J Egypt Public Health Assoc

Vol. 82 No. 1 & 2, 2007

Microbial Study of Meningitis and Encephalitis Cases Heba S. Selim*, Mohamed A. El-Barrawy*, Magda E. Rakha**, Samuel L. Yingst***, Magda F. Baskharoun* * Microbiology Department, High Institute of Public Health, Alexandria University. ** Ministry of Health 1st Under Secretary. *** Virology Research Program. U.S. Naval Medical Research Unit No. 3(NAMRU-3).

ABSTRACT Meningitis and/or encephalitis can pose a serious public health problem especially during outbreaks. A rapid and accurate diagnosis is important for effective earlier treatment. This study aimed to identify the possible microbial causes of meningitis and/or encephalitis cases. CSF and serum samples were collected from 322 patients who had signs and symptoms suggestive of meningitis and/or encephalitis. Out of 250 cases with confirmed clinical diagnosis, 83 (33.2%) were definitely diagnosed as bacterial meningitis and/or encephalitis cases (by using CSF culture, biochemical tests, latex agglutination test, and CSF stain), 17 (6.8%) were definitely diagnosed as having viral causes ( by viral isolation on tissue culture, PCR and ELISA), and one (0.4%) was diagnosed as fungal meningitis case (by India ink stain, culture, and biochemical tests). Also, there was one encephalitis case with positive serum ELISA IgM antibodies against Sandfly scilian virus. N.meningitidis, S. pneumonia and M. tuberculosis were the most frequently detected bacterial agents, while Enteroviruses, herpes simplex viruses and varicella zoster viruses were the most common viral agents encountered. Further studies are needed to assess the role of different microbial agents in CNS infections and their effective methods of diagnosis.

Key Words: Bacterial, Viral, Fungal, Meningitis, Encephalitis.

Corresponding Author: Dr. Heba S. Selim. High Institute of Public Health Microbiology Department E.mail: [email protected]

J Egypt Public Health Assoc

Vol. 82 No. 1 & 2, 2007

INTRODUCTION Infection of the central nervous system manifested as meningitis and/or encephalitis can pose a serious public health problem especially during outbreaks. These infections are associated with significant morbidity and mortality despite advances in antimicrobial therapy. A rapid and accurate diagnosis is important for effective earlier treatment of serious infections. (1) Meningitis is defined as inflammation of the membranes surrounding the brain and spinal cord, including the dura, arachnoid and pia mater. Encephalitis is an inflammation of the brain parenchyma. Meningo-encephalitis is inflammation of the brain and meninges. (2) Meningitis and encephalitis can be caused by a wide range of bacteria, viruses, fungi, protozoa, chemical agents and drugs.(3) Bacterial meningitis is principally a disease of early childhood with more than 50 per cent of cases occurring in children less than five years of age.(4) N. meningitidis is the leading infectious cause of death in childhood. Outside the neonatal period, the majority of cases of bacterial meningitis have been due to either N. meningitidis, Streptococcus pneumoniae or Haemophilus influenzae type b (Hib).(5)Less

frequently

encountered bacterial causes includes Mycobacterium tuberculosis, Listeria monocytogenes, Staphylococcus aureus, Group B haemolytic Streptococci, Escherichia coli and

other Coliforms , Salmonella species, and

Spirochetes.(6) Viruses play a major role in meningo-encephalitis. In the United States, the annual number of central nervous system infections that occur as a result of viral agents far exceeds that of infections caused by bacteria, yeast, molds and protozoa combined.(7) The most common viral causes include; DNA viruses: herpes simplex virus, Epstein- Barr virus

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(EBV), varicella zoster virus (VZV), cytomegalovirus (CMV)], and adenovirus;

RNA viruses: influenza virus, enteroviruses,

rabies,

arboviruses (Rift Valley fever virus, Japanese B encephalitis, St lauis encephalitis virus, West Nile encephalitis virus, Sandfly fever virus, lymphocytic choriomeningitis virus, Eastern, Western, and Venezuelan equine encephalitis viruses), reovirus, and retroviruses (HIV).(8) The main fungal causes of meningitis and encephalitis are Cryptococcus neoformans, Candida species, and Aspergillus species.

