A Prospective Study of Etiology of Childhood Acute Bacterial Meningitis, Turkey

Vaccines to prevent bacterial meningitis in this region must provide reliable protection against serogroup W-135.

A cute bacterial meningitis is one of the most severe infectious diseases, causing neurologic sequelae and accounting for an estimated 171,000 deaths worldwide per year (1,2). Although most disease occurs in infants, the societal impact is also important because of the continued high incidence in healthy older children and adolescents. Despite many new antibacterial agents, bacterial meningitis fatality rates remain high, with reported rates between 2% and 30% (3,4). Furthermore, permanent sequelae, such as epilepsy, mental retardation, or sensorineural deafness are observed in 10%-20% of those who survive (5,6).
The 3 most common etiologic agents are Haemophilus infl uenzae type b (Hib), Streptococcus pneumoniae, and Neisseria meningitidis, which account for 90% of reported cases of acute bacterial meningitis in infants and children >4 weeks of age (7,8). Hib meningitis is a disease affecting primarily young children; most of the cases occur in children 1 month to 3 years of age (3,8). The use of Hib conjugate vaccines has reduced the incidence of, or even virtually eliminated, invasive Hib disease in some industrialized countries (7,8). S. pneumoniae is a major cause of childhood bacterial meningitis in countries where Hib disease has been eliminated by vaccination (9). It is the second most frequently reported cause of septic meningitis in some European and sub-Saharan African countries, after meningococcal cases (4,9).
N. meningitidis is now considered to be the leading cause of bacterial meningitis in many regions of the world, causing an estimated 1.2 million cases of bacterial meningitis and sepsis worldwide each year (10,11). Meningococci are classifi ed into 13 serogroups based on the antigenic properties of their capsular polysaccharide; however, nearly all disease is caused by 5 serogroups: A, B, C, W-135, and Y. The epidemiology of N. meningitidis varies by sero-group; currently, serogroups A, B, and C account for >90% of meningococcal disease worldwide (12). However, the epidemiologic landscape is constantly changing, and with increasing international travel and cross-border migration, the epidemiology of this disease will remain dynamic. Currently, serogroups A and C predominate throughout Asia and Africa, whereas serogroups B and C are responsible for most cases in Europe and North America (11,(13)(14)(15)(16)(17)(18). In several countries, including the United States, the proportion of disease caused by serogroup Y has increased over the past decade, where it now accounts for approximately one third of meningococcal cases (19). Serogroup W-135 has also recently emerged in some parts of the world, primarily in the Middle East and Africa, in some instances causing large epidemics (20).
The annual Hajj pilgrimage to Mecca is a major international event; ≈2 million people from around the world gather in one place, where the extreme crowding provides an ideal environment for transmission of meningococcal carriage. On several occasions, meningococcal disease outbreaks have subsequently spread worldwide by returning pilgrims. A major serogroup A meningococcal disease epidemic occurred in the 1980s, affecting Muslim pilgrims initially, followed by populations in other Middle Eastern and African countries (21). After this epidemic, Hajj pilgrims were vaccinated with a bivalent (A and C) meningococcal polysaccharide vaccine before entering Saudi Arabia. With the emergence of serogroup W-135 meningococcal disease among Hajj pilgrims in the Middle East during 2000 and 2001 (20), vaccine recommendations for pilgrims were changed to quadrivalent (A, C, W-135, and Y) meningococcal polysaccharide vaccine in 2002 (22).
Global surveillance of confi rmed meningococcal cases, including surveillance of the diversity of causative strains, is essential to managing disease and developing vaccines. This study was undertaken to determine the current etiology of bacterial meningitis in Turkey, with particular emphasis on serogroup distribution of meningococci. Turkey is a predominantly Muslim country, and as such epidemics originating at the Hajj may have an effect on the national epidemiology. Although limited epidemiologic studies are available, cases of invasive meningococcal disease as well as carriage of serogroup W-135 have been reported in Turkey (23)(24)(25). This fi nding is in contrast to Western Europe, where the incidence of W-135 disease remains low. Turkey has no surveillance system for bacterial meningitis, and exact rates of meningococcal disease and serogroup distribution are unknown. Reliable surveillance data from countries such as Turkey are vital to understand, and better anticipate, the constantly changing landscape of bacterial meningitis and meningococcal disease.

