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Accurate etiologic diagnosis of meningitis in developing countries is needed to improve clinical care and optimize disease prevention strategies. Cerebrospinal fluid (CSF) culture and latex agglutination are the current standard diagnostic methods but lack sensitivity.
We prospectively assessed the utility of the Binax NOW® S. pneumoniae immuno-chromatographic test (ICT) compared to culture in five countries conducting bacterial meningitis surveillance in Africa and Asia. Most CSF was collected from patients 1 to 59 months old.
A total of 1173 CSF samples from suspected meningitis cases were included. The ICT was positive in 98.6% of culture-confirmed pneumococcal meningitis cases (N=69) and negative in 99.3% of culture-confirmed bacterial meningitis cases caused by other pathogens (N=125). Using culture and latex agglutination testing alone, pneumococci were detected from 7.4% of patients in Asia and 15.6% in Africa. The ICT increased pneumococcal detection, resulting in similar identification rates across sites, from 16.2% in Nigeria to 20% in Bangladesh. ICT detection in culture-negative cases varied by region (8.5% in Africa vs. 18.8% in Asia, p<0.001), prior antibiotic use (24.2% with prior antibiotics vs. 12.2% without, p<0.001) and WBC count (9.0% for WBC=10-99, 22.1% for WBC=100-999 and 25.4% for WBC≥1000; p<0.001 for trend).
The ICT provided substantial benefit over latex agglutination and culture in Asian sites, but not in African sites. With the addition of the ICT, the proportion of meningitis attributable to pneumococcus was similar in Asia and Africa. These results suggest that previous studies have underestimated the proportion of pediatric bacterial meningitis due to pneumococcus.
Streptococcus pneumoniae (or pneumococcus) is a major cause of bacterial meningitis in the developing world [1-4], including recently described epidemic disease in Sub-Saharan Africa [5, 6]. Safe and effective vaccines to prevent pneumococcal meningitis now exist [7-10] and many developing countries will introduce vaccine in the near future. As part of vaccine introduction, countries will monitor impact of vaccine against various outcomes, including meningitis. Additionally, in the African meningitis belt, patients with suspected acute bacterial meningitis are treated with a single dose of oily chloramphenicol (a long-acting preparation of chloramphenicol in an oil suspension, recommended by the World Health Organization as first-line treatment for presumptive bacterial meningitis in the African meningitis belt during the epidemic meningitis season) on the assumption that their illness is caused by meningococcus . This treatment is sub-optimal for pneumococcal meningitis. Accurate etiological tests appropriate for developing country situations will assist with measuring vaccine impact and improving clinical care.
Culture-based identification of the etiologic agents causing acute bacterial meningitis in developing countries may have poor sensitivity for a variety of reasons . These include antibiotic pretreatment, specimen contamination, lack of transport media, delays in transportation from outlying areas, inconsistent availability of laboratory supplies, and challenges in training and motivating laboratory staff . Antigen detection of cerebrospinal fluid (CSF) specimens provides a useful adjunct to culture-based diagnosis [14, 15]. It allows swifter feedback than culture and often permits accurate etiological diagnosis among meningitis patients who have been pre-treated with antibiotics. Latex agglutination testing is widely used, but the test must be interpreted by an experienced reader and is therefore not usually considered a ‘bed-side’ technique. Moreover, latex agglutination kits have a relatively short shelf-life, particularly in tropical climates.
The Binax NOW® antigen test for Streptococcus pneumoniae is an in vitro rapid immuno-chromatographic test (ICT) for the detection of S. pneumoniae antigen in the urine of adult pneumonia patients and the CSF of meningitis patients of all ages. The tool has demonstrated high sensitivity and specificity when testing CSF from patients with culture-confirmed meningitis [16, 17]. Moreover, it was as sensitive as PCR in a study of children with purulent CSF in Bangladesh . In this study, we aimed to further evaluate the sensitivity and specificity of the Binax NOW® ICT among patients with meningitis in five field sites in South Asia and in Africa, to test whether the ICT consistently increased the yield of pneumococcal cases among patients with meningitis in these settings, and to assess its utility among patients with and without prior antimicrobial treatment.