(9)

The

range of patients at risk for invasive fungal infections continues to expand beyond the normal host to encompass patients with the acquired

immunodeficiency

immunosuppressed

due

to

syndrome therapy

for

(AIDS), cancer

and

those

and

organ

transplantation. As the population at risk continues to expand, so also does the spectrum of opportunistic fungal pathogens infecting these patients also continue to increase. (10) The diagnostic criteria for bacterial meningitis include at least one of the following: cerebral spinal fluid (CSF) positive bacterial culture, CSF positive Gram's stain with negative cultures ( Gram stain revealing only gram-negative diplococci was classified as Neisseria meningitidis even if

CSF cultures remained negative in the presence of clinical

manifestations), positive bacterial culture or Gram's stain from blood, petechial lesion or sputum in presence of clinical manifestations of meningitis, positive antigen detection with latex agglutination, or counter immunoelectrophoresis (CIE) in the CSF or blood, CSF profile demonstrating significant cytochemical changes, such as white blood cells ≥ 1000 mm3, neutrophils ≥60%, protein ≥100 mg/dl, and glucose ≤ 50mg/dl in the presence of clinical manifestations of meningitis.(11)

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The laboratory diagnosis of confirmed case of viral meningitis and/or encephalitis is based on one of the following: fourfold or greater rise in serum antibody titer, isolation of virus or demonstration of viral antigen or genomic sequences in tissue, blood, CSF, or other body fluid, or specific IgM antibodies by enzyme immunoassay antibody captured CSF or serum. (12) The present work aimed at identification of the possible bacterial, viral, and fungal causes of meningitis and encephalitis in El-Behera, Kafr El-Sheikh, and Alexandria Governorates.

MATERIAL AND METHODS This study was carried out on 322 cases with acute febrile illness, and neurological signs suggesting meningitis and/or encephalitis admitted to Damanhour, Alexandria and Kafr El-Sheikh fever hospitals during the period from April 2004 to September 2005. Their ages ranged from 2 months to 75 years old. A questionnaire sheet was completed for each patient including personal data and medical history, also consent was taken from each patient. CSF and blood samples were collected from all patients from the three mentioned hospitals, and were subjected for:

A- CSF examination CSF samples were divided into 4 portions: the first CSF portion was examined for macroscopic appearance to detect any physical abnormalities, cytological count of white blood cells (WBCs) and chemical examination for sugar and protein. After centrifugation of the second CSF portion the supernatant was decanted and about 0.5 ml was left behind to suspend the sediment for direct microscopic examination

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(by Gram, methylene blue, Zeihl-Neelsen, and India ink stains), and bacterial culture on: 1. Blood agar and chocolate agar plates incubated at 37°C for 72 hours in 5-10% CO2. 2. Mac Conkey’s agar, incubated at 37°C for 72 hours. 3. Three slopes of Lowenstein Jensen medium two at 37°C and one at 25°C for 8 weeks, and checked weekly for evidence of growth. 4. Sabouraud’s

dextrose

agar(with

chloramphenicol

only,

as

cycloheximide inhibits Cryptococcus neoformans) and brain heart infusion media for fungal detection, incubated aerobically at 37°C and 28°C. All isolated colonies were identified biochemically. The third portion was used for performing direct bacterial antigen detection by

latex agglutination test (Remel wellcogen bacterial

antigen) The latex particles are coated with the appropriate rabbit antibody, as labelled for each organism. The streptococcus and H.influenzae reagents are specific to group B and type b, respectively and the N.meningitidis polyvalent reagent reacts with group A, C, Y and W135 antigens: The fourth portion of CSF samples was examined in NAMRU-3 laboratory for the detection of viral causes (arboviruses, herpesviruses, enteroviruses) by 1. Viral isolation on tissue culture, 6 cell lines were used:(Vero E6 clone, BHK-21, HEp2, C6/36, MRC-5 and LLCMK2).Each cell culture cells were splitted in tissue culture tubes, inoculated with the samples, incubated and checked daily for 14 days for the presence of cytopathic effect (CPE) characteristic of each group of viruses. If CPEs were detected, second passage in cell lines were

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performed and examined daily for CPE for 14 days. If CPE was detected, polymerase chain reaction ( PCR) and immunoflorescent assay (IFA) were done for confirmation. 2. PCR to detect viral nucleic acids : QIAamp DNA Mini kits and QIAamp RNA Mini Kits were used for purification of viral DNA and RNA from the samples, then different protocols were applied for the detection of different viruses Primer name

