S. pneumoniae, H. influenzae, group A βhemolytic streptococci
Head trauma; postneurosurgery
Staphylococcus
aureus,
Staphylococcus
epidermidis, aerobic gram-negative bacilli
(including P. aeruginosa)
The likely etiologic agents of bacterial meningitis vary according to the age
and underlying disease status of the patient.28
Haemophilus influenzae
H. influenzae was previously isolated in 45% to 48% of all cases of bacterial
meningitis in the United States. The number of cases of H.influenzae type b
meningitis since the introduction of vaccination has decreased more than 90%.
However, in developing countries, because of vaccine expense, the results are not as
dramatic.28In India the incidence of Hib meningitis is reported to be 6%-44%.29,30
The overall mortality rate is 3% to 6%. Most episodes of meningitis previously
occurred in infants and children younger than 6 years (peak incidence of 6 to 12
months),with 90% of cases caused by capsular type B strains. Isolation of this
organism in older children and adults should suggest the presence of certain
underlying conditions, including sinusitis, otitis media, epiglottitis, pneumonia,
diabetes mellitus, alcoholism, splenectomy or asplenic states, head trauma with CSF
leak, and immune deficiency.28Household contacts of those with H.influenzae type b
disease have a two percent risk of developing invasive disease,a relative risk 600
times greater than that for the general population.31
Neisseria meningitidis
N. meningitidis most commonly causes meningitis in children and young adults and is
associated with an overall mortality rate of 3% to 13%.Meningococci of serogroups
B, C, and Y account for most of the endemic disease. Disease caused by serogroups
A and C may occur in epidemics; group Y strains may be associated with pneumonia.
Respiratory tract infections, with viruses such as influenza virus, may play a role in
the pathogenesis of invasive meningococcal disease. Nasopharyngeal carriage of
N. meningitidis is an important factor that leads to the development of invasive
disease. Patients with deficiencies in the terminal complement components (C5, C6,
C7, C8, and perhaps C9), the so-called membrane attack complex, have a markedly
increased incidence of neisserial infection although mortality rates in patients with
meningococcal disease are lower than those in patients with an intact complement
system. An increased risk of invasive meningococcal disease has also been described
with dysfunctional properdin. Meningococcal meningitis occurs in approximately
39% of persons with late complement component deficiencies and 6% of those with
properdin deficiencies.28The risk of household contacts of those with meningococcal
meningitis is 2.2 per thousand per year. This risk is almost 1000 times greater than
that for the population as a whole .32Almost half of the cases tend to occur within
three days of illness in the index case. The increased risk continues to persist for 2 to
4 weeks.3
Streptococcus pneumoniae
S. pneumoniae, the most frequently observed etiologic agent of bacterial meningitis in
the United States, now accounts for 47% of the total cases. the mortality rate ranges
from 19% to 26%.Of the more than 90 known pneumococcal serotypes, 18 are
responsible for 82% of the cases of bacteremic pneumococcal pneumonia, with a
close correlation between bacteremic subtypes and those implicated in meningitis.
Patients often have contiguous or distant foci of pneumococcal infection such as
pneumonia, otitis media, mastoiditis, sinusitis, and endocarditis. Serious infection
may be observed in patients with various underlying conditions (e.g., splenectomy or
asplenic
states,
multiple
myeloma,
hypogammaglobulinemia,
alcoholism,
malnutrition, chronic liver or renal disease, malignancy, and diabetes mellitus. The
pneumococcus is the most common etiologic agent of meningitis in patients who have
suffered basilar skull fracture with CSF leak.28
Listeria monocytogenes
L. monocytogenes causes 8% of cases of bacterial meningitis in the United States and
carries a mortality rate of 15% to 29%.Serotypes 1/2b and 4b have been implicated in
up to 80% of meningitis cases caused by this organism. Listerial infection is most
common in infants younger than 1 month (up to 10% of cases), adults older than 60
years, alcoholics, cancer patients, those receiving corticosteroid therapy, and
immunosuppressed adults. Other predisposing conditions include diabetes mellitus,
liver disease, chronic renal disease, collagen-vascular diseases, and conditions
associated with iron overload. Recently, Listeria meningitis has been reported in a
patient with Crohn’s disease after treatment with infliximab. Listeria meningitis is
found infrequently in patients with HIV infection despite its increased incidence in
patients with deficiencies in cell-mediated immunity. Adults less than 50 years of age
who present with Listeria meningitis should be screened for HIV infection.
Meningitis can also occur in previously healthy adults. Outbreaks of Listeria infection
have been associated with the consumption of contaminated coleslaw, raw vegetables,
milk, and cheese, with sporadic cases traced to contaminated cheese, turkey franks,
alfalfa tablets, and processed meats, thus pointing to the intestinal tract as the usual
portal of entry.28
Streptococcus agalactiae
Group B streptococcus is a common cause of meningitis in neonates,with 52% of all
cases in the United States reported during the first month of life overall mortality rate
ranges from 7% to 27%.Group B streptococcus has been isolated from the vaginal or
rectal cultures of 15% to 35% of asymptomatic pregnant women. Colonization rates
do not vary during pregnancy, and carriage may be chronic (40%), transient, or
intermittent. The route of delivery does not influence transmission .Most cases of
neonatal meningitis are caused by subtype III organisms and occur after the first week
of life. Group B streptococcus can also cause meningitis in adults. Risk factors in
adults include age greater than 60 years, diabetes mellitus, pregnancy or the
postpartum state, cardiac disease, collagen-vascular diseases, malignancy, alcoholism,
hepatic failure, renal failure, previous stroke, neurogenic bladder, decubitus ulcers,
and corticosteroid therapy; in one review of group B streptococcal meningitis in
adults, no underlying illnesses were found in 43% of patients.28
Aerobic Gram-Negative Bacilli
Aerobic gram-negative bacilli (e.g., Klebsiella species, Escherichia coli, Serratia
marcescens, Pseudomonas aeruginosa, Salmonella species) have become increasingly
important as etiologic agents in patients with bacterial meningitis.These agents may
be isolated from the CSF of patients after head trauma or neurosurgical procedure and
may also be found in neonates, older adults, immunosuppressed patients, and patients
with gram-negative septicemia. Some cases have been associated with disseminated
strongyloidiasis in the hyperinfection syndrome . In patients with E. coli meningitis,
75% of cases are caused by strains possessing the K1 antigen. Almost half of pregnant
women have this organism isolated on rectal culture, and as many as 75% of their
infants will be colonized during the first days of life.28
Staphylococci
Meningitis caused by Staphylococcus aureus is usually found in early
postneurosurgical or post-trauma patients and in those with CSF shunts; other
underlying conditions include diabetes mellitus, alcoholism, chronic renal failure
requiring hemodialysis, injection drug use, and malignancies.Other sources of
community-acquired
S.
aureus
meningitis
include
patients
with
sinusitis,
osteomyelitis, and pneumonia. Mortality rates have ranged from 14% to 77% in
various series. Staphylococcus epidermidis is the most common cause of meningitis in
patients with CSF shunts28.
Other Bacteria
A review of 28 cases of nocardial meningitis revealed predisposing conditions in
approximately 75% of patients. Anaerobic meningitis is unusual and is generally
associated with contiguous foci of infection , in many cases, more than one organism
may be recovered. Enterococci are unusual etiologic agents of bacterial meningitis;
most adult patients have underlying illnesses. Despite the frequency with which the
viridans streptococci cause bacteremia, they are unusual causes of meningitis. Group
A streptococcal meningitis is also unusual, generally seen in association with
pharyngitis, otitis media, and sinusitis Diphtheroids, particularly Propionibacterium
acnes, have become important etiologic agents of meningitis in patients with CNS
shunt infections.28
CHARACTERISTICS OF ORGANISMS ENCOUNTERED IN
PYOGENIC MENINGITIS33, 34
Streptococcus pneumoniae:
Morphology: Typical gram positive (1 µm. in diameter) lanceolate shaped cocci
arranged in pairs with broad ends in apposition. Short chains are often found in
specimens (sputum, pus) and young culture.
 Capsulated – capsule surrounds each pair.
 In old culture, capsule is usually lost and short chains of
more rounded
diplococci with gram variable appearance may be obtained.
 Non-motile and non-sporing.
Cultural characteristics:
On blood agar, after incubation for 18 hrs, the colonies are small (0.5-1 mm), dome
shaped, glistening with an area of narrow zone of green discolouration (αhaemolysis) around them.On further incubation – the colonies become flat with raised
edges and central umbonation, so that concentric rings are seen on the surface when
viewed from above – draughtsman or carrom coin appearance. Under anaerobic
condition growth is better and haemolysis resemble β-haemolytic type due to oxygen
labile haemolysin O.
Biochemical reaction:
Ferment several sugars, forming acid only. Fermentation is tested in Hiss’s serum
water or serum agar slopes.
 Fermentation of inulin.
 Bile soluble .
 Sensitive to optochin
 Catalase and oxidase negative.
Virulence Factors:
1. Colonization and Migration
a) Protein adhesin
b) Secretory IgA protease
c) Pneumolysin
2. Tissue destruction
a) Teichoic peptidoglycan fragments.
b) Pneumolysin
c) H2O2
d) Phosphoryl choline
3. Phagocytic survival:
a) Capsule
b) Pneumolysin
Haemophilus influenzae:
 Haemophilus meaning blood loving
 They are characterized by their requirement of one or both of two accessory
growth factors X and V present in blood.
 It is the first free living organism whose complete genome has been
sequenced.
.
Morphology: Gram negative, small (1 µm x 0.3 µm) non motile, non sporing bacillus
exhibiting considerable pleomorphism. The bacillus has fastidious growth
requirements. The accessory growth factors X and V present in blood are essential for
growth. Factor X, a heat stable factor is hemin or other porphyrins required for
synthesis of chytochrome and other heme enzymes such as catalase and peroxidase
involved in aerobic respiration. V factor is a co-enzyme, Nicotinamide adenine
dinucleotide (NAD) which acts as a hydrogen acceptor in the metabolism of the cell.
Colony: Uncapsulated strains of
H. influenzae
produce smooth, small and
translucent colonies in 24 hrs at 370 C in 5-10% CO2. Capsulated strains form large,
more mucoid colonies with a mouse nest odour and are non haemolytic on rabbit or
horse blood agar.
Satellitism: Colonies of H. influenzae appear large and well developed alongside the
streak of Staphylococcus aureus, due to the availability of factor V released by
Staphylococcus aureus into the surrounding medium and is termed satellitism.
Biochemical reactions:
Sugars like glucose and xylose are fermented with acid production, Catalase and
oxidase are positive, Nitrates are reduced to Nitrites. 8 biotypes are identified on the
basis of Indole production, urease and ornithine decarboxylase activity. Biotype-I is
most frequently responsible for meningitis.
Antigenic properties:
The major antigenic determinant of the capsulated strains is the capsular
polysaccharide based on which H. influenzae strains are classified by Pittman into 6
capsular types – a, b, c, d, e and f. Type b is most commonly encountered in
meningitis in humans. Type b capsular polysaccharide has unique chemical structure
containing the pentose sugar ribose and ribitol, instead of hexoses and hexosamines as
in the other 5 serotypes. The capsular PRP antigen of Hib induces IgG, IgM, IgA
antibodies which are bactericidal, opsonic and protective. Hib PRP is therefore
employed for immunization. Outer membrane protein antigens of Hib have been
classified into 13 subtypes and are of epidemiological value.
Niesseria meningitidis :
Morphology: These are gram negative oval or spherical cocci, 0.6-0.8 µm in size,
typically arranged in pairs, with the adjacent sides flattened. They are non motile and
fresh isolates are capsulated.
Cultural characteristics: Meningococci are fastidious organisms requiring media
enriched with blood, serum or ascitic fluid. They are strict aerobes with an optimum
temperature of 35-360 C and pH 7.4-7.6. On solid media colonies are small (1 mm
diameter) translucent, round, convex, bluish grey with a smooth glistening surface and
entire edges. Colonies are typically lenticular in shape, butyrous in consistency and
easily emulsifiable. Growth is poor in liquid media showing granular turbidity with no
surface growth weak haemolysis occurs on blood agar.
Biochemical reactions: They are catalase and oxidase positive. Glucose and maltose