Study Design
From February 16, 2005, through February 15, 2006, active surveillance of acute bacterial meningitis among children admitted to 12 participating hospitals was undertaken. Turkey is divided into 7 geographic areas ( Figure 1). Twelve health centers in 9 cities located in all of these 7 geographic regions were selected to represent the population characteristics of the country. Two centers from each of the 3 biggest cities and 1 center from each of the other cities were included. Each health center served as a referral center for its region in the fi eld of pediatric diseases. The centers serve ≈32% of the entire pediatric population of Turkey. Approval was obtained from the ethical committees of the participating centers and Ministry of Health.
In each hospital, suspected cases of acute bacterial meningitis were identifi ed by a pediatrician, based on the following criteria: any sign of meningitis (fever [  The number of suspected meningitis cases included in the study per region is shown in boldface, with the region-specifi c estimated incidence rate of laboratory-confi rmed meningitis (per 100,000 population) shown in parentheses. In total, 408 children were diagnosed with suspected acute bacterial meningitis. Bacterial meningitis was confi rmed by PCR, culture, or latex agglutination test in 243 cases. Region-specifi c incidence rates ranged from 1.0 to 10.9/100,000 population.
if the CSF had 1) >10 leukocytes/mm 3 in the CSF, and/or 2) higher CSF protein levels than normal for the patient's age, and/or 3) lower CSF glucose levels than normal for the patient's age. In addition to these patients, all who had a positive CSF culture, PCR, Gram stain, or antigen detection test result were also included in the study. No neonates (<1 month of age) were included in the study since the pathogens of neonatal meningitis were expected to be different than those of the older case-patients (3).

CSF Cultures and Bacterial Isolates
CSF cultures, Gram stain, and latex agglutination tests (Wellcogen Bacterial Antigen Kit, Lenexa, KS, USA) were performed in the local hospitals. A CSF sample (minimum 0.5 mL) from each patient was kept at -20°C until transportation to the Central Laboratory (Hacettepe University, Ankara, Turkey) for PCR analysis. CSF samples and, if available, bacterial isolates were sent to the Central Laboratory, where all isolates were recultured on chocolate and blood agars and grown at 37°C in 5% CO 2 . Suspected meningococcal colonies were characterized by Gram stain, oxidase test, and rapid carbohydrate utilization test (Gallerie Pasteur, Pasteur Merieux, Lyon, France). The microbiology laboratory records were crosschecked in each hospital for missing data. The phenotypic determination, based on the antigenic formula (serogroup: serotype:serosubtype) of meningococcal isolates, was performed by standard methods in the Meningococcal Reference Unit, Health Protection Agency, Manchester, United Kingdom (18,(20)(21)(22)(23)(24)(25)27).

DNA Isolation
All samples collected in the Central Laboratory were kept at -80°C and were thawed immediately before each test. Bacterial colonies were suspended in 500 μL sterile double-distilled water and vortexed. Bacterial suspensions and CSF were boiled for 3 min at 100°C, then centrifuged for 5 min at 10,000 × g, and the supernatant was retained. DNA concentration was estimated spectrophotometrically, and 15 μL (≈50 ng) was used in each fi nal reaction mixture.

