Incorporation of real-time PCR into routine public health surveillance of culture negative bacterial meningitis in São Paulo, Brazil

Abstract

Real-time (RT)-PCR increases diagnostic yield for bacterial meningitis and is ideal for incorporation into routine surveillance in a developing country. We validated a multiplex RT-PCR assay for Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae in Brazil. Risk factors for being culture-negative, RT-PCR positive were determined. The sensitivity of RT-PCR in cerebrospinal fluid (CSF) was 100% (95% confidence limits, 96.0%-100%) for N. meningitidis, 97.8% (85.5%-99.9%) for S. pneumoniae, and 66.7% (9.4%-99.2%) for H. influenzae. Specificity ranged from 98.9% to 100%. Addition of RT-PCR to routine microbiologic methods increased the yield for detection of S. pneumoniae, N. meningitidis, and H. influenzae cases by 52%, 85%, and 20%, respectively. The main risk factor for being culture negative and RT-PCR positive was presence of antibiotic in CSF (odds ratio 12.2, 95% CI 5.9-25.0). RT-PCR using CSF was highly sensitive and specific and substantially added to measures of meningitis disease burden when incorporated into routine public health surveillance in Brazil.

Figure 1. Proportion of culture-negative and culture-unknown 481 CSF specimens that were RT-PCR positive, by cerebrospinal fluid (CSF) white blood cell (WBC) count (leukocytes/mm³).

Numbers above bars represent the number of specimens tested. Includes 188 patients with unknown culture results.

Figure 2. Proportion of culture-negative and culture-unknown 452 CSF specimens that were RT-PCR positive, by cerebrospinal fluid (CSF) percent neutrophils.

Numbers above bars represent the number of specimens tested. Includes 188 patients with unknown culture results.

Figure 3. Relationship between minimum CSF WBC count used as a cut-off to determine what specimens are tested by RT-PCR and 1) the proportion of the total RT-PCR positives (n = 122) that are identified (dashed line) and 2) the proportion of all specimens (n = 481) that would be tested (solid line).

For example, if only those specimens with a CSF WBC of at least 1000 had been tested, about 34% of the 481 CSF specimens would have been tested, which would have detected about 92% of the 122 specimens that were RT-PCR positive. Data were censored at a CSF WBC cut-off of 10,000. Data are for culture-negative and culture unknown specimens. Includes 188 patients with unknown culture results.