are utilised, but not sucrose or lactose, producing acid but no gas. Indole and H2S are
not produced, nitrates are not reduced.
Antigenic properties: Based on capsular polysaccharide antigens meningococci are
classified into 13 serogroups of which Group A, B and C are the most important.
Group A > Causes epidemics
Group C > Causes localised outbreaks
Group B > Causes both epidemics and localised outbreaks
Groups 29-E, W-135 and Y frequently cause meningitis.
Virulence factors: Surface structures perhaps pili, facilitate attachment to mucosal
epithelial cells and invasion to the submucosa. Once in the blood, survival is mediated
by production of a polysaccharide capsule. Endotoxin release mediates many of the
systemic manifestations of infections such as shock.
Enterobacteriaceae: Members of this family exhibit general, morphological and
biochemical similarities and are grouped together under a large and complex family:
 They are non sporing, non acid fast, Gram negative bacilli.
 They may or may not be capsulated.
 They are motile by peritrichate flagellae or non-motile.
 They are aerobic and facultative anaerobic, grow in ordinary media, ferment
glucose producing acid and gas or acid only, reduce nitrates to nitrites.
 Catalase positive and oxidase negative.
Escherichia coli : These are gram negative straight rod (1-3 x 0.4-0.7 ųm) arranged
singly or in pairs. Motile with peritrichate flagella.
Cultural characteristics: aerobe with a temp range of 10-400 C. Colonies are thick,
large, greyish white, moist, smooth, opaque or partially translucent discs on ordinary
media. On Mac Conkey agar – colonies are bright pink due to lactose fermentation.
Biochemical reactions: Glucose, lactose, mannitol, maltose sugars are fermented
with production of acid and gas. Four biochemical tests widely employed in the
identification of Enterobacteriaceae are the Indole, Methyl red (MR), Vogesproskauer (VP) and Citrate utilisation tests IMViC. E-coli is IMViC + + - -
Antigenic structure : Serotyping of E.coli is based on 3 antigens – somatic O,
capsular K and flagellar H antigen. The antigenic pattern of a strain is recorded as the
member of the particular antigen it carries, as for ex: 0111:K58:H2.
Virulence factors: The virulence factors of E.coli as a cause of extra intestinal
infections are several including endotoxin, capsule production and pili that mediate
attachment to host cells. Envelope K antigens afford protection against phagocytosis
and E.coli strains responsible for neonatal meningitis carry K1 envelope antigen.
S. agalactiae: Group B streptococcus have been recognized as the leading cause of
early and late onset septicemia and meningitis in neonates and as an important cause
of invasive infections in elderly or immunocompromised adults. Its ability to survive
in the host is to a high degree dependent on its ability to resist phagocytosis.
Morphology: Streptococci are gram positive cocci, 0.5-1.0 µm x 1-2µm in size
arranged in chains or pairs. Chain formation is most pronounced in broth media.
Human pathogen strains of S.agalactiae produce a polysaccharide capsule which
appears to confer virulence.
Cultural characteristics – Growth occurs on media enriched with blood, serum or a
fermentable carbohydrate. It is an aerobe and a facultative anaerobe, growing best at
370C. On blood agar colonies are small (0.5-1 mm), circular, semitransparent, low
convex discs with an area of alpha-haemolysis around them. They produce an orange
red pigment which presumably is a beta-carotenoid. In liquid media such as glucose
or serum broth growth occurs as a granular turbidity with a powdery deposit. No
pellicle is formed.
Biochemical reactions – The ferment sugars producing acid but no gas.
Catalase negative.
Not soluble in 10% bile,
Hippurate hydrolysis and Camp test positive.
Virulence factors
Clinical isolates produce a polysaccharide capsule. A total of 9 different capsular
serotypes (Ia, Ib, II, III-VIII) has been demonstrated. Although invasive infection may
be associated with all the serotypes, strains with serotype III capsule predominate
among isolates from neonatal infections. All capsular serotypes are polysaccharides
composed of galactose and glucose. Antibodies to the nine capsular serotypes confer
type specific protection.
Camp factors and Haemolysin
Strains of streptococci produce a diffusible substance that completes the lysis of sheep
erythrocytes exposed to Sphingomyelinase C such as Staphylococcal β toxin or α
toxin of C.perfringens. Its role as a virulence factor is supported by its ability to bind
immunoglobulin G and M and has also been referred to as protein B. Haemolysin of
S.agalactiae which is considered as virulence factor has been cloned and sequenced,
is not related to streptolysins of S.pyogenes.
Other factors – S.agalactiae produce several M. like proteins which binds various
host proteins, a C5a peptidase and hyaluronidase.
GRAM NEGATIVE NON-FERMENTERS
Pseudomonas aeruginosa : It is a slender Gram negative bacilli. 1.5-0.3 µm x 0.5
µm, actively motile by a polar flagellum. They are non capsulated. They are
ubiquitious, mostly saprophytic, being found in water, soil or other moist
environments.
Cultural characteristics: It is an obligate aerobe, grows at a wide range of
temperature 6-420 C. On ordinary media, they produce large, opaque, irregular
colonies with a distinctive, musty, mawkish or earthy smell. On blood agar, they are
hemolytic and produce Non-lactose fermenting colonies on Mac Conkey agar. In
broth, it forms a dense turbidity with a surface pellicle.
Virulence factors: These include exotoxin A, endotoxins, proteolytic enzymes,
alginate and pili.
Biochemical reactions: Metabolism is oxidative and non fermentative. Glucose is
utilised oxidatively forming acid only. IMViC are negative. Catalase and oxidase
positive.
Acinetobacter: is strictly aerobic, non motile, gram negative cocco bacillary rods that
are oxidase negative, nitrate not reduced and do not ferment sugars. They are non
motile and 1-1.5µm x 1.5-2.5µm in size. Often appearing in pairs, mimicking
neisseriae in appearance. Acinetobacter is subdivided as follows: Glucose oxidising,
non haemolytic clinical strains as A.baumannii, Glucose negative, non-hemolytic
strain as A. lwoffi and the haemolytic strain as A. hemolyticus.
On Blood Agar: Smooth, opaque, raised, creamy colonies.
A. baumannii forms pinkish colonies on Mac Conkey medium. Acid without gas is
formed in glucose, arabinose, and xylose. Characteristic reaction is the formation of
acid in 10% but not 1% lactose.
A.lwoffi – Forms yellow colonies on the Mac Conkey medium, does not acidify
sugars and some strains are oxidase positive. They are widely distributed in nature,
including the hospital environment, may become established as a part of skin and
respiratory flora of patients hospitalised for prolonged periods.
Entercoccus species: can be found in soil, food, water and as normal flora in animals,
birds, insects and humans. In humans and other animals it inhabits the gastrointestinal
tract and the genitourinary tract. Enterococcus faecalis is one of the most common
bacteria isolated from the gastro-intestinal tract of humans.
Morphology: Enterococci may be spherical or oval and arranged in pairs at an angle
to each other (like spectacles) and in short chains. They are gram positive and nonmotile.
Colony Characteristics: Colonies on blood agar are 1-2 mm in diameter, small,
cream or white, smooth, entire and alpha or beta or non-hemolytic. On Mac Conkey
agar, they produce tiny deep pink colonies.
Virulence factors: Little is known about virulence. Adhesins, cytolysins and other
metabolic capabilities may allow these organisms to proliferate as nosocomial
pathogens. In addition, enterococci are capable of acquiring and exchanging genes
that encode resistance to antimicrobial agents. And multi drug resistance also
contributes to proliferation.
Biochemical : Enterococci possess several distinctive features that help in their
identification from streptococci:
 Ability to grow in the presence of 40% bile.
 Salt resistant, grow in the presence of 6.5% NaCl
 Heat resistant, grow at 450 C.
 Grow at a pH of 9.6
E. faecalis is identified by its ability to ferment mannitol, sucrose, sorbitol and
aesculin. It grows on tellurite blood agar producing black colonies.
Listeria monocytogenes: It is widely distributed in nature and has been isolated from
a wide range of mammals, birds, fish, ticks and crustacea. It occurs as a saprophyte in
soil, water and sewage.
Morphology: Listeria monocytogenes is a small, coccoid, gram positive bacillus,
with a tendency to occur in chains. Rough forms may be seen as long filaments. It
exhibits a characteristic, slow, tumbling motility at 250C and is non motile when
grown at 370 C. It is aerophilic or microaerophillic.
Cultural characteristics: Growth is improved with 5-10% CO2. Greater success of
isolation is achieved if samples are stored in nutrient broth at 40C and subcultures
done at weekly intervals for 1-6 months (cold enrichment). Grows best between 300 C
and 370 C colonies are non haemolytic on blood agar.
Virulence factors: Listeriolysin, a hemolytic and cytotoxic toxin that may allow
survival within phagocytes.
Biochemical reactions: Listeria monocytogenes ferments glucose, maltose, rhamnose
and alpha methyl D-mannoside, producing acid without gas, catalase positive, grows
in 0.1% potassium tellurite, 10% salt and at pH 9.6.
Many serovars have been recognised. Most human infections are caused by Servovar
1/2a or 1/2b and 4b. Monocytosis is a feature of human listeriosis, hence the name
monocytogenes.
*****
PATHOGENESIS:
The neurotropic potential of the most common bacterial causes of meningitis
(S. pneumoniae, H. influenzae, N. meningitidis and E. coli) relates to their ability to
evade several host defences.35
TABLE-2: Pathogenic sequence of Bacterial Neurotropism.35
NEUROTROPIC STAGE
HOST DEFENCE
STRATEGY OF PATHOGEN
Colonization or mucosal
invasion
Secretory Ig A
Ig A protease secretion
Ciliary activity
Ciliostasis
Mucosal epithelium
Adhesive pili
Intravascular survival
Complement
Polysaccharide capsule
Crossing of blood brain
barrier
Cerebral endothelium
Adhesive pili
Survival within CSF
Poor opsonic activity
Bacterial replication
In infants and children, many of the organisms that cause meningitis colonize
the upper respiratory tract (nasopharynx). Viral infections of the upper respiratory
tract commonly precede invasion of the blood stream. Pneumococci, meningococci
and Hib all secrete IgA proteases that cleave the proline rich hinge region of IgA
rendering it non functional facilitating bacterial attachment to the epithelium. Binding
to the epithelium is dependent on the presence of pili on the surface of the bacteria.
After mucosal invasion and attachment the pathogen enters blood stream and survives
complement mediated host defence by virtue of its capsular polysaccharide. After
bacteremia, pathogens penetrate blood brain barrier (BBB) to enter the subarachnoid
space – the step that is least understood in the pathogenesis of meningitis. Bacterial
penetration of the blood brain barrier is poorly understood, but it appears to be
dependent on a sufficient bacterial inoculum (>1000 colony-forming units per
millilitres of blood) aided by multiple inflammatory mediators liberated by invading
pathogens and the host’s own immune cells. The vulnerable sites of BBB are choroid
plexus and cerebral capillaries, surface bacterial proteins facilitate invasion and
organisms reach subarachnoid space.26, 35, 36, 37, 38.
Meningitis can also develop by direct extension of infection from a paranasal
sinus or from the middle ear through the mastoid to the meninges. Severe head trauma
with a skull fracture, CSF rhinorrhoea, or both, can lead to meningitis, usually caused
by S. pneumoniae. Bacteria can be directly inoculated into the CSF by congenital
dural defects (dermal sinus or meningomyelocele), neurosurgical procedures (such as
CSF diversion shunts), penetrating wounds, or extension from a suppurative
parameningeal focus.26
Once bacteria reach the CSF, they are likely to survive because humoral
defences such as immunoglobulin, complement activity and opsonic activity are
undetectable in the CSF.35
PATHOPHYSIOLOGY
Bacterial meningitis is almost always a systemic disease, with bacteria gaining
entry to the CNS through the blood stream.38once the meningeal pathogens have
entered the CNS, they replicate rapidly and liberate active cell wall or subcapsular
surface components –i.e., lipoteichoic acid and peptidoglycan fragments of grampositive organisms, and lipopolysaccharide of gram-negative organisms. Antibiotics
that act on cell walls cause rapid lysis of bacteria, which can initially cause enhanced
release of these active bacterial products into the CSF. These potent inflammatory
substances can stimulate macrophage-equivalent brain cells (astrocytes and
microglia), cerebral capillary endothelia, or both, to produce cytokines such as tumor
necrosis factor (TNF), interleukin-1 (IL-1), interleukin-6, interleukin-8, platelet
activating factor, nitric oxide, arachidonic acid metabolites (prostaglandin and
prostacycline), and macrophage derived proteins. The cytokines activate adhesion
promoting receptors on cerebral vascular endothelial cells and leukocytes, attracting
neutrophils to these sites.26, 35, 36, 37,38,39,40