Enterovirus

Entero-IN-F

CCCCTGAATGCGGCTAAT

Genosys

5` UTR

146bp

Enterovirus

Entro-IN-R

ATTGTCACCATAAGCAGCCA

Genosys

5` UTR

146 bp

HSV-1&2

HSV-IN-F

GCGCCGTCAGCGAGGATAAC

Genosys

gp D

281 bp

HSV-1&2

HSV-IN-R

AGCTGTATASGGCGACGTG

Genosys

gp D

281 bp

VZV

VZV-IN-F

ACCTTAAAACTCACTACCAGT

Genosys

ORF 29

208 bp

VZV

VZV-IN-R

CTAATCCAAGGCGGGTGCAT

Genosys

ORF 29

208 bp

Alpha

Alpha FOR1

YAGAGCDTTTTCGCAYSTRGCH W

Genosys

ns P1

434 bp

Alpha

Alpha REV1

ACATRAANIGNGTNGTRTCRAAN CCDAYCC

Genosys

ns P1

434 bp

FL1-cfD2-250 AA ATG ATG GGR AAR AGR GAR AA

Genosys

NS5 gene

252 bp

AAR GGH AGY MCD GCH ATH TGG T

Genosys

NS5 gene

214 bp

Genosys

Latent cycle gene

296 bp

Epstein-Bar EBV20MEN10 AGACAATGGACTCCCTTAGC virus

Genosys

Latent cycle gene

296 bp

RVF

RVF-NS-3a

ATGCTGGGAAGTGATGAGCG

Genosys

S segment

699 bp

RVF

RVF-NS29

GAT TTG CAG AGT GGT CGTC

Genosys

S segment

699 bp

Flavi Flavi

FL3-FS778

Epstein-Bar virus

Sequence

EBV10MEN9 AAGGAGGGTGGTTTGGAAAG

Manufacturer

Genomic Product size region

Virus

In this study the following PCR techniques were used: 1. Multiplex PCR for enteroviruses, HSV, and VZV. 2. Reverse-transcriptase PCR (RT-PCR) for Alpha viruses. 3. Nested-PCR for Flaviviruses and RVF viruses. 4. PCR for detection of EBV.

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The PCR products were separated by electrophoresis on 2% agarose gel, stained with ethidium bromide, and photographed under UV light.

B- Blood examination Sera were separated for detection of possible viral etiologic agents and examined in NAMRU-3 laboratory for: 1. Virus isolation on tissue culture. 2. PCR for detection of viral nucleic acids. 3. Enzyme-linked immunosorbent assay (ELISA) for detection of specific antiviral antibodies (IgM and IgG antibodies against RVF, WN, Sindibis (SIN), and Sandfly Scilian (SFS) viruses). RESULTS After examination of the 322 CSF samples for bacterial agents, 83 were definitely diagnosed as bacterial meningitis / encephalitis cases by one or more of the mentioned methods (CSF culture, latex agglutination test, and CSF stain). One case was diagnosed as fungal meningitis by India ink stain, culture, urease test, and brown pigment (melanin) on TOC medium. Seventeen viral meningitis / encephalitis cases were identified by CSF viral isolation on tissue culture and PCR. One serum IgM positive sample for Sandfly Scilian virus was detected by ELISA technique. Table (1a) and Figure (1) shows that out of 322 suspected meningitis/encephalitis cases, 165 (51.2%) were finally diagnosed as meningitis, 79 (24.5%) were finally diagnosed as encephalitis, 6 (1.9%) were diagnosed as meningo-encephalitis, and 72 (22.4%) cases were diagnosed as non meningitis / encephalitis, with other final clinical diagnosis(as vascular causes, brain tumors, and disease associated or

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complicated). Table (1b) shows that among 165 clinically diagnosed meningitis cases, 72 (43.6%) were due to bacterial causes, 10 (6.1%) due to viral causes, and one case (0.6%) was fungal meningitis. Out of 79 clinically diagnosed encephalitis cases, 10 (12.7%) cases were due to bacterial causes, and 6 (7.6%) were due to viral causes. Among 6 clinically diagnosed meningo-encephalitis cases, one case (16.7%) was of bacterial origin, and one case (16.7%) was viral. The results were statistically significant (MCE

P