The study was conducted at five sites with existing surveillance for acute bacterial meningitis: Centre Hospitalier Universitaire Yalgado Ouédraogo in Ouagadougou and Centre Médical avec Antenne Chirurgicale (CMA) of Kaya district, Burkina Faso; Kilifi District Hospital in Kilifi, Kenya; University College Hospital in Ibadan, Nigeria; Dhaka Shishu Hospital in Dhaka, Bangladesh; and Aga Khan University in Karachi, Pakistan [Table 1]. At the time of the study, Haemophilus influenzae type b vaccine was in use for routine infant immunization only in Kenya (since 2001) and Burkina Faso (since January 2006).
The primary target population was children 1 to 59 months of age with a clinical suspicion of acute bacterial meningitis, although older persons were included as well. In particular, Burkina Faso actively recruited all patients with suspected acute bacterial meningitis regardless of age.
Cerebrospinal fluid (CSF) was collected from all study subjects and cultured using standard microbiological procedures . Latex agglutination (LA) testing was performed according to the manufacturer's instructions using Pastorex® (Biorad, Hercules, CA) in Burkina Faso and Wellcogen S. pneumoniae and H. influenzae type b kits in Bangladesh, Kenya, Nigeria and Pakistan (Remel, UK). Testing for prior antimicrobial treatment was conducted by plating a Micrococcus ATCC strain (K. rhizophila ATCC 9341) sensitive to most common antibiotics on sheep blood agar plates, placing a disk with 10 μl of CSF on the bacterial lawn, and measuring the diameter of growth inhibition after overnight incubation at 37°C. Any zone of inhibition was considered indicative of the presence of antibiotics.
The Binax NOW® immuno-chromatographic test was performed on all CSF specimens with at least 10 WBC/mL or with a positive culture result. The test consists of a hinged device in which rabbit anti-pneumococcal antibody is adsorbed onto a nitrocellulose membrane (the sample line), and goat anti-rabbit IgG is adsorbed onto the same membrane as a second stripe (the control line). A second set of rabbit anti-pneumococcal antibodies are conjugated to gold particles dried onto an inert fibrous support. The technician performing the assay dips a swab into the CSF sample and inserts it into the test device, adds a citrate buffer to facilitate antigen flow and closes the device. If pneumococcal antigen is present in the specimen, it binds to the gold-conjugated rabbit antibodies and the resulting complex is captured by the immobilized rabbit IgG stripe, forming the sample line. In addition, immobilized goat anti-rabbit IgG captures excess conjugated rabbit antibody, forming the control line. Results are read visually after 15 minutes, with the appearance of the control line only signifying a negative test and the appearance of both the control and sample lines, a positive test.
Suspected acute bacterial meningitis was defined as fever (temperature ≥38°C) and at least one of the following meningeal signs: convulsions, bulging fontanelle in children <12 months of age or stiff neck; poor sucking or irritability; prostration or lethargy; or petechial or purpural rash. CSF with 10-99 WBC per mL and non-turbid appearance was considered abnormal but non-purulent. Purulent CSF was defined as 1) visual turbidity or 2) a CSF WBC count of at least 100/mL.
Data were entered locally, de-identified and sent to the PneumoADIP at Johns Hopkins University for analysis. In this paper, we report on data from patients who had an ICT done. We calculated the proportion of specimens testing positive by culture or LA by age, prior antibiotic use and study site, and the proportion of specimens testing positive with the Binax NOW® ICT by age, culture and LA results, prior antibiotic use, white blood cell (WBC) count, and study site. We used chi-square tests to compare proportions across strata and set a cut-off of 0.05 for statistical significance. All analyses were conducted in Stata 9.2 (StataCorp, College Station, TX).
CSF was collected as part of routine care and therefore countries did not require informed consent for the study. Nevertheless informed consent was obtained from all participants in Nigeria and an information sheet explaining the purpose of the study was provided to patients or their guardians in Burkina Faso. The study was approved by local Ethical Review Committees at all sites and by the Johns Hopkins Institutional Review Board. The Binax ICT kits were provided to PneumoADIP at a reduced cost by Binax, Inc; otherwise, this company did not provide study support and no staff from the company had access to study data or to advance copies of the manuscript.