Subsequently, leukocytes penetrate the intercellular junctions of the capillary
endothelium and release proteolytic products and toxic oxygen radicals. These events
result in injury to the vascular endothelium and alteration of BBB permeability.
Depending on the potency and duration of the inflammatory stimuli, the alterations in
permeability result in penetration of low-molecular-weight serum proteins into CSF
and in vasogenic edema. Additionally, large number of leukocytes enters the sub
arachnoid space and release toxic substances that contribute to the production of
cytotoxic edema. As a result of high protein and cell content, the increased CSF
viscosity contributes to generation of interstitial edema. The accumulation of
inflammatory cells, protein, and other material within the CSF interferes with
functioning of the arachnoid villi and blocks the resorption of CSF from the
subarachnoid space. Similarly, the ventriculitis that often accompanies meningitis
may occlude the cerebral aqueduct and lead to hydrocephalus, further exacerbating
interstitial edema and elevating intracranial pressure. Early and common findings on
CT scan in children with bacterial meningitis are ventriculomegaly and enlargement
of subarachnoid spaces. All these inflammatory events, if not modulated promptly and
effectively, eventually cause alteration of CSF dynamics (brain edema, intracranial
hypertension), of brain metabolism, and of cerebrovascular autoregulation (decreased
cerebral blood flow).26, 36, 39
Meningitis, sepsis and proinflammatory cytokines accompanying them may
induce septic shock and global cerebral hypoperfusion. The purulent exudates,
through which arteries and veins pass in the subarachnoid space, may lead to
vasospasm and vasculitis with secondary thrombosis, ischemia, and infarction.
Occasionally, necrotizing arteritis may induce subarachnoid hemorrhage. Increased
intracranial pressure in meningitis induces brain pathologic changes via at least two
mechanisms. First, elevation of intracranial pressure reduces cerebral perfusion
pressure and can lead to cerebral ischemia. Second if the intracranial pressure rises
substantially, as it often does in bacterial meningitis, cerebral herniation may occur.
Cerebral herniation is commonly the ultimate cause of death in fatal cases of bacterial
meningitis in children.39
Current research focuses on delineating the mechanisms involved in neuronal
injury, possibly through the participation of potential mediators such as reactive
oxygen and nitrogen substances, excitatory amino acids, metalloproteinase, and
cellular apoptosis mediated by caspases. Genetic targeting or pharmacological
blockade of these molecular pathways, or both, might prevent the irreversible focal or
diffuse brain damage frequently associated with meningitis.26,37
Fig-1:
PATHOPHYSIOLOGIC
CASCADE
IN
BACTERIAL
MENINGITIS.36
Endothelial damage
Pro inflammatory
cytokines
Permeability of blood
brain barrier
Vasogenic
edema
Leukocyte attraction &
CSF entrance:
meningeal inflammation
Interstitial
edema
↑ Intra cranial
pressure
Septic shock and
systemic effects of
inflammatory
cytokines
↓ Global perfusion
Neuronal injury
Apoptosis
Cytotoxic
edema
Cerebral
vasculitis
↓ Cerebral blood flow
ischemia
Release of
excitatory amino
acids
Clinical features:
Infants and children with bacterial meningitis have acute onset of a febrile
illness (fever, lethargy, and anorexia), that can be accompanied by meningeal
inflammation (nausea, vomiting, headache, photophobia, and nuchal rigidity, apathy
and tripod phenomenon) and encephalopathy (altered consciousness, stupor, coma,
seizures) and focal neurologic signs.38,41 An antecedent history of upper respiratory
infection or gastrointestinal infection with acute rhinitis, cough, vomiting and
diarrhoea is often present and the neurologic findings can be overshadowed.
Recognizing the point at which an upper respiratory or gastrointestinal infection has
evolved into an infection of nervous system is difficult.40The key to an early
recognition is to have high index of suspicion of meningitis whenever a child is sick.
In young infants the specific signs of meningeal inflammation are often minimal or
absent and non specific manifestations such as paradoxical irritability, lethargy and
poor feeding may be the early, or even only, findings.3,41,42
Bacterial meningitis (BM) generally presents in two ways, the first pattern is
insidious in which the condition develops over several days and the other is acute and
sometimes fulminant, in which the features of sepsis and meningitis develop in a few
hours. The level of consciousness on admission predicts ultimate morbidity and
mortality; comatose patients have a poorer outcome than do patients who are only
irritable or lethargic. Children with a preadmission illness of >48 hours do better than
those with symptoms for 24 hours or less. Normal consciousness at admission is a
good prognostic sign in BM. Occasionally, the first sign of illness is a convulsion that
can recur during progression of the disease. Seizures occur in 30% of cases of BM at
some stage of their illness.3, 5, 36, 40
On examination, infants have high fever, bulging anterior fontanelle, altered
sensorium, hypoactive or hyperactive deep tendon reflexes. Older children exhibit
signs of meningeal inflammation Kernig’s (flexing the hips to right angles and then
attempting to extend the legs at knees – full extension is impossible in meningitis)
and Brudzinski’s signs (elicited by rapidly flexing the neck- immediate flexion of the
legs at the hips and knees results). Less commonly frank coma, pupillary changes,
cranial nerve dysfunction, or an altered respiratory pattern (e.g., Cheyne-Stokes
respirations, sustained hyperventilation, or hypoventilation or apnoea) is noted. Focal
neurologic deficits are present in 20% of children with BM. Papilledema is
uncommon in ABM.3,40 Cerebral edema and increased intra cranial pressure are the
rule in ABM.3 BM may show signs of circulatory failure (e.g., shock, oliguria and
hypotension), as a part of systemic nature of the disease. 36,38
Petechial and purpuric eruptions associated with shock like state are usually
indicative of meningococcemia, although the rash can be seen less commonly in
Haemophilus influenzae b meningitis also. The presence of a chronically draining ear
or a history of head trauma with or without skull fracture is most likely to be
associated with pneumococcal meningitis.36, 38
Other foci of infection such as otitis media, pneumonia, cellulitis and septic
arthritis are present in variable proportion of children. A persistently high level of
ADH, despite low serum osmolality, is commonly seen in meningitis and is labelled
the Syndrome of Inappropriate ADH secretion (SIADH). This contributes to increased
intra cranial pressure and may be partly responsible for seizures and other acute
manifestations of meningitis.3
Laboratory methods for diagnosis of bacterial meningitis:
Collection and Transport of Clinical Specimens
The collection of clinical specimens is important in the isolation and identification of
bacterial agents that cause meningitis. It is recommended that clinical specimens be
obtained before antimicrobial therapy is begun to avoid loss of viability of the
etiological agents. Treatment of the patient, however, should not be delayed while
awaiting collection of specimens. CSF and blood should be processed in a
bacteriology laboratory as soon as possible.43
Usually, 3 tubes of CSF are collected for chemistry, microbiology, and cytology. If
only one tube of fluid is available, it should be given to the microbiology laboratory.
If more than one tube (1 ml each) is available, the second or third tube should go to
the microbiology laboratory.43
Transport of Clinical Specimens
S. pneumoniae, H. influenzae and N. meningitidis are fastidious and fragile bacteria.
They are more reliably isolated if the clinical material is examined as soon as possible
after collection.43
CSF
As soon as the CSF has been collected, it should be transported to the microbiology
laboratory, where it should be examined as soon as possible (within one hour from the
time of collection) . CSF should not be exposed to sunlight or extreme heat or cold. If
N. meningitidis is suspected to be the cause of the illness, and a delay of several hours
in processing specimens is anticipated, incubating the CSF (with screw-caps
loosened) at 350C in a 5% CO2 atmosphere (or candle-jar) may improve bacterial
survival. If sameday transport to the laboratory is not possible, CSF should be
inoculated aseptically into a Trans-Isolate (T-I) medium with a syringe and held
overnight at, or close to, 350C. T-I is a biphasic medium that is useful for the primary
culture of meningococci and other etiological agents of bacterial meningitis from CSF
and blood samples. It can be used as a growth medium as well as a holding and
transport medium. 43
Inoculation of Primary Culture Media
Once the CSF has arrived at the microbiology laboratory, centrifuge it for 20 minutes
at 2000 rpm. Draw off the supernatant with a Pasteur pipette. When antigen detection
by latex agglutination is planned, save the supernatant. Sediment must be either
vigorously vortexed or well mixed. Use one or two drops of sediment to prepare the
Gram stain and use 1 drop to streak the primary culture media.
Note: If less than 1 ml of CSF is available, do not centrifuge. Use it for the Gram
stain and plate it directly.43
The best medium for S. pneumoniae is a Blood agar plate(BAP), which is a trypticase
soy agar (TSA) plate containing sheep or horse blood (5%). Human blood is not an
acceptable substitute.43
For H. influenzae, a Chocolate agar plate(CAP) supplemented with haemin and a
growth supplement such as IsoVitaleX, supplement B, or Vitox should be used. An
acceptable alternative to achieve growth of H. influenzae on BAP is cross-streaking
the medium with Staphylococcus aureus or applying a filter paper/disk saturated with
X and V factors to the surface of the medium, after the medium had been inoculated.
H. influenzae forms satellite colonies along the length of the staphylococcal growth or
produces a halo of growth around the XV strip/disk. N. meningitidis grows on both
BAP and CAP, as will S. pneumoniae. If only one type of plate is available, choose
CAP, because all three etiological agents can grow on it. A bacteriological loop is
used to streak or thin the bacteria into single colonies. The agar plates should be
incubated in a 5% CO2 incubator or candle-jar. A backup broth (e.g. brain heart
infusion broth) should be inoculated with some of the pellet and also incubated.[66]
When T-I medium was used for transport, after 24 hours of incubation, with a sterile
needle and syringe, transfer 100 μl of the liquid portion of T-I onto BAP and CAP,
and streak for isolation. Mac Conkey agar is for identification of lactose fermenting
and non-lactose fermenting gram negative bacilli.43
Gram Stain
A presumptive diagnosis of bacterial meningitis caused by H. influenzae, S.
pneumoniae, and N. meningitidis can be made by Gram stain of the CSF sediment or
by detection of specific antigens in the CSF by a latex agglutination test. Positive
results of either or both tests can provide evidence of infection, even if cultures fail to
grow. N. meningitidis may occur intra- or extra-cellularly in the polymorphonuclear
leukocytes and will appear as Gram-negative, coffee-bean-shaped diplococci .
S. pneumoniae are lanceolate, Gram-positive diplococci sometimes occurring in short
chains. H. influenzae are small, pleomorphic Gram-negative rods or coccobacilli with
random arrangements .43
T-I Medium
(a) After 24 hours of incubation, using a sterile needle and syringe, transfer 100 μl of
the liquid portion of T-I onto BAP and CAP, streak the plate, and incubate at 350 C in
a CO2 atmosphere for up to 48 hours.
(b) Check for purity of the growth by performing Gram stain.
(c) If no growth occurs, subculture the T-I medium at 3 days and again at 7 days.43
Macroscopic Examination of Colonies
N. meningitidis grows on BAP, but H. influenzae does not. H. influenzae appears as
large, flat, colorless-to-grey opaque colonies. No haemolysis or discoloration of the
medium is apparent. On BAP, young colonies of N. meningitidis are round, smooth,
moist, glistening and convex, with an entire edge. Some colonies appear to coalesce
with their neighbours. Growth is greyish and unpigmented. Older cultures become
more opaquely grey and sometimes cause the underlying agar to turn dark. Wellseparated colonies can grow from about 1 mm in diameter in 18 hours to as large as 4
mm, with a somewhat undulating edge, after several days . S. pneumoniae appears as
small, greyish, moist (sometimes mucoid), watery colonies with a greenish zone of
alpha-haemolysis surrounding them on BAP and CAP. Young pneumococcal colonies
appear raised, similar to viridans streptococci. However, once the culture ages 24
hours to 48 hours, the colony becomes flattened, and the central portion becomes
depressed. Thus, a microscope (30x-50x) or a 3x hand lens can be a useful tool in
differentiating
pneumococci
from
alpha-haemolytic
viridians
streptococci.
Presumptive identification can be made on the basis of the growth on BAP and CAP,
and on the basis of the microscopic morphology of the organisms .43
Identification of N. meningitidis

