Review
Current challenges in the accurate identification of Streptococcus pneumoniae and its serogroups/serotypes in the vaccine era

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Abstract

Streptococcus pneumoniae is a major cause of pneumonia, meningitis and other invasive diseases resulting in high mortality and morbidity among children under the age of five. Inaccurate identification of S. pneumoniae masks the exact estimation of disease burden and could delay treatment options. This is the common problem most frequently faced in developing countries due to several reasons that include poor infrastructure, insensitive operational procedures and lack of expertise. Inconsistent methods for phenotypic detection often delay the early identification and confirmation of S. pneumoniae. For serotyping S. pneumoniae, Quellung method is the gold standard which can be performed only on viable isolates, needs expertise and is expensive. Therefore, the data available on disease burden and serotype prevalence is not truly estimated in most of the developing countries, in turn, the use of available pneumococcal vaccines have been restricted. This current review deliberates an overview on advantages and limitations of routinely used phenotypic tests for S. pneumoniae identification. Also discussed in this review are the roles and current challenges faced by various molecular identification and serogroup/serotype identification methods of S. pneumoniae, including PCR, real time PCR, sequence analysis of different specific genes of S. pneumoniae, PCR combined with RFLP, MALDI-TOF, MLST, MLSA and WGS.

Introduction

Streptococcus pneumoniae belongs to the mitis group of streptococci that normally (Facklam, 2002) exists as a commensal flora of the upper respiratory tract (URT) (Gray et al., 1980). It is one of the major causes of acute otitis media, pneumonia and in its severe (invasive) form, causes bacteremia and meningitis (Advisory Committee on Immunization Practices, 2000, Musher, 1992) in children leading to high morbidity and mortality, especially in developing countries (CDC, 2013). Among the mitis group, other closely related species such as S. mitis, S. oralis, and S. pseudopneumoniae have also been associated with human disease (Bochud et al., 1994, Douglas et al., 1993, Keith et al., 2006, Rolo et al., 2013). Hence it is very crucial to identify them accurately for an early diagnosis and better treatment (Roberts et al., 1979, Bochud et al., 1994, Elting et al., 1992, Carratalá et al., 1995). Misidentification of causative agents not only affects the true burden of disease surveillance but also falsely increases the pneumococcal antimicrobial resistance rate since other closely related species such as S. mitis are associated with a high rate of penicillin and multidrug resistance (Simões et al., 2010, Wester et al., 2002). In addition, precise identification followed by serotyping is needed for the effective formulation of conjugate vaccine which is based on most prevalent serotypes. Development and implementation of an appropriate vaccine are always driven by proper laboratory identification of the disease causing agents. The current review discusses the different sides of routine culture-based identification of S. pneumoniae which involves insensitive bile solubility and optochin susceptibility testing. This review will also consider the uses and limitations of various molecular assays used for identifying and serotyping of S. pneumoniae.

Section snippets

The role of a conventional method for identification and serotyping

The routine culture-based identification of S. pneumoniae involves bile solubility and optochin susceptibility testing (Lund and Henrichsen, 1978). However, these tests are not completely reliable due to the reports that indicate the presence of bile insoluble (2%) (Richter et al., 2008), optochin resistant strains (10%) of S. pneumoniae (Kellogg et al., 2001). Moreover, the presence of similar biochemical properties in a significant proportion of other closely related Streptococcus spp. makes

Role of molecular methods

In the last decade, different molecular assays were developed using an array of pneumococcal specific targets, like - pneumolysin (ply) (McAvin et al., 2001), autolysin (lytA) (Corless et al., 2001), pneumococcal surface antigen A (psaA) (Morrison et al., 2000), manganese-dependent superoxide dismutase (sodA) (Kawamura et al., 1999), and penicillin binding protein (pbp) (O'Neill et al., 1999) to name a few. But subsequent detection of these pneumococcal targets in viridans group streptococci (

MALDI-TOF

MALDI-TOF Mass Spectrometry (MS) is considered as a rapid tool for identification of bacteria in diagnostic laboratories (Van Veen et al., 2010). MALDI-TOF can identify the streptococcal genus, but could not differentiate the pneumococci and the mitis group. Recent studies have reported selective peaks in MALDI-TOF-MS pattern of S. pneumoniae and S. mitis group using ClinProTools™. Ikryannikova et al. (2013) identified a specific peak of 6949 m/z in S. mitis isolates alone but not seen in S.

Methods for accurate identification of serogroup/serotype of S. pneumoniae from nasopharyngeal specimens

Surveillance of S. pneumoniae serogroup/serotype is important in both disease, carriage and vaccine strategies. Each serotype differs in their disease-causing potential, age group infected, and geographic distribution (Shouval et al., 2006; Scott et al., 1996, Hausdorff et al., 2005). The direct impact of a vaccine can be determined by the reduction in vaccine serotypes causing invasive disease. The first molecular method recommended by CDC for serotyping of S. pneumoniae isolates was

Enhanced capturing of multiple serogroup/serotype of S. pneumoniae from nasopharyngeal sample

Since S. pneumoniae is part of the normal flora of the nasopharynx, carriage studies help to determine the serotypes circulating in the population as well to determine serotype replacement after vaccination. Colonization can be with a single serotype or with more than one serotype. For multiple serotype detection, five colonies from the primary culture have to be serotyped in order to identify the additional serotype which is in 50% proportion. If the second serotype is in 25% proportion 11

Conclusion

The routine use of culture based identification such as bile solubility and optochin susceptibility is not reliable due to the presence of similar properties in closely related Streptococcus spp. of respiratory sites. To overcome insensitivity of phenotypic methods, various different molecular methods (such as real time and conventional PCR targeting ply, lytA, psaA and sodA) have been developed to accurately identify and differentiate S. pneumoniae from other closely related species.

Acknowledgement

The authors would like to thank Dr. R.S. Suresh Kumar Mr. James John and Mr. Francis Yesurajan for their constructive comments on the manuscript.

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