Recruitment began on March 1st 2006 in Bangladesh and July 1st 2006 at all other sites, and continued through June 30th 2007. All patients with suspected bacterial meningitis and WBC count≥10/mL or a positive CSF culture were included into the study, except those for whom the quantity of CSF remaining after all routine tests were completed was insufficient for ICT testing (211 cases in Bangladesh, 5 in Burkina Faso, 7 in Nigeria and 37 in Pakistan). We enrolled 1173 patients of which 859 were under 5 years of age: 415 in Bangladesh (<5: 358, 86%), 354 in Burkina Faso (<5: 121, 34%), 78 in Kenya (<5: 68, 87%), 37 in Nigeria (<5: 37, 100%) and 289 in Pakistan (<5: 275, 95%). Infants (i.e., age less than 1 year) represented 65% to 73% of children age <5 years enrolled in Bangladesh, Kenya and Nigeria, compared to 34% in Burkina Faso and 52% in Pakistan. Of all subjects, 61% were male: 55% in Burkina Faso, 58% in Pakistan, 62% in Kenya,60% in Nigeria, and 69% in Bangladesh. Enrollment followed no clear seasonal patterns at any of the sites except for Burkina Faso, which lies in the African meningitis belt and, consistent with historical patterns, experienced a sharp rise in cases from December 2006 to April 2007, with peak recruitment in March.
In Burkina Faso, a majority (61%) of patients had a WBC count ≥1000/mL, whereas most patients had WBC counts <100/mL at the four other sites [Figure 1]. The proportion of CSF specimens that were purulent ranged from 44.6% in Bangladesh to 81.6% in Burkina Faso. The distribution of WBC count was similar across age groups at all sites (data not shown).
The prevalence of prior antibiotic use as detected by CSF testing for antimicrobial activity varied from 12% in Pakistan to 53% in Bangladesh, and ranged from 30% to 35% at all three African sites. Prior antibiotic use prevalence did not vary by age group in Burkina Faso, Nigeria or Pakistan. It was significantly higher in infants than in older children in Kenya (39% vs. 11%, p=0.02) and Bangladesh (60% vs. 36%, p<0.001). Prior antibiotic use increased with WBC count in Bangladesh (WBC=10-99: 48%, WBC=100-999: 53%, WBC≥1000: 69%, p=0.02); no significant trends were found at other sites.
Of the 1168 CSF cultures, 209 (17.9%) were positive for a bacterial organism: 19 were H. influenzae (9.1%), 69 S. pneumoniae (33.0%), 106 N. meningitidis (50.7%) (all but one coming from Burkina Faso) and 15 other pathogens (7.2%) [Table 2]. Overall culture isolation rates were 11.1% in infants (range: 3.1%-41.5%), 16.6% in 1 to 4 year-olds (range: 2.3%-57.5%), 30.6% in 5 to 14 year-olds (range: 0.0%-53.2%) and 30.9% in patients 15 years or older (Burkina Faso only). Yields were highest in Burkina Faso, where elevated rates of meningococcal meningitis were observed between December and April 2007, coinciding with the epidemic meningitis season. In Africa, 39.9% of CSF specimens yielded an etiological agent on culture compared to 3.2% in Asia. Pneumococcus was identified by culture in 13.0% of CSF specimens in Africa and 1.1% of specimens in Asia (13.3% and 1.3% among children age <5 years, p<0.001) [Table 3].
The addition of latex agglutination greatly increased Hib and pneumococcal detection in both Asian countries but had limited benefit over culture in African countries [Table 2]. With the addition of latex agglutination, pneumococcus was identified from 16.0% of subjects in Africa and 6.3% in Asia (16.4% and 6.5% among children age <5 years, p<0.001) [Table 3].
The proportion of patients with a positive ICT ranged from 16.2% to 20% depending on the geographic site considered (p=0.96 for homogeneity). Using CSF culture as the gold standard, the ICT was positive in 98.6% (95% confidence interval (CI): 92-100%) of patients with a culture positive for pneumococcus and was negative in 99.3% (95% CI: 96-100%) of patients with a culture positive for another pathogen. The ICT greatly increased the detection of pneumococcus over culture alone in Asian but not in African sites (p<0.001) [Table 3 and Figure 2]. Differences between regions were strongest in patients with elevated white blood cell count (WBC≥100/mL) or without prior antibiotic use.