.Kovac’s Oxidase Test

Identification of the N. meningitidis Serogroup

Carbohydrate Utilization by N. meningitidis – Cystine Trypticase Agar
Method

Commercial Identification Kits.43
Identification of S. pneumoniae

Susceptibility to Optochin

Inulin fermentation

Bile Solubility Test

Slide Agglutination Test.43
Identification of H. influenzae

Identification of the H. influenzae Serotype

Identification of X and V Factor Requirements

X, V and XV Paper Disks or Strips.43
Enterobacteriaceae

Nitrate reduction test

Indole production test

Citrate utilization test

Methyl red test

Voges proskauer test

Triple sugar iron agar medium

Urea hydrolysis test

Fermantation of glucose,lactose,sucrose,maltose,arabinose,xylose,mannitol.

Lysine and ornithine decarboxylase test

Arginine dihydrolase test.34
Bacterial Antigen detection:
In the early 1970’s, immuno-diagnostic methods for the detection of bacterial antigens
in CSF emerged to fill the need for prompt identification of common agents of
Bacterial meningitis. Numerous assays, including counter immunoelectrophoresis,
co-agglutination and latex particle agglutination were rapidly developed to serve as
adjuncts to routine culture and gram staining. General recommendations and
instructions typical for the detection of soluble bacterial antigens are provided here.
For best results, test the supernatant of the centrifuged CSF sample as soon as
possible. If immediate testing is not possible, the sample can be refrigerated (between
20C and 80C) up to several hours, or frozen at –200C for longer periods. Reagents
should be kept refrigerated between 20C and 80C when not in use. Product
deterioration occurs at higher temperatures, especially in tropical climates, and test
results may become unreliable before the expiration date of the kit. Latex suspensions
should never be frozen.43
Latex agglutination test uses the technique of absorbing anti serum for a specific
antigen onto latex particles of uniform diameter, generally 0.8 microns. When the
sensitised latex particles are placed in a solution such as CSF containing antigen
against which the anti-serum is directed, agglutination of the particles occurs and is
visible on inspection. Latex agglutination tests require, in addition to the patient
specimen, known positive and negative control specimens to be mixed with the
sensitized latex particles. In addition, control latex particles which have not been
sensitized are added with patient specimen and with negative and positive controls in
order to detect non-specific agglutination. 43
ANTIMICROBIAL THERAPY
Empiric antimicrobial therapy for bacterial meningitis .45,46
Current international recommendations for empiric therapy according to the age,
immunocompetence and country status are given in Table 3.
Table -3: Empiric therapy for bacterial meningitis in developed and
developing countries.
Patient group
Immunocompetent
children: age < 3
months
Likely etiology
Developed countries
Group B
Streptococcus
E. coli
L. monocytogenes*
Antimicrobial choice
Developed
Developing
countries
countries
ampicillin plus
cefotaxime or
ceftriaxone
ampicilllin
+gentamicin
Developing countries
S. pneumoniae
E. coli
Immunocompetent
children: age > 3
months - 18 years
Heamophilus influenza
S.pneumoniae
N.meningitidis
cefotaxime or
ampicillin +
ceftriaxone ** chloramphenicol
Immunodeficient
L. Monocytogenes
Gram negative
Organisms
ampicillin plus
ceftazidime
Neurosurgical
problems and head
trauma
S.aureus
Gram negative
organisms
S. pneumoniae
vancomycin plus
third generation
cephalosporins
* In infants age 1-3 months Staphylococcus aureus, H. influenzae, N. meningitidis and
Salmonella species also occur in developing countries
** For resistant S. pneumoniae the American Academy of Pediatrics recommends
vancomycin plus cefotaxime or ceftriaxone as empiric therapy
Table-4:Recommendations for Pathogen-specific antimicrobial therapy of
children with Bacterial Meningitis. 37
Bacteria
Antibiotic of choice
Neisseria meningitidis Penicillin
G
Other useful antibiotis
or Cefotaxime or ceftriaxone
ampicillin
Haemophilus
Cefotaxime
influenzae
ceftriaxone
or Ampicillin,
Chloramphenicol
Streptococcus
pneumoniae
S.pneumoniae
Penicillin-susceptible
(MIC<0.1g/ml)
PenicillinG,
Cefotaxime or ceftriaxone
S.pneumoniae
Penicillinintermediate
(MIC=0.1-1.0g/ml
Cefotaxime
S.pneumoniae
Penicillin resistant
(MIC>1.0 g /ml)
Cefotaxime or
ceftriaxone
plus vancomycin
Cefepime or meropenem
S.pneumoniae
Cephalosporinresistant
(MIC>0.5 g /ml)
Cefotaxime or
ceftriaxone
plus vancomycin
Meropenem? New
fluoroquinolones?
Listeria
Ampicillin+
Trimethoprim
sulfamethoxazole
monocytogenes
gentamicin
ampicillin
or Cefepime or meropenem
ceftriaxone
Streptococus
PencillinG
agalactiae
gentamicin
Enterococcus
Ampicillin +
aminoglycoside
Enterobacteriaceae
Cefotaxime
+ Ampicilin + gentamicin
Vancomycin
or Cefepime or meropenem
ceftriaxone
Pseudomonas
Ceftrazidime +
aminoglycoside
Cefepime or Meropenem
aeruginosa
Supportive and adjunctive treatment is equally important: adequate
oxygenation, prevention of hypoglycaemia and hyponatraemia, anticonvulsants for
seizures, measures to decrease intracranial hypertension and to prevent fluctuation in
cerebral blood flow, controlling fever to reduce metabolic demands, maintaining
arterial blood pressures within normal limits are crucial measures. Dexamethasone
used 0.15mg/kg every 6 h for 4 days for amelioration of meningeal inflammation.36
Prognosis:
Prognosis is predicted by many factors: 37
1. Age of patient (increased mortality in neonatal period)
2. Duration and type of illness before effective antibiotic therapy is instituted.
3. Type of causative organism.
4. Member of bacteria or the quantity of active bacterial products in CSF at the time
of diagnosis.
5. Intensity of the host’s inflammatory response.
6. Time needed to sterilize CSF culture.
Bad prognostic factors include: 47
1. Age less than 3 years.
2. History of partial treatment
3. Presence of coma or convulsions.
4. Pneumococcal meningitis
5. CSF cell count below 1000 cells per cumm
VACCINES
Antibodies directed against the bacterial capsular components of H.influenzae
,N.meningitides and S.pneumoniae play a major role in development of immunity
against these organisms. Immunisation with the Haemophilus ,pneumococcal and
meningococcal conjugate vaccines has had a significant impact on the incidence of
invasive disease in children caused by these organisms.26
The routine use of conjugate Hib vaccine in children has been associated with a
reduction of more than 99% of invasive disease, including meningitis, in developed
countries. Rates of Hib disease have been affected modestly in other areas of the
world where the vaccine is not routinely available.26
The heptavalent conjugate pneumococcal vaccine, PCV7, was approved for routine
use in infants from 2000. Initial clinical trials showed a reduction of more than 90% in
invasive pneumococcal infection in children. subsequent clinical studies have
confirmed the efficacy of conjugated pneumococcal vaccine in
children and a
concomitant reduction in the incidence of invasive pneumococcal disease in adults,
attributed to reduced circulation of bacteria. Children older than 2 years of age who
are at risk of developing invasive pneumococcal disease , such as children with sickle
hemoglobinopathy, should receive the conjugate vaccine followed by the 23–valent
polysaccharide vaccine. This includes patients with cochlear implants.26
A quadrivalent meningococcal polysaccharide vaccine against serogroups A,C,Y, and
W-135 strains is recommended in the united states for high-risk children older than 2
years ,such as children with asplenia or terminal complement deficiencies . In 2000,
the Advisory Committee on Immunization Practices recommended that health care
providers inform all college students about the risks of meningococcal disease in this
population and to make this vaccine available to individuals who want to reduce their
risk of meningococcal disease, which is highest in freshmen living in dormitories
.Immunogenicity of the vaccines developed against serogroup B meningococci is
poor. A major problem of vaccine development of this serogroup is the homology of
this bacterium’s capsular polysaccharide with components of human neural tissue.
Current research is ongoing to improve the immune response to vaccines designed
against this serogroup, which is endemic in North America and Europe. A
meningococcal serogroup C conjugate vaccine is routinely being administered in the
Unites Kingdom and Canada. The Vaccines and
Related Biological Products
Advisory Committee of the US Food and Drug Administration voted to recommend
licensure of a quadrivalent conjugate meningococcal vaccine(groups A,C,Y, and W135) for protection against invasive meningococcal disease in adolescents and adults
age 11 to 55 years. Clinical trials with this vaccine showed modest immunogenicity in
infants.26
Maternal immunization with GBS (Streptococus agalactiae) conjugate vaccine may
represent a future strategy to reduce neonatal GBS disease. Maternal administration of
prophylactic antibiotics has an impact on preventing only early-onset GBS disease. 26
NEED FOR THE STUDY
 Bacterial meningitis is a serious disease and is potentially associated with a high
rate of mortality, risk of chronicity and neurological deficits. Early
implementation
of
appropriate
antimicrobial
therapy
requires
prompt
identification of the infecting pathogen.12
 The production of clinically useful bacteriological report depends on the time it
takes for the organism to grow under test and may result in a delay of 18 hours or
longer in case of culture .Even after such a delay, cultures may fail to yield growth
because of the previous antimicrobial treatment.48
 Etiological diagnosis of bacterial meningitis by CSF culture in India, in the best of
the laboratories, is achieved only in 25 to 40%. CSF gram stain is also positive
only in about 25 to 30%. Hence, tests with shorter turn around time and good
sensitivity and specificity are important for early and accurate diagnosis. The latex
particle agglutination test is highly sensitive and specific, simple to perform, no
special equipment is required, technically easy, results are available in 10
minutes10 and finally there is no alteration of results due to prior antibiotic
therapy6.
This study is carried out to establish the diagnostic value of latex particle
agglutination test for the rapid specific etiological diagnosis of bacterial meningitis.
MATERIALS AND METHODS.
This prospective study was done in the department of Microbiology, Karnataka
Institute Of Medical Sciences hospital , Hubli ,during the period of December-2008
to November-2009. A total of 100 clinically suspected cases of pyogenic meningitis
in children below 5 years of age, constituted the study group.
Inclusion Criteria: Children below five years of age who were admitted with the
clinical suspicion of bacterial meningitis.
Exclusion Criteria: Children developing meningitis following head trauma or
neurosurgical procedure or children developing meningitis following hospital
admission.
Specimen Collection
CSF samples were collected by paediatricians in NICU and emergency wards on
immediate arrival of the case with clinical suspicion of meningitis, prior to
administration of antibiotics whenever possible. About 1-2 ml of CSF was collected
in sterile container, by lumbar puncture done with all aseptic precautions.49
Informed consent was obtained from the parents for performing Lumbar puncture.
Transport
Sample was immediately sent to the laboratory and processed without delay on
arrival. Samples collected during night hours were kept in the incubator and processed
without delay the following day.49
 Macroscopic appearance of CSF was observed for volume / turbidity / purulent
(pus) and whether blood stained.
 CSF was alicoted into two sterile test tubes. One of the tubes was centrifuged at
1500 to 3000 x g for 20 minutes and the sediment was used to inoculate culture
media first and then direct smear was made for examination by gram stain to
prevent contamination. The other tube was used for bacterial antigen detection.
 Cell count was done from the remaining uncentrifuged CSF.49
Cytological examination
Cell count was done by charging the counting chamber with CSF and counting the
leucocytes. A film of centrifuged deposit stained by methylene blue was examined for
differential count and the predominant cell type found out.49
Culture: The sediment of the centrifuged CSF was inoculated on to the following
media:
 Chocolate agar plate incubated at 370 C in 5-10% CO2.
 Mac Conkey agar incubated aerobically at 370 C.
 BHI broth incubated aerobically at 370 C.49
Inoculated primary plates were incubated for 48 to 72 hours. The plates were
examined daily for 72 hours before discarding as negative. BHI broth was incubated
for 7 days and examined daily for presence of growth or turbidity and was considered
negative at the end of 7 days of incubation. Tubes showing turbidity were subcultured
on Chocolate agar and Mac Conkey agar . Tubes that remained non turbid were
subcultured on the 7th day before discarding. Any growth on the above mentioned
media were identified on the basis of their colony morphology, cultural
characteristics, Gram stain and biochemical reactions according to standard
techniques.34
Antibiotic susceptibility testing was done by standard Kirby bauer disk diffusion
method and controls were put up according to CLSI guidelines.50
Direct Smear
Smears are made by placing 1 or 2 drops of sediments of CSF on an alcohol rinsed
slide, allowing the drop to form a large heap and air dried. Air dried smears were
fixed by heating and stained by Gram’s stain and observed for the presence of pus
cells and organisms.49
Tests used for the identification of the isolates:
1) Streptococcus pneumoniae:

