Polymerase chain reaction for diagnosis and serogrouping of meningococcal disease in children

Abstract

A prospective study was performed including all children younger than 18 years with the clinical diagnosis of invasive meningococcal disease (IMD) hospitalized at the University Hospital Sant Joan de Déu in Barcelona, Spain, from January 2001 to December 2006. During the study period, 168 meningococcal disease cases were reported. Microbiologic confirmation was obtained in 118 cases. Forty-six (38.9%) of 118 cases were only detected by polymerase chain reaction (PCR); 6 patients were culture positive and PCR negative (5%). Serogroup B predominated in the 6-year period with 83.1% of the strains. A significant decrease in serogroup C was observed in the last 3 years of the study (P = 0.029), and less common serogroups, such as serogroup A and W135, emerged. Serogroup distribution of patient diagnoses only by real-time PCR showed a similar distribution: serogroup B, 85.7%; serogroup C, 7.1%; and nontypeable serogroups, 7.1%. In conclusion, real-time PCR is more rapid and sensitive than culture for diagnosis and serogrouping of IMD.

Introduction

Invasive meningococcal disease (IMD) is an important public health problem because of its high morbidity and mortality rates. Invasive meningococcal disease is a global problem with endemic or epidemic patterns and seasonal variations. Although epidemics can occur in any part of the world, the “African meningitis belt” concentrates the largest number of meningococcal outbreaks. Nevertheless, several waves of IMD have been described in Europe, especially in the Republic of Ireland, United Kingdom, and Spain (Stephens, 2007, World Health Organization, 2003). Nowadays, in developed countries, mortality from meningococcal infection is approximately 10% (Morton and Swartz, 2004), and IMD is one of the main infectious causes of death in children. In the most affected countries of sub-Saharan Africa, attack rates vary from 100 to 800 cases per 100 000 population or higher during epidemic episodes (WHO, 2003). The rate of IMD in the European Union in 2005 varied from 0.31 cases per 100 000 in Poland, 2.32 cases per 100 000 in Spain, and 5.14 cases per 100 000 in the Republic of Ireland, with an estimated mean incidence of 1.3, approximately (WHO, 2007). Epidemiologic factors and the absence of uniformity in notification guidelines for IMD may explain the differences in number of cases reported. In addition, the difficulty of microbiologic confirmation of Neisseria meningitidis may underestimate the threat of the disease because sensitivity of bacterial culture can be low.

An IMD case can be classified as a confirmed case when clinically compatible disease is confirmed by laboratory, or as a suspected clinical case, with compatible signs but no microbiologic confirmation. The ideal situation for case definition would be always confirmation of the presence of the bacteria. Thus, owing to the heterogeneous declaration of meningococcal infection, there is no certainty that every reported case is a true one unless the pathogen is identified.

Conventional methods of microbiologic diagnosis, such as microscopy and culturing techniques, are becoming insufficient to confirm IMD. Meningococci can only be isolated from blood cultures in 50% of untreated patients with invasive meningococcal infection, and this rate drops to 5% when antibiotics are administered before patient admission (Cartwright et al., 1992). When the sample is cerebrospinal fluid (CSF), the sensitivity of the microscopy and culture in untreated meningitis patients is higher (80–90%), but prior antimicrobial therapy also reduces microbiologic confirmation (Bryant et al., 2004). Globally, the low sensitivity of these methods, which is about 40% to 63% (Bryant et al., 2004, Muñoz-Almagro et al., 2003, Richardson et al., 2003, Taha et al., 2005), and the delay in meningococci identification, which takes 2 or 3 days of culturing, make it necessary to establish more efficient techniques.

Molecular methods such as polymerase chain reaction (PCR)-based techniques allow reliable rapid diagnosis with higher sensitivity and specificity (Bryant et al., 2004, Muñoz-Almagro et al., 2003, Richardson et al., 2003, Taha et al., 2005). In addition, PCR is a very useful method in those cases in which antibiotic treatment has been administered before the collection of samples, because detection of the microorganism does not require viable bacteria. Real-time PCR, the latest technique in microbiologic molecular methods, detects and quantifies the DNA amplification at the end of each cycle through fluorescence emission. This technique enjoys all the advantages of traditional PCR while also being more sensitive and specific, yet without requiring additional procedures for detection of amplified products. In addition, PCR and real-time PCR are molecular techniques that permit meningococcal serogrouping and the development of surveillance systems of meningococcal infection with reliable epidemiologic data.

Serogroup classification of meningococcal strains is a useful tool for global management of invasive meningococcal infection in situations where vaccination might be possible (Carrol et al., 2000, Taha and Fox, 2007). To date, up to 13 N. meningitidis serogroups have been described, designated by capital letters. The 5 major meningococcal serogroups associated with disease are A, B, C, Y, and W-135. Currently available conjugate vaccines are effective in preventing disease caused by serogroups A, C, Y, and W-135, but vaccines that protect against the serogroup B disease are still in development. Furthermore, serogroup characterization allows monitoring of the trends of the disease. In this way, public health decisions and interventions in the management of outbreaks are based on the knowledge of previously reported cases.

We submit the contribution of PCR and real-time PCR toward the microbiologic confirmation and serogrouping of IMD in a pediatric population from January 2001 to December 2006.