The ICT identified pneumococci in 15.8% (95% CI: 13.5-18.2%) of culture-negative CSF specimens; this proportion varied by prior antibiotic use (24.2% with prior antibiotics vs. 12.2% without, p<0.001), region (8.5% in Africa vs. 18.8% in Asia, p<0.001), and WBC count (9.0% for WBC=10-99/mL, 22.1% for WBC=100-999/mL and 25.4% for WBC≥1000/mL; p<0.001 for trend).
Regional differences persisted after stratifying by prior antibiotic use and WBC count. Among culture-negative, antibiotic-negative cases, the ICT identified pneumococcus in 1.1% of children <5 years of age in Africa and in 15.1% in Asia (p<0.001). Comparable values in patients 5 years old and above were 5.5% and 34.6% (p<0.001). Similar trends were seen in culture-negative, antibiotic-positive cases, though the regional differences were not statistically significant in either age group (all ages: 19.4% in Africa vs. 26.4% in Asia, p=0.19). Among culture-negative patients with a WBC count of 10-99/mL, the ICT was positive in 5.1% of cases in Africa and 10.1% in Asia (p=0.10), compared to 9.7% and 26.4% for WBC counts of 100-999/mL (p=0.01) and 11.7% vs. 39.8% for WBC counts ≥1000/mL (p<0.001). These values did not vary by age group.
Although the ICT identified pneumococci for a higher percentage of culture-negative CSF specimens as WBC count increased, the absolute number of newly identified pneumococcal infections did not vary by WBC count because of the greater number of children with lower CSF WBC counts: 46 pneumococcal meningitis cases were detected in the 10-99 WBC/mL group, compared to 54 in the 100-999 WBC/mL group and 51 in the ≥1000 WBC/mL group.
The ICT was positive in 99% (95% CI: 94-100%) of cases with an LA test positive for pneumococcus and negative in 100% (95% CI: 98-100%) of specimens with an LA test positive for Hib, meningococcus or other pathogens. In children <5 years old, 0% (95% CI: 0-5%) of latex-negative cases tested positive by ICT in Africa compared to 14.6% (95% CI: 11.6-17.9%) in Asia (p=0.001). For older children (5 to 14 years of age), these values were 3.7% (1 of 27) in Africa and 14.3% (9 of 63) in Asia (p=0.14). Too few adults were enrolled in Asia for meaningful comparisons within this age group.
Similar to the comparison with culture results, differences between regions were observed irrespective of prior antibiotic use or WBC count. Among patients pre-treated with antibiotics, 5.6% of latex-negative cases were positive by ICT in Africa versus 17.8% in Asia (p=0.07); among patients who were not pre-treated, these values were 6% and 14%, respectively (p=0.001). Among persons with a CSF WBC count of 10-99/mL, 0% of those who were LA-negative had a positive ICT in Africa compared to 9% in Asia (p<0.001); among persons with a CSF WBC of 100-999/mL, these values were 10.3% and 19.5% (p=0.24) while among those with a CSF WBC≥1000/mL, these values were 5.0% and 35.0% (p<0.001). The proportion of latex-negative specimens testing positive by ICT increased with WBC count (WBC=10-99/mL: 7.6%; WBC=100-999/mL: 18%; WBC≥1000/mL: 23.0, p<0.001 for trend). There were no differences in ICT case detection among latex-negative cases by prior antibiotic use (with antibiotics: 15.5%, without antibiotics: 11.4%, p=0.15).
When compared to culture and latex agglutination, the Binax NOW® S. pneumoniae immuno-chromatographic test was >99% sensitive for the diagnosis of pneumococcal meningitis and yielded no false-positive results in Hib or meningococcal cases in these five African and Asian settings. It identified additional pneumococcal cases among patients with a negative CSF culture and latex agglutination test, primarily in Asian sites. Compared to Africa, previous studies in Asia have reported a confirmed etiology – including pneumococcus – for a substantially lower proportion of suspected bacterial meningitis cases [1, 20-26]. However, in our study we found that the addition of the Binax ICT resulted in a similar proportion of meningitis cases due to pneumococcus at all sites, regardless of location in Asia or Africa. This occurred despite differences in patient population characteristics such as age distribution, prior antibiotic use and CSF WBC count, and in environmental characteristics such as Hib vaccine use, presence of meningococcal epidemics, prevalence of other meningitis-like syndromes, and HIV prevalence. These findings indicate that in Asia, where latex agglutination and culture failed to identify the majority of pneumococcal meningitis cases, the Binax ICT has a role in disease burden and vaccine impact evaluations, as well as in clinical management of meningitis patients. In Africa, the ICT may also prove valuable in settings without adequate staff or laboratory capacity to perform culture and latex agglutination.