Gram positive cocci in pairs

Catalase test-negative

Optochin susceptible

Bile soluble34
2) Haemophilus influenzae:

Gram negative pleomorphic bacilli

Oxidase positive

Satellitism-positive34
3)EnterobacteriaceaeA)Escherichia coli
 Indole produced

Methyl red test-positive

Citrate not utilized

Urea not hydrolysed

Triple sugar iron agar –acid slant by acid butt with production of gas ,no H2S
produced34
B) Klebsiella pneumoniae

Indole not produced

Methyl red test-negative

Citrate utilized as a sole source of carbon

Urea hydrolysed

Triple sugar iron agar-acid slant by acid butt with production of gas ,no H2S
produced34
C) Citrobacter koseri

Indole produced

Methyl red test-positive

Citrate utilized as a sole source of carbon

Urea not hydrolysed

Triple sugar iron agar-alkaline slant by acid butt with production of gas ,no
H2S produced34
4) Pseudomonas aeruginosa:

Oxidase positive

Hugh and Liefson’s OF medium-oxidative

Growth at 420C –positive

Denitrification of nitrates and nitrites-positive34
5 )Acinetobacter baumannii:

Non-motile

Oxidase negative

Hugh and Liefson’s OF medium-oxidative

10% lactose-utilised with production of acid34
LATEX PARTICLE AGGLUTINATION TEST
CSF samples were tested for bacterial antigen detection using BD DIRECTIGEN
MENINGITIS COMBO TEST ,bacterial antigen kit, a latex test to detect antigens of
5 organisms:
– E coli K1 antigen
– N. meningiditis ABCY or W 135 antigen
– Streptococcus pneumoniae antigen
– Group B streptococcus antigen
– H. influenzae type b antigen
Meningococcus group B antigen being structurally and immunologically related to
E.Coli K1 antigen is provided as a single test latex reagent and depending on the age
of the child, a positive reaction in a neonatal specimen would suggest E.Coli K1
infection and in older children meningococcus group B is a more likely infection and
correlating with direct smear examination of CSF.
Procedure: CSF is preheated at 1000C in a water bath for 3 minutes, cooled to room
temperature and centrifuged to remove the proteinitious material that would cross
react with the antigen (to minimize non specific reactions). The supernatant is then
used for LAT.
Disposable reaction cards containing six separate circles of different test latex
reagents are provided with the kit. One drop of (50µl) CSF is placed on the separate
circle of the reaction card and one drop of five different test latex reagents are added
to the separate circles, mixed thoroughly and rotated on a mechanical rotator at a
speed of 100+/-2 rpm for 10 minutes and observed for agglutination. Positive and
negative controls and control latex tests supplied by the manufacturer were put up
simultaneously.
RESULTS
This study was taken up in the department of Microbiology, Karnataka Institute of
Medical Sciences Hospital, Hubli for a period of one year from Dec-2008 to Nov2009. 100 cases of children below 5 years of age, who presented with signs and
symptoms of meningitis,were investigated for laboratory diagnosis of Acute bacterial
meningitis.
During the study period there were 1743 children aged <5 years admitted to the
pediatric ward, of which 100 cases were suspected to have meningitis. After the CSF
examination, 31 cases were diagnosed to have bacterial meningitis. Thus the hospital
frequency of ABM was 1.7%.
TABLE- 5: Age and sex distribution of cases (n=100).[percentage
values in parenthesis]
AGE
MALE
FEMALE
TOTAL
1-28days
15 (62.50)
9 (37.50)
24(23)
29days-1yr
22 (59.45)
15(40.54)
37(37)
1yr-3yrs
14 (77.77)
4(22.22)
18(18)
3yrs-5yrs
11 (52.38)
10(47.61)
21(21)
Total
62 (62)
38 (38)
100
Fig-28: Age and sex distribution of cases
90
77.77
Percentage
80
70
62.5
59.45
60
52.38
47.61
50
37.5
40
40.54
male
30
female
22.22
20
10
0
1-28days
29d-1yr
1-3yr
3-5yrs
Age group
In the present study, of the 100 cases, 62(62%) were male and 38(38%) were female ,
making a male to female ratio of 1.63:1. 61(61%) children were below one year of
age ,of which 24(24%) were neonates. About 21 (21%) cases were in the age group of
3yrs-5yrs and 19(19%) were between 1yr -3yrs of age.
TABLE-6: Clinical presentation of cases.(n=100).[Percentage values in
parenthesis]
Clinical feature
NO.of cases
Fever
88(88%)
Seizure
70(70%)
Altered sensorium
56(56%)
Refusal of feeds
37(37%)
Vomiting
36(36%)
Meningeal signs
16(16%)
Fever was the predominant symptom , seen in 88(88%) cases, followed by seizure in
70(70%), altered sensorium in 56(56%) and vomiting in 36(36%) cases. Signs of
meningeal irritation were present only in 16(16%) cases.
TABLE-7: Laboratory confirmed cases of ABM as per WHO criteria
(n=31).
Tests
No.of cases positive
Gram stain+LAT+Culture
3(9.67%)
Gram stain+LAT
9(29.03%)
Gram stain+Culture
5(16.12%)
Culture+LAT
3(9.67%)
Only Gram stain
5(16.12%)
Only Culture
4(12.90%)
Only LAT
2(6.45%)
Total
31
Fig-29:Laboratory confirmed cases of ABM
6.45
9.67
12.9
GS+LAT+CULTURE
GS+LAT
29.03
16.12
GS+CULTURE
CULTURE+LAT
ONLY GS
ONLY CULTURE
ONLY LAT
9.67
16.12
GS=Gram stain, LAT=Latex particle agglutination test.
As per WHO criteria 51, 31(31%) cases were laboratory confirmed as cases of Acute
Bacterial Meningitis.
TABLE-8: CSF Cell count among the laboratory confirmed cases of
ABM (n=31).
CSF cell count/mm3
Total
6 – 50
12 (38.70%)
51 – 500
15 (48.38%)
501 – 1000
02 (6.45%)
>1000
02 (6.45%)
Total
31
CSF cell count was most commonly in the range of 51-500 cells /cumm i.e. in 15
(48.38%) cases, 12(38.70%) cases were in the range of 6-50 cells/cumm, 2(6.45%)
were in the range of 501-1000 cells/cumm and 2(6.45%) cases had a cell count of
> 1000 cells /cumm.
TABLE-9: Distribution of cases according to age and causative agent
among the laboratory confirmed cases of ABM (n=31). [percentage
values in parenthesis]
Etiological
1d-28d(%)
28d-1yr(%)
agent
1yr-
3yrs-
3yrs(%)
5yrs(%)
Total(%)
H.influenzae
3(33.33%)
6(66.66%)
-
-
9(29.03%)
S.pneumoniae
-
1(14.28%)
2(28.57%)
4(57.14%)
7(22.58%)
GBS
-
2(100%)
-
-
2(6.45%)
K.pneumoniae 1(33.33)
1(33.33%)
-
1(33.33%)
3(9.67%)
E. coli
1(50%)
1(50%)
-
-
2(6.45%)
C. koseri
-
-
1(100%)
-
1(3.22%)
A. baumannii
1(100%)
-
-
-
1(3.22%)
P. aeruginosa
1(100%)
-
-
-
1(3.22%)
Species
3(60%)
1(20%)
1(20%)
-
5(16.12%)
4(12.90%)
5(16.12%)
31(100%)
Unidentified
Total
10(32.25%) 12(38.70%)
Species Unidentified=only gram stain positive, GBS=Group B Streptococcus.
Haemophilus influenzae was common in children less than 1 year of age and
S. pneumoniae was most common in children more than 1 year of age. 2 cases of
Group B streptococcus were above 28 days of age.
Fig-30: Results of CSF Gram stain among the
laboratory confirmed cases of ABM
GPC in pairs
8(25.80%)
no micro
organisms
9(29.03%)
GPC in chains
1(3.22%)
Gram
negative
bacilli
13(41.93%)
GPC=Gram positive cocci, GNB=Gram negative bacilli
Gram stain was positive in 22(70%) cases of ABM, 8(25.80%) were gram positive
cocci in pairs, 1(3.22%) was gram positive cocci in chain and 13(41.93%) were gram
negative bacilli.
Fig-31:Results of CSF culture among the
laboratory confirmed cases of ABM
4(12.90%)
3(9.67%)
16(51.61%)
3(9.67%)
2(6.45%)
S.pneumoniae
H.influenzae
K.pneumoniae
E.coli
C.koseri
A.baumanii
P.aeruginosa
No growth
1(3.22%)
1(3.22%)
1(3.22%)
CSF culture results in the present study showed that 15(48.38%) cases of ABM were
culture positive. S.pneumoniae was the commonest organism isolated,4(12.90%) in
cases ,followed by H.influenzae and K.pneumoniae i.e. 3(9.67%) cases each. C.koseri,
A.baumannii and P.aeruginosa were isolated in 1(3.22%) case each. About
16(51.61%) cases were culture negative.
Fig-32: Results of CSF LAT among laboratory confirmed
cases.
Negative
14(45.16%)
H.influenzae
8(25.80%)
S.pneumoniae
7(22.58%)
GBS
2(6.45%)
GBS=Group B Streptococci.
CSF LAT was positive in 17(54.83%) cases of ABM. 8(25.80%) were H.influenzae
type b, 7(22.58%) were S.pneumoniae and 2(6.45%) were Group B Streptococci.
TABLE-10:Comparison
of
CSF
Gram
stain,
Culture
and
LAT(n=31).[percentage values in parenthesis]
TESTS
Positive
Negative
Total
Gram stain
22(70.96)
09 (29.03)
31(100)
Culture
15 (48.38)
16(51.61)
31(100)
LAT
17 (54.83)
14 (45.16)
31(100)
Fig -33:Etiological agents identified in CSF by all methods.
25
22
Total number
20
15
16
17
14
15
positive
10
negative
9
5
0
Gram stain
Culture
LAT
Test
The comparative analysis of Gram stain, culture and LAT in the present study showed
that culture was positive in 48.38%, Gram stain in 70.96% and LAT in 54.83% of the
31 ABM cases. CSF Gram stain could identify the maximum number of cases of
ABM 70.96%(22) ,followed by LAT ,54.83%(17) cases.
TABLE-11 :Etiological agents identified in CSF by various methods
(n=31) [percentage values in parenthesis]
Organisms
Total
identified
Gram stain
Culture
positive
positive
LAT positive
H.influenzae
9(29.03)
7(77.77)
3(33.33)
8(88.88)
S.pneumoniae
7(22.58)
5(71.42)
4(57.14)
7(100.00)
GBS
2(6.45)
1(50)
0
2(100.00)
Other GNB
8(25.80)
4(50)
8(100)
0
Species
5(16.12)
5(100.00)
0
0
31
22(70.96)
15(48.38)
17(54.83)
unidentified
Total
H.influenzae was the commonest isolate in the present study positive in 9(29.03%)
cases, followed by S.pneumoniae in 7(22.58%) cases. 2 cases of Group B
Streptococcus were identified by LAT only. Overall fastidious bacteria like
H.influenzae. S.pneumoniae and GBS were more often identified by LAT compared
to Culture.
TABLE -12: CSF LAT results compared with administration of
antibiotics prior to CSF collection among the laboratory confirmed
cases of ABM.(n=31)
Prior
LAT positive
LAT negative
Antibiotics
Total (%)
(n=31)
No
%
No
%
Given
12
38.70
11
35.48 23 (74.19)
Not given
5
16.12
3
9.67
Total
17
54.38
14
45.16
p>0.05
08 (25.80)
31
Prior antibiotic administration did not have statistically significant association with
LAT results.
TABLE-13: CSF culture results compared with administration of
antibiotics prior to CSF collection among the laboratory confirmed
cases of ABM.(n=31)
Prior
Culture positive Culture negative Total (%)
antibiotics
(n=31)
No
%
No
%
Given
11
35.48
12
38.70
23 (74.14)
Not given
4
12.90
4
12.90
08(25.80)
Total
15
48.38
16
51.61
31
p>0.05
The association between CSF culture results and prior antibiotic administration was
not statistically significant.
TABLE-14: CSF Gram stain smear results compared with
administration of antibiotics prior to CSF collection among the
laboratory confirmed cases of ABM(n=31).
Prior
Gram stain +
Gram stain -
antibiotics
Total (%)
(n=31)
No
%
No
%
Given
16
51.61
7