PCR Amplifi cation
For the simultaneous identifi cation of bacterial agents, single tube, multiplex PCR assay was performed. The specifi c gene targets were ctrA, bex, and ply for N. meningitidis, Hib, and S. pneumoniae, respectively (28). In each assay, the fi nal reaction mixture of 50 μL contained 15 μL (≈50 ng) DNA, 1× PCR buffer, 3 mmol/L MgCl 2 , 200 μmol/L of each dNTP (AB Gene, Epsom, UK), 0.6 μmol/L of each corresponding oligonucleotide primer (Sigma Aldrich, Seezle, Germany) as described (28) and 1 U of Taq polymerase (AB Gene). The PCR was performed by using a DNA thermal cycler (Perkin-Elmer Cetus, Emeryville, CA, USA model 9600) under the following conditions: a fi rst cycle of denaturation at 95°C for 5 min followed by 35 cycles of 95°C for 25 s, 57°C for 40 s, and 72°C for 60 s.
Among the samples positive for N. meningitidis, serogroup prediction (A, B, C, W-135, and Y) was based on the oligonucleotides in the siaD gene for serogroups B, C, W-135, and Y and in orf-2 of a gene cassette required for serogroup A (28). For serogroup determination, amplifi cation reactions (50 μL) contained 15 μL of DNA, 60 mmol/L Tris-HCl (pH 8.8), 17 mmol/L (NH4) 2 SO 4 , 5 mmol/L Mg-Cl 2 , 0.5 mmol/L of each dNTP, 0.3 μmol/L corresponding oligonucleotides, and 1 U of Taq polymerase. The PCR conditions were as follows: denaturation at 94°C for 3 min, followed by 35 cycles of 92°C for 40 s, 55°C for 30 s, and 72°C for 20 s in a DNA thermal cycler. A fi nal extension cycle at 72°C for 10 min was then performed (29).
All amplicons were analyzed by electrophoresis on standard 3% agarose gels and visualized by using UV fl uorescence. A negative control consisting of distilled water and a positive control consisting of a reference strain (S.

Statistical Analysis
Continuous variables were compared by the Student t test and categorical variables with χ 2 or Fisher exact tests. A 2-tailed p value <0.05 was considered signifi cant. All statistical analysis was performed with SPSS version 11.5 (SPSS Inc, Chicago, IL, USA).

Meningitis Cases
In total, 408 children were hospitalized with a clinical diagnosis of meningitis during the study period (Figure 1), and a CSF sample from each patient was obtained. The distribution of these suspected cases according to the geographic regions was as follows: 109 (26.7%) in Southern Anatolia, 74 (18.1%) in Aegean region, 79 (19.6%) in Central Anatolia, 53 (13.0%) in Marmara region, 43 (8.7%) in Eastern Anatolia, 24 (5.9%) in Black Sea region, and 26 (6.4%) in Mediterranean region. The mean age of the 408 children was 4.8 years (standard deviation 4.1 years), and the boy-to-girl ratio was 1.5:1. Of 408 patients diagnosed with acute bacterial meningitis, 20 (4.9%) died and 14 (5.7%) of these deaths were among patients with laboratory-confi rmed cases.