ICT yields in culture-negative, latex-negative and all specimens increased with CSF WBC count. This supports the contention that the ICT is identifying true cases of pneumococcus and not yielding false positives, which is further supported by negative test results with culture- and latex-proven non-pneumococcal cases. Despite the higher percentage of CSF with pneumococcus identified among patients with higher WBC count, our study found that the total number of pneumococcal cases identified remained similar at all categories of CSF WBC count evaluated (at least 10 per mm3). One implication of this finding is that studies focusing only on purulent meningitis, usually defined as at least 100 WBCs per mm3, will miss a substantial proportion of pneumococcal disease burden.
ICT use led to greater increases in pneumococcal yields compared to culture alone in patients with prior antimicrobial treatment than in those without, regardless of region. Patients who have received antibiotics prior to lumbar puncture are less likely to have viable organisms in their CSF for isolation on culture, enhancing the value of non-culture based tools such as latex agglutination and the Binax ICT in this group.
In the African sites, the ICT provided little benefit over culture and even this small advantage disappeared when latex agglutination was added. The increased utility of the ICT in Asia suggests an underperformance of culture and latex agglutination in that region. There are several possible explanations for this finding. Our study was conducted in two urban centers of Asia where children may access care earlier in the course of disease, including presenting to a hospital and receiving a lumbar puncture, than do children at the three African sites. The prevalence of previous antibiotic use did not differ by site. Nevertheless, differences in the availability, timing, type and dosing of antibiotics and patient compliance may lead to higher CSF antibiotic levels in Asia; the current study could not evaluate this hypothesis. These differences in access to care and antibiotic use patterns could lead to lower antigenic loads in Asia, limiting the ability of culture or latex agglutination to detect pneumococcus. Regardless of the reason, it appears that in sites with good laboratory facilities and highly trained microbiologists, a majority of cases can be identified currently by conventional methods alone in Africa, whereas more sensitive tools such as the Binax ICT are needed in Asia. These findings may not hold in non-research settings with limited laboratory facilities and poorly trained staff. Results also may change over time as meningitis epidemiology changes.
Epidemiological studies in Africa have consistently documented high incidence rates of laboratory-confirmed bacterial meningitis [1, 6, 26-29], but most studies conducted in Asia have shown very low rates [20, 21, 30, 31]. Our results suggest that differences in bacterial meningitis incidence between Asia and Africa – as determined by traditional surveillance approaches – may result in part from differences in detection using current standard methods. In contrast to surveillance studies from Asia, vaccine-probe studies (i.e., the use of vaccine in a randomized controlled trial to evaluate disease burden rather than vaccine efficacy) conducted in Indonesia  and Bangladesh  found very high incidences of bacterial meningitis. For example, the former found an estimated bacterial meningitis incidence ten-fold higher using the probe study design compared to that estimated from use of latex agglutination and culture test results. Our study sheds light on why these results occurred. We recommend that the ICT be used as an adjunct to culture in pneumococcal meningitis surveillance projects in Asia to ensure optimal case identification. In Africa, the ICT may be useful in settings with poor laboratory capacity or very high levels of antibiotic use. However, the ICT cannot replace latex agglutination in either of these settings, since commercially available latex agglutination kits test for Hib and meningococcus in addition to pneumococcus, which is particularly useful in the African meningitis belt.
The authors wish to thank Faisal Imram, Désiré Ilboudo, Maxime Kienou, Régina Idohou, Shazia Azeem and Kirimi Anampiu for help with data collection and data management.
This work was sponsored by the PneumoADIP at Johns Hopkins University. The PneumoADIP is funded in full by the GAVI Alliance and The Vaccine Fund.