43.47
23 (74.19)
Not given
6
19.35
2
21.74
08 (25.80)
Total
22
70.96
9
29.03
31
p>0.05
Statistically significant association between Gram stain and prior antibiotic
administration was not observed in the present study.
TABLE-15:Accuracy indices of CSF Gram stain against CSF culture
as gold standard.
Culture positive
Culture negative
Total
Gram stain+
8
14
22
Gram stain-
7
71
78
Total
15
85
100
On comparing CSF Gram stain and CSF Culture ,Gram stain showed a sensitivity of
53.33%, specificity of 83.52%, positive predictive value of 36.36% and negative
predictive value of 91.02%.
TABLE-16:Accuracy indices of CSF LAT against CSF Culture as
gold standard.
Culture positive*
Culture negative
Total
LAT positive
6
11
17
LAT negative
3
80
83
Total
9
91
100
*Positive Culture for H.influenzae , S.pneumoniae & E.coli.
When CSF LAT and CSF Culture were compared, LAT had a sensitivity of 66.66%,
specificity of 87.91%, positive predictive value of 35.29% and negative predictive
value of 96.38%.
TABLE-17:Table showing comparison of sensitivity , specificity,
PPV& NPV of CSF Gram stain and CSF LAT with culture as gold
standard.
Sensitivity
Specificity
PPV
NPV
Gram stain
53.33%
83.52%
36.36%
91.02%
LAT
66.66%
87.91%
35.29%
96.38%
LAT was found to have a higher sensitivity i.e. 66.66% when compared to Gram stain
53.33%, however the specificity and NPV of LAT was marginally higher i.e. 87.91%
and 96.38% ,compared to Gram stain i.e. 83.52% and 91.02%. The PPV was
comparable.
Table 18: Resistance pattern of the isolates.
Organism
No.
A
Ci
Ce
Ca
Cpm
C
Ak
Pt
tested
H.influenzae
3
1(33.33)
0
0
-
0
0
0
0
S.pneumoniae
4
1(25.00)
0
0
-
0
0
0
-
K.pneumoniae
3
3(100)
1(33.33)
1(33.33)
-
0
0
0
0
E.coli
2
2(66.66)
0
0
-
0
0
0
0
C.koseri
1
1(100)
0
0
-
0
0
0
0
A.baumannii
1
1(100)
1(100)
1(100)
-
0
0
0
0
P.aeruginosa
1
-
1(100)
1(100)
0
0
-
0
0
The antimicrobial sensitivity pattern of our isolates showed that all Gram positive and
many of the Gram negative organisms were 100% sensitive to amikacin and third
generation cephalosporins (cefotaxime & ceftriaxone). Ampicillin was the least
effective.
TABLE-19:Mortality rate among the laboratory confirmed cases of
ABM.(n=31)[percentage values in parenthesis]
ORGANISMS
TOTAL CASES
CASE FATALITY
H.influenzae
9(34.61)
5(55.55)
S.pneumoniae
7(26.92)
1(14.28)
Group B Streptococcus
2(7.69)
2(100.00)
K.pneumoniae
3(11.53)
2(66.66)
E.coli
2(7.69)
1(50.00)
C.koseri
1(3.84)
0(00.00)
A.baumannii
1(3.84)
0(00.00)
P.aeruginosa
1(3.84)
1(100.00)
Species Unidentified
5(16.12)
2(40.00)
Total
31
14(45.16)
Species Unidentified=only gram stain positive
The case fatality rate in the present study was 45.16%(14/31) .Maximum case fatality
was observed in case of Group B streptococcus100%(2/2) & P.aeruginosa 100%(1/1)
meningitis ,followed by K.pneumoniae and H.influenzae in 66.66%(2/3) and
55.55%(5/9) respectively.
DISCUSSION
Acute bacterial meningitis is a medical emergency, which warrants early diagnosis
and aggressive therapy. Most often therapy for bacterial meningitis has to be initiated
before the etiology is known. The choice of initial antimicrobial therapy in
community acquired acute bacterial meningitis (CAABM) is based on the most
common pathogen prevalent in a particular geographical area , its antibiotic sensitivity
pattern and the age group affected .6
Though the common pathogens associated with CAABM are S.pneumoniae, H.
Influenzae and N.meningitidis, the aetiological agents and their relative frequency
may vary in different geographical areas. Some changing trends in the epidemiology
of CAABM have also been reported worldwide over the past few decades.6 For
instance, the incidence of meningitis caused by Hib has decreased markedly in areas
of the world where Hib conjugate vaccines are routinely used.26S.pneumoniae is the
major cause of childhood bacterial meningitis in countries where Hib disease has been
eliminated by vaccination.25
An accurate laboratory confirmation of the etiology in acute bacterial meningitis is
essential to provide optimal patient therapy, appropriate case contact management,
and reasoned public health actions. Prospectively, it also provides information upon
which to base decisions regarding immunization programs, especially for countries
without routine vaccination against the main acute bacterial meningitis pathogens.
Although bacterial culture is considered to be the standard method, the negative effect
of prior antimicrobial drug use on its sensitivity necessitates nonculture techniques for
diagnosis.25
There is currently considerable interest in applying immunodiagnostic procedures to
the rapid detection of microbial antigens in clinical specimens. This approach to the
diagnosis of infectious diseases has been spurred by the development of techniques
that are simple and, depending on the quality of the reagents used, can be both
sensitive and specific. Immunological detection of bacterial antigens is particularly
applicable to cerebrospinal fluid (CSF) obtained from bacterial meningitis patients,
since high concentrations of antigen are generally found in these specimens.23
The present study was undertaken to study the bacteriological profile of meningitis
and also to do a comparative evaluation of Gram stain, culture and LAT, in clinically
suspected cases of acute bacterial meningitis, in children below 5 years of age. A
total of 100 cases of children below 5 years of age with clinical suspicion of
meningitis were tested for ABM.
Age and Sex distribution of cases:
The reported frequency of occurrence of ABM in hospital admissions among children
below 5 years of age is 0.5 – 2.5 %, although the incidence in the general population
is not known.4,5In our study ABM constituted 1.7% of all pediatric ward admissions
among children below 5 years of age. The distribution of patients according to age
and sex, noted in our study was 62/100 (62.00%) males and 38/100 (38.00%) females.
The male to female ratio was 1.63:1(Table-5)
Male to Female ratio:
Table-20 showing Comparative studies of male to female ratio.
Studies
Year
Ratio
Bijay Mirdha et al 18
1991
1.5 : 1
Deivanayagan et al 10
1993
1.75 : 1
Bandaru Rao et al 8
1998
1.2 : 1
Ciji N. George et al 52
2002
1.5 : 1
Nanditha et al 4
2002
1.07 : 1
Present Study
2009
1.63 : 1
The male and female ratio in our study correlated with the studies of
Deivanayagan et al10 and Ciji N. George et al52 who have reported 1.75:1 and 1.5:1 in
their studies. All the studies show male preponderance including present study.
61(61%) children were below one year of age ,of which 24(24%) were neonates.
Similarly, Chinchankar N,et al.,4in their study noted 42 of 58 cases (77%) ,
Deivanayagam N, et al.,10reported 44 of 55 cases (80%) and Kabra SK, et al.,5found
450 of 852 cases (52%) of ABM to be below one year of age.
Distribution of symptoms:
Patients presented with high grade fever, seizures and altered sensorium in 88
(88%), 70 (70%), and 56(56%) cases respectively. 37(37%) cases had refusal of feeds.
About 36(36%) cases also presented with vomiting . Only 16 (16%) cases had
meningeal signs viz neck rigidity and Kernig’s sign.(Table-2) Similar observations
were made by other workers.4
The distinct clinical syndromes of CNS include acute bacterial meningitis,
viral meningitis ,encephalitis, focal infections such as brain abscess and subdural
empyema, and infectious thrombophlebitis1. In young infants the specific signs of
meningeal inflammation are often minimal or absent and non specific manifestations
such as irritability, lethargy and poor feeding may be the early or even the only
findings.3 Altered sensorium and seizures are late features.4A small number of
children (15%) may fail to exhibit any meningeal signs during their entire illness
inspite of having meningeal inflammation as demonstrated by CSF pleocytosis.53
Clinical diagnosis of bacterial meningitis is often difficult. Hence a high index of
suspicion is necessary to suspect meningitis and perform lumbar puncture.4
WHO criteria 51
WHO criteria defines a proven case of bacterial meningitis as : A case that is
laboratory-confirmed by growing (i.e. culturing) or identifying (i.e. by Gram stain or
antigen detection methods) a bacterial pathogen in the CSF or from the blood in a
child with a clinical syndrome consistent with bacterial meningitis.
As per the above criteria 31/100(31%) cases were laboratory confirmed as bacterial
meningitis in present study.(Table:7)
Gross appearance of CSF:
The CSF appeared turbid in 13/31(41.93%) laboratory confirmed cases of ABM.
In 18/31(58.06%) cases of ABM, CSF was not turbid. Nandita chinchankar et al4.,
noted CSF turbidity in 85% cases and N Deivanayagan ,et al.,10 in 56.4% cases in
their study. Thus CSF turbidity was not reliable in ruling out acute bacterial
meningitis.
Cell Count
Table-21 showing comparative studies of Cell count in ABM
Studies
Year
Percentage of neutrophils
Kalra et al 54
1985
68.4%
Deivanayagan et al 10
1993
60%
Nanditha et al 4
2002
70%
Present Study
2009
64.5%
CSF cell count was most commonly in the range of 51-500 cells/cumm i.e. in
15(48.38%) cases of ABM.12(38.70%) cases were in the range of 650cells/cumm.(Table:8), 8(25.80%) of these had a normal CSF picture, of which
6(19.35%) were neonates and 2(6.45%) were beyond 1 month of age. Upto 15% of
neonates may have bacterial meningitis in the absence of CSF pleocytosis.55 In
3(9.67%) cases there was lymphocyte predominance and this could be due to partial
treatment of the cases as this leads to change in the CSF picture.4
In the present study, Neutrophils were seen predominantly in 20(64.5%) cases of
ABM and correlates with the studies of Deivanayagan et al10 and Kalra et al54 .
TABLE 22: Comparison of etiological agents isolated/identified in CSF with
other studies(n=26).[ values within the parentheses indicate percentage]
Studies
H.influenzae S.pneumoniae Group
B Others
streptococci
Chinchankar N, 14 (26)
21 (38.88)
0
9 (16.66)
5 (10)
4 (8)
0
21 (42)
28 (24.56)
15 (13.15)
2 (1.75)
10 (8.7)
7 (19.44)
22 (61.11)
0
1 (2.7)
5 (10.41)
1 (2)
27
et al4
Mirdha BR,et
Al18
Deivanayagam
N, et al.10
Das BK, et al.21
Finlay FO, et al19 15 (31.25)
(56.25)
Sahai S, et al22
17 (17)
12 (12)
0
6 (6)
Vishwanath G ,et 9(22.5)
7(17.5)
1(2.5)
15(37.5)
7 (22.58)
2 (6.45)
8 (25.80)
al.56
Present study
9(29.03)
* Excluding 5 cases which were positive only on Gram stain and could not be
speciated.