Laboratory-Confi rmed Meningitis Cases and Etiology
Of the 408 cases, bacterial meningitis was confi rmed by PCR, culture, or latex agglutination test in 243 (59.6%) patients. Regional incidence rates of laboratory-confi rmed meningitis were estimated as ranging from 1/100,000 population in the Black Sea region to 10.9/100,000 population in the Southeast Anatolia region (Figure 1). Nationwide, the highest incidence was in children 1-12 months of age and was slightly more common in boys. The boy-to-girl ratio of the confi rmed cases was 1.3:1, and the age distribution was as shown in Figure 2.
Overall, the diagnosis of acute bacterial meningitis was confi rmed with CSF culture in 41 (17%) of 243 cases, with latex agglutination test in 56 (23%), and with PCR in 243 (100%) ( Table). Latex agglutination test was positive in 37 cases for N. meningitidis, in 10 cases for Hib, and in 9 cases for S. pneumoniae.
Where data were available, 7 (17%) of 41 cases with positive CSF culture and 111 (54.9%) of 202 cases with negative CSF culture had a history of use of antimicrobial agent(s) before lumbar puncture, which may account for the relatively low diagnosis rate by using this technique. N. meningitidis was reported in 23 cases, S. pneumoniae was reported in 12, and Hib was reported in 6 cases as positive in CSF culture.
Blood PCR analysis was by far the most reliable method of confi rming bacterial meningitis, accounting for all confi rmed cases with 243 positive results. In these PCR-positive samples, 138 (56.5%) were attributable to N. meningitidis, 55 (22.5%) to S. pneumoniae, and 50 (20.5%) to Hib (Figure 3). Of the 408 patients, 118 (48.5%) of 243 cases with positive PCR and 96 (58.2%) of 165 cases with negative PCR had received antibacterial drugs in the week before CSF sampling.
In the evaluation of the bacterial agents among the 7 different geographic regions of the country, N. meningitidis was the most common cause of acute bacterial meningitis in all regions except the Mediterranean region, located on the southern coast of Turkey. Here S. pneumoniae were the prominent bacteria, and N. meningitidis were detected in only 2 cases.
Comparison of the incidence of N. meningitidis, S. pneumoniae, and Hib among different age groups demonstrated that N. meningitidis was the prominent bacterial agent causing acute bacterial meningitis, especially in chil-dren <7 years of age ( Figure 2). The highest incidence was detected during the fi rst year of life for all 3 bacteria.
CSF fi ndings were recorded for 368 (90.2%) of 408 CSF samples sent to the Central Laboratory. As a mean, in PCR-negative samples, CSF protein level was signifi cantly lower (70.

Meningococcal Epidemiology
Among the samples that were positive for N. meningitidis following PCR analysis, serogroup W-135 was the cause of most infections; 59 (42.7%) cases were serogroup W-135, 43 (31.1%) were serogroup B, 3 (2.2%) were serogroup Y, and 1 (0.7%) was serogroup A. There were no cases with a positive result for serogroup C (this was also 1092 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 7, July 2008  the case following analysis of CSF culture-positive samples) and in 32 (23.2%) N. meningitidis-positive samples the serogroup could not be determined by the PCR assay. Analysis by age reveals the greatest meningococcal disease incidence is in children <3 years of age, particularly infants <1 year of age. The numbers of cases caused by the 2 most common N. meningitidis serogroups (serogroups W-135 and B) were similar in the most vulnerable age groups (<3 years of age), but W-135 was more common in children 4-16 years of age (Figure 4).
Etiologic and meningococcal serogroup distribution among the different geographic regions is illustrated in Figure 5. N. meningitidis serogroup W-135 was more prominent than the other meningococcal serogroups in the Southeast Anatolia, Aegean (Western Turkey), Eastern Anatolia, and Black Sea regions. N. meningitidis serogroup B was much more common in the Marmara region (northwestern Turkey), and in the Central region; the numbers of serogroup B and serogroup W-135 cases were similar. The Mediterranean region had 2 N. meningitidis-positive samples; both were nongroupable by PCR analysis.

