Development for Clinical Use of a Multiplexed Immunoassay Using Sputum Samples for Streptococcus pneumoniae: a Non-Culture-Based Approach for Serotype-Specific Detection

ABSTRACT The multiplexed immunoassay (MIA) is an automated, monoclonal antibody-based serotyping assay that uses culture lysates of Streptococcus pneumoniae. This study describes the development and validation of applying MIA directly to sputum samples for the serotype-specific detection of S. pneumoniae. Sputum optimization involved liquefaction and fractionation. The subjects included 173 adult patients from whom both pneumococcal isolates cultured from sputum samples and the corresponding sputum samples were available at the Korea University Hospital from March 2012 to June 2015. Pneumococcal lysates and the sputum fraction were separately evaluated by MIA with a set A reaction to identify 27 serotypes (24 vaccine serotypes and serotypes 6C, 6D, and 11E). MIA results were validated by multiplex PCR (mPCR). Among the 173 patients analyzed, the pneumococcal isolate MIA detected a single set A serotype in 104 patients, and the corresponding sputum MIA showed concordant results with additional multiple serotypes in 21 patients. For the remaining 69 patients whose pneumococcal isolates were not determined to be set A serotypes by the pneumococcal isolate MIA, the corresponding sputum MIA identified additional set A serotypes (single serotypes, n = 17; multiple serotypes, n = 4). Serotypes 3 and 11A/D/F were the most commonly detected serotypes in both the pneumococcal isolate and sputum MIA analyses. However, serotype 8 was the most prevalent serotype detected only by the sputum MIA. The results of mPCR, performed for validation, showed a high concordance with the results of the sputum MIA. In conclusion, MIA using sputum samples enables the accurate, rapid, direct, and serotype-specific detection of S. pneumoniae, which may improve postvaccination serotype surveillance.

Streptococcus pneumoniae capsule determines disease severity in experimental pneumococcal meningitis

Streptococcus pneumoniae bacteria can be characterized into over 90 serotypes according to the composition of their polysaccharide capsules. Some serotypes are common in nasopharyngeal carriage whereas others are associated with invasive disease, but when carriage serotypes do invade disease is often particularly severe. It is unknown whether disease severity is due directly to the capsule type or to other virulence factors. Here, we used a clinical pneumococcal isolate and its capsule-switch mutants to determine the effect of capsule, in isolation from the genetic background, on severity of meningitis in an infant rat model. We found that possession of a capsule was essential for causing meningitis. Serotype 6B caused significantly more mortality than 7F and this correlated with increased capsule thickness in the cerebrospinal fluid (CSF), a stronger inflammatory cytokine response in the CSF and ultimately more cortical brain damage. We conclude that capsule type has a direct effect on meningitis severity. This is an important consideration in the current era of vaccination targeting a subset of capsule types that causes serotype replacement.

Trimethoprim-Sulfamethoxazole Prophylaxis and Antibiotic Nonsusceptibility in Invasive Pneumococcal Disease

ABSTRACTAmong 5,043 invasive pneumococcal disease (IPD) isolates identified through South African national surveillance from 2003 to 2007, we estimated the effect of trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis on antimicrobial resistance. Patients on TMP-SMX prophylaxis were more likely to have a pneumococcal isolate nonsusceptible to TMP-SMX, penicillin, and rifampin. TMP-SMX nonsusceptibility was associated with nonsusceptibility to penicillin, erythromycin, and rifampin and multidrug resistance. This study informs empirical treatment of suspected IPD in patients with a history of TMP-SMX use.

Site-Specific Mutagenesis Analysis of PBP 1A from a Penicillin-Cephalosporin-Resistant Pneumococcal Isolate

ABSTRACT Our mutagenesis study has investigated all amino acid mutations in the penicillin-binding domain of PBP 1A from Hungarian pneumococcal isolate 3191 to determine the importance of every mutation in the development of penicillin and cefotaxime resistance. Our data reveal that mutations at amino acid positions 574 to 577 and position 539 cause penicillin and cefotaxime resistance.

Detection of pneumococci in respiratory secretions: clinical evaluation of gentamicin blood agar

The use of sheep blood agar containing 5 microng of gentamicin per ml has been suggested as a means of selectively isolating Streptococcus pneumoniae from respiratory secretions. We have tested this method, in parallel with standard methods, on 844 respiratory specimens in a clinical laboratory and have confirmed that the yield of pneumococci can be increased approximately 40% by using agar containing gentamicin. However, since the antibiotic suppresses the growth of staphylococci, group A streptococci, and gram-negative bacilli, gentamicin agar cannot be used as a replacement for the standard method. The requirement for duplicate plating raises the cost per additional pneumococcal isolate to prohibitive amounts. Although the method is useful in studies designed to isolate only pneumococci, it cannot be recommended for the routine clinical laboratory. An unanticipated observation from our study is that the yield of pneumococci in respiratory secretions can be increased 10-fold simply by screening sputum for the presence of leukocytes using the Gram stain. This is in agreement with results reported from other laboratories.