H.influenzae was the most common etiological agent of ABM in the present
study accounting for 9/31( 29.03%) cases. All the 9 cases were below 1 year
of age. S.pneumoniae was the next most common etiological agent accounting
for 7/31(22.58%) cases of ABM. Similar results were observed by Vishwanath
G, et al.56, N Deivanayagan ,et al10 and Sahai S ,et al 22 ,in their studies.

K.pneumoniae was the third most common causative agent of ABM in the
present study , accounting for 3/31(9.67%) cases .1(33.33%) was a neonate
and the other 2(66.66%) were more than 1 month of age.

Group B Streptococcus was the etiological agent in 2/31(6.45%) cases of
ABM in the present study (Table:9). Similar isolation rates have been reported
by other workers 10,56. Although Group B Streptococcal meningitis is known to
occur in neonates, recently there have been reports of invasive Group B
Streptococcus (GBS) disease in children beyond early infancy and nonpregnant adults associated with diabetes mellitus, liver disease, peripheral
vascular disease , neoplastic diseases, wound infections ,elective abortion
especially septic incomplete abortion , dermal sinus, and in HIV-positive
patients28. M Sanjeev et al., has reported a case of uncomplicated meningitis
due to GBS in a previously healthy 5 year old boy.57 Similarly both the cases
in the present study were beyond one month of age and both were without any
obvious predisposing factor.

2 children beyond 1 month of age , presented with E.coli and C.koseri
meningitis, and both gave a positive history of Chronic suppurative otitis
media(CSOM).

E.coli was the causative agent in another case of neonatal meningitis.

Acinetobacter baumannii and Pseudomonas aeruginosa were the causative
agents in one case each of neonatal meningitis(3.22%).

No case of meningitis due to N.meningitidis was noted in the present study,
since N.meningitidis isolation is uncommon as long as an outbreak does not
occur.24

In about 5/31(16.12%) cases of ABM the organism could not be speciated as
they failed to grow in the subsequent culture nor they were LAT positive.
They were identified only on Gram stain, 3 were gram positive cocci in pair
and 2 were gram negative bacilli.
Gram Stain
Table-23 showing comparative studies of percentage positivity of Gram stain in
ABM
Studies
Year
Percentage
Benedict et al 58
1982
64%
Bijay R Mirdha et al 18
1991
74%
Deivanayagan et al10
1993
30%
Bandaru rao et al8
1998
85.7%
Nanditha et al 4
2002
67%
Shiva Prakash et al 69
2004
36.36%
Col.Prasad et al 60
2005
21.05%
Present Study
2009
70.96%
In the present study, percentage positivity of Gram stain was 70.96%. 29.03% of
cases were negative on Gram stain.(Fig:30) Our study correlates with studies of the
other Indian authors, Nanditha chinchankar et al4 and Bijay R Mirdha et al18, who
reported 67% and 74% Gram stain positivity respectively.
The probability of visualising bacteria on gram stained CSF smear is dependent on the
number of organisms present; 25% are positive with < 1000 colony forming units
(CFU) per ml of CSF, 60% with 1000 – 10,000 CFU/ml and 97% with > 10,000
CFU/ml.5 Prior use of antibiotics, technique used for smear preparation, staining
technique and the observer’s skill and experience are the other determinants of smear
positivity. Despite the low Gram stain positivity from CSF sample and the fact that a
negative Gram stain does not rule out infection, the importance of a positive smear
cannot be over emphasized , especially in developing countries when financial
constraints limit the use of other rapid diagnostic tests to diagnose the potentially fatal
infection.6
In addition to being a low cost test, it also provides a clue to the possible etiology thus
making empirical therapy more focussed, especially in view of the low sensitivity and
long turn around time of culture reports . Nevertheless the major limitation of gram
stain is the inability to speciate the organism and to ascertain the antibiotic
susceptibility of the isolate.
In the present study ,Gram stain provided the evidence of bacteria in 22(70.96%) lab
confirmed cases of ABM. Of these 22, 8 samples yielded growth on culture ,while 14
were culture negative. 9 of the culture negative cases were positive by LAT. Thus in a
total 5 cases which were both culture and LAT negative , the etiologic pathogen was
detected only by Gram stain.(Table:14)
.
Culture
Table-24 showing comparative studies of culture isolation in ABM
Studies
Sippel et al 23
Year
1984
Percentage
72.83%
Kalra et al 54
1985
77.6%
Harcharan Singh et al 9
1988
42.8%
Bijay R Mirdha et al 18
1991
66.66%
Bandaru Rao et al 8
1998
66.2%
Deivanayaganm et al 10
1993
22%
Tankhiwala et al 61
2001
36%
Nandhita et al 4
2002
50%
Present Study
2009
48.38%
In the present study, culture detected 48.38% of lab confirmed cases of ABM. 51.61%
of cases were culture negative(Fig:31). Our study correlates with studies of the other
Indian authors,Nanditha chinchankar et al4and, Harcharan Singh et al9 who reported
50% and 42.8% culture positivity respectively.And was higher than Deivanayagam et
al.10and Tankhiwala et al61
Majority of our isolates were gram negative organisms 73.33% and only 26.66%
were gram positive organisms. Bandaru Rao et al8 in their study have reported
predominance of gram negative bacteria (63.3%), while gram positive bacteria were
found only in 36.7% of cases. This correlates with the gram negative and gram
positive isolation in the present study.
Reasons as reviewed in other studies for low CSF culture yield are:

Low bacterial load.22

Use of antimicrobial agents prior to CSF collection.5

Poor culture media.5

Poor culture facility such as non availability of special media, stored in
unsatisfactory conditions, samples refrigerated before plating, delayed and
faulty inoculation, lack of transport media and inadequacy in processing of
CSF specimens.10,22


Autolytic enzymes.18
Lack of 24 hour facility for processing CSF samples.6
Therefore, prompt inoculation of CSF on culture media plates at bedside by
residents rather than transporting the CSF specimen to laboratory, if
transported then using transport media is recommended.5,7
Inspite of the above limitations , the utility of culture in terms of species identification
and the ability to perform antimicrobial susceptibility testing makes it superior to
Gram stain and LAT.
LAT
Table-25 showing comparative studies of percentage positivity of LAT in ABM
Studies
Year
Percentage
Sippel et al 23
1984
83.95%
Harcharan Singh et al 9
1988
100%
Bijay R Mirdha et al 18
1991
100%
Deivanayagan et al 10
1993
84%
Nanditha Chinchanakar et
al 4
Das K. et al 21
2002
78%
2003
83%
Shivaprakash et al 59
2004
69.09%
Present Study
2009
54.83%
In the present study, Bacterial antigen detection test could detect 17 lab confirmed
cases of ABM (54.83%) viz, 8 H.influenzae, 7 S.pneumoniae and 2 Group B
Streptococci. 45.16% were LAT negative.(Fig:32)
No case of N.meningitidis was detected in the present study as its isolation is
uncommon as long as an outbreak does not occur.24Besides the detection of
N.meningitidis Group B antigen by immunological techniques continues to pose a
problem and this has been attributed to the poor immunogenicity of this particular
antigen.62
Comparative studies of Harcharan Singh et al9 Bijay Mirdha et al18 quote a higher
detection rate of LAT in their studies because majority of organism in their study
comprised of meningococci, pneumococci and Hib that were negative for culture, as
compared to our study where 35.48% of organism were Non-fermenters and
Enterobacteriaceae other than E.coli , that were culture isolated but the reagents to
detect them, were not included in the panel of the kit.
In developing countries like India where a majority of neonatal meningitis is caused
by Enterobacteriaceae , culture is superior to LAT in neonatal meningitis as the latter
is not designed to detect Enterobacteriaceae other than E.coli, besides, the cost of
LAT is the major limiting factor.
Effect of prior antibiotic administration on CSF Gram stain , Culture and LAT:
In the present study, CSF Gram stain could identify maximum number of
cases(16/31) of acute bacterial meningitis when compared to CSF LAT(12/31) and
CSF Culture(11/31) ,in cases who had received the antibiotics prior to lumbar
puncture(Table:11,12,13). These values therefore ,do not support the claim that the
LAT is of greater value than Gram staining in arriving at a diagnosis once antibiotic
treatment has started. Similar findings were reported in another study.19
Although not statistically significant ,LAT detected a marginally higher number of
cases i.e. 38.70% in comparison to Culture i.e. 35.48% in cases who had received
antibiotics prior to CSF collection.
Comparing CSF Gram stain and CSF Culture:
Accuracy indices of Gram stain against Culture showed a sensitivity of 53.33%,
specificity of 83.52% positive predictive value of 36.36% and negative predictive
value of 91.02% in the present study(Table:15)
Hristeva et al.,55have reported the sensitivity of Gram stain to be 86%, specificity 99%
, positive predictive value 46%, and negative predictive value of 99.9% in their study.
In another study done by Mark I Neuman et al.,63 the reported sensitivity of Gram
stain is 67%, specificity 99.9% , positive predictive predictive value of 60% and
negative predictive value of 99.9%.
The frequency of bacterial meningitis in high enough among patients with an
organism seen on Gram stain to warrant presumptive treatment, pending the results of
CSF Culture. On the other hand , the positive predictive value is low enough that
unless there are other factors that increase the risk of bacterial meningitis (eg:
antibiotic pretreatment, very low CSF glucose), the presence of organisms alone
would not require a complete empiric course of therapy.63
The low positive predictive value of Gram staining is a consequence of the low
prevalence of meningitis in the population. Clinicians should be aware that diagnostic
tests performed in a population with a low prevalence of disease are likely to generate
many false positive results and have a low positive predictive value. 55
In the present study, inspite of the low PPV of 36.36% ,the test had a moderate
sensitivity of 53.33% .With a specificity and NPV as high as 83.52% and 91.02%
respectively, a negative Gram stain should reassure the clinician. Additionally the
low cost of Gram stain and rapid availability of results makes it an attractive option.
Similarly, if the culture is negative the clinician must also make a determination as to
whether the culture is falsely negative.63 Therefore in view of the above facts,
additional tests like CSF chemistries , cell count and antigen detection should be
considered to make a diagnosis in order to avoid false positive results. In the present
study 5 cases that were Gram stain positive and negative on Culture and LAT, had
raised cell count and thus treated as cases of ABM.
Comparing CSF LAT and CSF Culture.
Accuracy indices of LAT against culture in the present study showed a sensitivity of
66.66%, specificity of 87.91%, positive predictive value of 35.29% and negative
predictive value of 96.38%(Table:16). P A M Camargos et al., have reported the
sensitivity and specificity of LAT to be 83.8% and 94.0% respectively.64
In another study, Paulo A M et al11., have reported the sensitivity and specificity of
LAT to be 93.0% and 100% respectively.
Among the 31 lab confirmed cases of ABM 16 cases were culture negative. Among
these 16 cases LAT was positive in 11 cases,6 were H.influenzae type b, 3
S.pneumoniae and 2 Group B Streptococcus(Table:10,11). The fastidious nature of
these bacteria and the effect of prior antibiotic therapy in most of our cases probably
explains the poor sensitivity of culture in these cases, conversely LAT was better at
identifying these bacteria .9 of these 11 cases were also positive by Gram stain but
2(6.45%) of the samples which did not show any evidence of pathogen either in Gram
stain or Culture ,were positive by LAT only, thus helping clinch the diagnosis. As is
evident by the results ,despite the moderate sensitivity of LAT in the present study,
LAT was superior to culture in identifying fastidious bacteria like H.influenzae,
S.pneumoniae and Group B Streptococci. LAT was only marginally superior to Gram
stain in identifying these cases.
Using CSF culture as a gold standard for calculating sensitivity, specificity, PPV and
NPV, has its own limitations, as CSF culture is less sensitive compared to serologic
tests. So, a culture negative and LAT positive test result that is taken as false positive
could actually be a true positive and therefore could influence the sensitivity,
specificity and PPV adversely. However, since culture is the gold standard we too
have used this for comparison of the tests.
LAT was positive in 6 of the 15 culture positive cases(Table:10,11) , 2 Hib & 4
S.pneumoniae. Among the 9 culture positive and LAT negative cases 6 were gram
negative bacilli whose reagents were not included in the panel of the kit. Hence LAT
failed to detect these organisms. Among the remaining 3 culture positive cases(1
H.influenzae and 2 Escherichia coli ) LAT failed to detect the corresponding antigens
in the CSF. The false negative LAT could be possibly because of low antigen titres in
the CSF.5It is possible that the antiserum in diagnostic LAT kit does not detect all the
capsular serotype prevalent in a geographical area or probably as yet unrecognised
serotypes are the causative agents in such cases.This probably explains the false
negative LAT in our study. False positive LAT results may occur in case of recent
immunization with Hib conjugate vaccine and infection with cross reacting
organisms.6
Despite the good specificity of LAT 87.91% in the present study, the low positive
predictive value 35.29% is of concern,especially in view of the high cost of the test.
Nevertheless because of the high NPV of 96.38%, a negative LAT fairly rules out
ABM in clinically suspected cases.
It is important to note that bacterial antigen testing (BAT) was originally designed to
be used in patients who demonstrated laboratory and clinical findings consistent with
meningitis. Despite these initial intentions, this test has been used much too often as a
screening tool in cases of suspected meningitis in patients whose CSF specimens have
normal chemistries and cell counts. The indiscriminate use of BAT without
consideration of the chemical and cytological profiles of the CSF is a misuse of
valuable resources.65 Systematic review of the performance of laboratory test against
clinical gold standards leads
to more rational use of laboratoty resources and
improves interpretation at the bedside. However several studies advocate the
usefulness of LAT, especially in pretreated cases and to differentiate partially treated
pyogenic meningitis from tuberculous meningitis which is rampant in India.6 Despite
its drawbacks, we found LAT to be simple, rapid procedure suitable to be used as an
adjunct laboratory test.
Antibiotic susceptibility pattern of the isolates.
The antimicrobial sensitivity pattern of our isolates showed that all Gram positive and
many of the Gram negative organisms were 100% sensitive to amikacin and third
generation cephalosporins (cefotaxime & ceftriaxone). Ampicillin was the least
effective(Table-18).These
findings
are
in
accordance
with
other
investigators.66,69There is an increasing resistance among the major pathogens which
cause meningitis to most of the traditional antimicrobial agents used empirically. A
high percentage of ampicillin resistant H.influenzae were isolated in the present
study. Similar findings have been observed by other workers.70,71 The isolates were
found to be 100% sensitive to Chloramphenicol in the present study. In our study we
found that majority of our isolates were highly sensitive to cefotaxime and
ceftriaxone. Recently ceftriaxone is used as drug of choice in many centres in treating
meningitis. Apart from having a broad spectrum of activity, ceftriaxone has excellent
penetration into the CSF with a ratio of achievable CSF concentration to minimum
inhibitory concentration levels in the range of 100:1.72A ratio of greater than 10:1 has
been suggested as the most critical factor in determining the success of therapy.73
Case Fatality Rate
Table-26 showing comparative studies of case fatality rate in ABM
Studies
Year
Percentage
Lata Kumar et al 47
1980
44%
Deivanayagan et al 10
1993
22%
Bandaru Narasinga rao et al 8
1998
13%
Nanditha Chinchanakar et al
2002
31.5%
2009
45.16%
4
Present Study
In the present study, the case fatality is 45.16% and is similar to the studies of other
Indian authors like – Nanditha Chinchankar et al4 and Lata Kumar et al.47
Mortality was highest in case of P.aeruginosa and Group B Streptococcal meningitis
where in all the positive cases expired. Followed by K.pneumoniae meningitis in
which 2/3(66.66%) cases expired. Case fataliy rate in case of H.influenzae meningitis
was 55.55% and 14.28% in case of Pneumococcal meningitis. (Table19)
CONCLUSION

