Development and evaluation of serotype-specific PCRs for detection of Streptococcus pneumonia in clinical specimens

Pneumococcal disease is a serious disease that causes sickness e.g. bacterial pneumonia (an infection of the lung) or meningitis (an infection of the covering of the brain) and death. Children younger than two years are at highest risk for serious disease. There are roughly 90 different types of pneumococcal bacteria and these can spread from person to person through close contact. The available pneumococcal conjugate vaccine for use in preventing pneumococcal disease in infants and toddlers can work only on seven of the ninety types. In addition, there are reports that the use of this seven-valent vaccine will surely kill the seven types of bacteria but there are concerns that the disease could re-emerge if children were infected with the other types of pneumococcal not covered in the original vaccine. Consequently, monitoring children who had received the seven-valent vaccine for possible replacement is very important. We are therefore developing new sensitive laboratory methods for distinguishing the different types of pneumococcal in clinical samples.

Pneumococcal conjugate vaccines are efficacious against invasive pneumococcal disease in young children (Black 2000) and have an effect against carriage of pneumococci (Klugman 2001). There are reports of increased rates of colonization by non-vaccine serotypes after vaccination (Obaro, 1996; Sprat, 200) and concerns have been expressed about possible consequences of serotype replacement in vaccinated communities (Obaro, 2002). Long term effects of vaccines that project against some and not all of pneumococcal serotypes are still uncertain. Therefore, it is important to have continuing surveillance of pneumococcal serotypes from invasive and carriage sources. Current methods are insensitive for laboratory diagnosis of pneumococcal disease, particularly pneumonia and are grossly inadequate for studying colonization by multiple serotypes of Streptococcus pneumoniae. True carriage rates of multiple serotypes are yet not known because the methods for detecting multiple serotypes have relied mainly on picking several colonies directly from culture place for serotyping. We recently developed a two-step multiplex PCR assay, which is capable of distinguishing 9 pneumococcal serotypes commonly found in The Gambia. We now propose to (1) test strains of other bacterial species and other pneumococcal serotypes for confirmation of specificity and sensitivity of developed serotype specific primers and (2) do a formal evaluation of the PCR assay for direct detection of pneumococcal sterotypes in clinical specimens.