Discussion
In this study, 243 cases of laboratory-confi rmed acute bacterial meningitis were recorded. Because our study centers provide service to 32% of the population of Turkey, extrapolation from the number of cases recorded suggests that 759 acute bacterial meningitis cases (excluding neonatal cases) per year occur in the whole country. The population of children 1 month through 16 years of age was calculated as 21.6 million. Therefore, the annual incidence of acute bacterial meningitis was estimated as 3.5 cases/100,000/ year. Although similar to incidence rates reported from other countries without routine vaccination against N. meningitidis, S. pneumoniae, and Hib (3,4,10,30), this value likely represents a lower limit estimate of the true disease incidence, given the inherent limitations of hospital-based surveillance. Furthermore, the specifi c role of these 3 most common bacterial causes of acute bacterial meningitis varies between regions.
An accurate laboratory confi rmation 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 (28,31). 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. Among nonculture diagnostic tests, PCR is the most accurate and reliable method, especially among patients with a history of anti-microbial drug use before spinal tap (32). This fi nding was evident in the present study, in which PCR analysis was the most sensitive method, confi rming 243 cases (59.6%) among 408 children meeting the case defi nition for bacterial meningitis (100% of all cases that were confi rmed by any method). Using other methods that are more sensitive may increase the rate of laboratory confi rmation.
Several reports review the rates of bacterial causes of acute bacterial meningitis from many different countries, based on CSF cultures. Some factors, such as previous antimicrobial drug treatment, interfere with the recovery of microorganisms from CSF. In our study, bacterial isolation was only possible in 41(16.8%) of 243 of confi rmed cases. However, the most important factor for this low positive  In patients with acute bacterial meningitis, blood cultures can be used in the etiologic diagnosis in up to 80% of cases since the bacteria generally invade meningeal membranes following bacteremia (3,10,33). In our study, however, only 12 (4.9%) of 243 case-patients who had blood culture tests returned positive results. This fi nding may also be related to the previous antimicrobial drug use.
Previous reports suggested that S. pneumoniae and N. meningitidis serogroups A and B would be the most common bacteria causing acute bacterial meningitis in Turkey (25,(34)(35)(36). Serogroup W-135 meningococcus was isolated for the fi rst time in Turkey in an asymptomatic healthy preschool child in 2001 (23), and the fi rst patient with meningitis caused by serogroup W-135 was reported in 2003 (24). In this study N. meningitidis, especially serogroup W-135, was responsible for most of the cases observed, with serogroup B the second most common. Only a small number of serogroup A or Y cases were noted, and no serogroup C cases were observed. These data are in contrast to those from many parts of Europe, where serogroups B and C dominate the epidemiologic landscape.  (38). Therefore, the rapid rise in the proportion of cases caused by serogroup W-135 may be attributable to transmission from pilgrims returning from the Hajj carrying this particular serogroup. Although not defi nitive, this conclusion is further supported by the fi nding that all serogroup W-135 isolates available for phenotypic characterization were identical to the Hajj-associated clone, W135:2a:P1.5,2 (20). Because strains with this serologic profi le have not been uniquely associated with the Hajj outbreak (39), additional typing data (e.g., multilocus sequence typing or multilocus enzyme electrophoresis), and epidemiologic investigations will be required to support this hypothesis. Therefore, it remains speculative that the increased W-135 disease in Turkey may be caused by spread of Hajj epidemic strain through carriage and transmission by pilgrims.
This study demonstrates the need for good quality, continued surveillance of bacterial meningitis cases, as well as the etiology and epidemiology of the causative bacteria. Only by accurately monitoring meningococcal epidemiology will effective vaccination policies be developed. The bacterial meningitis epidemiologic landscape is not static, and the causative agents change with time and across regions of the world. This study has demonstrated that the relative contribution of serogroup W-135 to the meningococcal disease incidence in Turkey is increasing, which is in contrast to the rest of Europe. Turkey may remain isolated in terms of W-135 disease incidence or it may represent the beginning of epidemiologic change in Eastern Europe. This possibility should be investigated in greater depth and monitored prospectively. Introduction of vaccines can dramatically reduce the meningitis disease incidence, but these vaccines must be targeted against the correct bacteria and, where relevant, the correct bacterial serogroup. The choice of meningococcal conjugate vaccines in Turkey will need to include coverage for serogroup W-135; introduction of such a vaccine would be helpful in protecting the Turkish population from this invasive bacterial meningitis. Moreover, it may also be prudent to switch the meningococcal vaccine used for pilgrims to conjugated vaccine to prevent the carriage of the microorganism by pilgrims.
This study was supported by Novartis Vaccines.
Dr Ceyhan is professor of pediatrics and pediatric infectious diseases at Hacettepe University, Ankara. His areas of interest include vaccines, epidemiology of vaccine-preventable diseases, and mechanisms of antibacterial resistance.