The incidence of bacterial meningitis was 1.7% in the population
studied.

Male were affected more commonly than the females making a male to
female ratio of 1.62:1.

Haemophilus influenzae was the most common etiological agent of
ABM in early childhood followed by Streptococcus pneumoniae.

The simple Gram stain smear of CSF was the most useful single test for
identifying bacterial meningitis. However in view of the low positive
predictive value of Gram stain, CSF Chemistries and LAT could
provide additional information.

LAT has an advantage over Gram stain in terms of species
identification. It was also more sensitive compared to conventional
Gram stain and Culture technique in identifying the fastidious
organisms like H.influenzae , S.pneumoniae and Group B Streptococcus.

CSF Culture is the gold standard and no test can replace the utility of
culture especially so in neonates as LAT does not identify
Enterobacteriaceae other than E.coli. Thus CSF Culture is crucial to the
diagnosis of neonatal meningitis regardless of the other laboratory
results.

The case fatality rate of ABM in early childhood is likely to be high due
to co-morbid conditions such as septic shock and raised intracranial
pressure. Early referral from peripheral health care facility and proper
intensive care management in the referral unit could improve the results.

No single test possessed the quality of an ideal diagnostic test for the
diagnosis of acute bacterial meningitis. Thus, the combination of these
three tests will yield more productive results rather than any of the tests
alone.

We also found that CSF latex agglutination test is a valuable tool in the
rapid etiological diagnosis of acute bacterial meningitis in early
childhood. However, it should not be used indiscriminately as a
screening tool in the routine diagnostic laboratory and its use should be
reserved for the detection of bacterial antigens in CSF specimens that
fail to reveal the organism on Gram stain. Besides the high cost of LAT
is a prohibitive factor in resource poor countries.
SUMMARY
This prospective study for a period of one year was conducted in the department of
Microbiology, Karnataka Institute of Medical Sciences Hospital, Hubli,from
December 2008 to November 2009.

Children below 5 years of age with signs and symptoms of meningitis
admitted in the Pediatric ward, KIMS hospital, Hubli, were included in the
study.

Aim of the study was: 1)To know the incidence of bacterial meningitis in
children below five years of age.2) To identify the specific etiological agent
causing bacterial meningitis in children below five years of age by CSF
culture.3) To detect bacterial antigens in CSF by latex particle agglutination
test.4) To compare conventional culture method and antigen detection by latex
particle agglutination test.

A total of 100 samples of CSF collected under strict aseptic precautions, were
subjected to cell count, Gram stain ,Culture and LAT.

Latex agglutination tests (LAT) for the detection of five bacterial antigens was
done using BD DIRECTIGEN Meningitis Combo Test kit, manufactured by
Becton and Dickinson Company, USA.

The BD DIRECTIGEN Meningitis Combo Test kit contained antibody coated
latex beads for the detection of Haemophilus influenzae type b, Streptococcus
pneumoniae, Escherichia coli K1, group B streptococcus and Neisseria
meningitidis groups A, B, C, Y and W135.

The frequency of ABM in the age group <5 years in hospital admissions noted
in our study was 1.7%.

The disease was more common in males with a male to female ratio of 1.63:1,
in the present study.

Cases presented with wide array of symptoms with fever being the most
common followed by seizures, altered sensorium ,refusal of feeds and
vomiting. Signs of meningeal irritation were present only in 16% of cases.

31 cases were declared as laboratory confirmed cases of ABM as per the
WHO criteria[147]

64.5% cases of ABM had predominant neutrophil count in their CSF. 9.67%
cases had predominant lymphocyte count. 25.80% cases had normal CSF cell
count among them 19.35% were neonates and 6.45% were beyond 1 month of
age.

An etiological agent was speciated by means of CSF Culture and CSF LAT in
26/31 (%) cases of ABM. Most common etiological agent was, H. Influenzae
in 9 cases,followed S. Pneumoniae in 7 cases ,Klebsiella pneumoniae in 3
cases Group B streptococcus and Escherichia coli in 2 cases each, and one
each of Citrobacter koseri ,Acinetobacter baumanii, and Pseudomonas
aeruginosa .Remaining 5 cases were only Gram stain positive ,3 smears
showed Gram positive cocci in pairs and 2 Gram negative bacilli.

H.influnzae was most common etiological agent of ABM in children below 1
year of age ,while S.pneumoniae was more common in children above
infancy.

Gram stain was positive in 22 of cases, Gram positive cocci in pairs in 8 cases,
Gram positive cocci in chains in 1 case and Gram negative bacilli in 13 cases.
Thus Gram stain could identify maximum number of cases of ABM in the
present study.

CSF Culture could identify 15 cases in the present study , 4 S. pneumoniae,3
H.influenzae, 3 K.pneumoniae, 2 E.coli, 1 each of C.koseri, A.baumanii, and
P.aeruginosa.

CSF LAT was positive in 17 cases , 8 H.influenzae, 7 S.pneumoniae and 2
Group B Streptococcus. Thus LAT was superior to Gram stain and Culture in
identifying the fastidious bacteria which are the most common causative
agents of ABM.

Accuracy indices of Gram stain against Culture showed a sensitivity of
53.33%, specificity of 83.52% ,positive predictive value of 36.36% and
negative predictive value of 91.02% in the present study.

Accuracy indices of LAT against culture showed a sensitivity of 66.66%,
specificity of 87.91%, positive predictive value of 35.29% and negative
predictive value of 96.38%, in the present study. Thus CSF LAT was more
sensitive than Gram stain. Specificity and negative predictive value were
marginally higher compared to Gram stain, while the positive predictive
values were comparable.

The association between prior antibiotic administration i.e., before the lumbar
puncture and the results of CSF Gram stain , Culture and LAT were not
statistically significant, with a marginally higher rate of positivity by LAT and
Gram stain compared to culture in these cases.

AST was done on all culture isolates by the Kirby Bauer disc diffusion method
. All Gram positive and many of the Gram negative organisms were 100%
sensitive to amikacin and third generation Cephalosporins (cefotaxime &
ceftriaxone). Ampicillin was the least effective. The isolates were found to be
100% sensitive to Chloramphenicol in the present study.

The rate of mortality in the present study was 45.16%.Maximum case fatality
was observed in case of Group B Streptococcus and P.aeruginosa meningitis..
PROFORMA
“EVALUATION OF THE CONVENTIONAL CULTURE METHOD AND
RAPID ANTIGEN DETECTION METHOD (LATEX PARTICLE
AGGLUTINATION TEST) FOR DIAGNOSIS OF BACTERIAL MENINGITIS
IN CHILDREN BELOW FIVE YEARS OF AGE”
Name of the patient:
Age:
Address:
Informant:
Clinical History:
Complaints
Duration of present illness:
History of medication:
Past history
Obstetric history:
Previous investigations (if any):
Signs of examination:
Nature of the samples sent
Time and date of collection:
Sex:
I.P.No.
CSF ANALYSIS: Cell count
Cell type
Sugar
Protein
MICROBIOLOGICAL STUDY:
SPECIMEN: Cerebro spinal fluid(CSF)
GROSS APPEARANCE:
MICROSCOPY:
1. GRAM STAIN:
CSF CULTURE:
1. BLOOD AGAR:
2. CHOCOLATE AGAR:
3. MAC CONKEY AGAR:
4. BRAIN HEART INFUSION BROTH:
RELEVANT BIOCHEMICAL TESTS:
ANTIBIOTIC SENSITIVITY TESTING:
SEROLOGY: Latex particle agglutination test:
REPORT:
Treatment:
Signature of candidate:
Signature of guide: