Multilocus Sequence Typing Directly on DNA from Clinical Samples and a Cultured Isolate To Investigate Linked Fatal Pneumococcal Disease in Residents of a Shelter for Homeless Men

Abstract

Two apparently linked fatal cases of pneumococcal meningitis were investigated. Pneumolysin PCR performed on blood and cerebrospinal fluid was positive in a culture-negative case. A second case yielded Streptococcus pneumoniae from blood culture. Multilocus sequence typing performed on DNA extracted from case 1 (specimens) and case 2 (isolate) revealed identical sequence types (ST53) substantiating the link between them. CASE REPORTTwo male residents of a shelter for homeless people, each with a history of excess alcohol consumption, both developed febrile illnesses. Case 1, aged 39, had a 2-week history of cough prior to his admission to hospital with clinical evidence of meningitis. This diagnosis was confirmed by microscopic examination of the cerebrospinal fluid (CSF), which revealed gram-positive cocci. Blood culture and CSF culture were negative. Both CSF and an EDTA blood sample were positive for pneumolysin on multiplex PCR testing by the Health Protection Agency Meningococcal Reference Unit, Manchester, United Kingdom (5). Case 1 died 2 days after admission to hospital. Case 2, aged 37, presented with fitting and decreased level of consciousness the day after case 1 was admitted. Blood cultures grew Streptococcus pneumoniae serotype 8. The CSF showed gram-positive cocci on microscopy but was culture negative. He also died. Neither man had been immunized against pneumococcal infection. The close associates of the two cases were immunized using polysaccharide vaccine. No further cases were detected among residents of the shelter.Probable linkage of these cases was further investigated by multilocus sequence typing (MLST) (13) using the standard scheme for S. pneumoniae (7), which involves sequencing segments of seven housekeeping genes and can be undertaken directly on clinical samples (8) as well as on pneumococcal isolates. We used a modified method to amplify and sequence MLST targets from both the isolate and clinical samples. Modification of the published methods was necessary to overcome problems encountered using the Beckman-Coulter Genetic analysis system, due to differences in chemistries between this and the previously described sequencing system.

Presence of Nonhemolytic Pneumolysin in Serotypes ofStreptococcus pneumoniaeAssociated with Disease Outbreaks

“…Serotype 8 pneumococci have also been responsible for such outbreaks [25,40]. In the present study of 121 pneumococcal isolates, nonhemolytic pneumolysin variants were found only in serotypes that have been associated with outbreak disease.…”

“…In addition, serotype 1 pneumococci have a high attack rate and are rarely isolated from nasopharyngeal carriage [23,24]. Serotype 8 pneumococci have also been associated with linked cases of disease [25].…”

Serotypes and clones causing invasive pneumococcal disease before the use of new conjugate vaccines in Catalonia, Spain

“…In this baseline study we detected ST53 as the main clone found in IPD caused by serotype 8. This clone has been reported as a virulent clone 38 and at present is the dominant clone in serotype 8-related IPD in Scotland. 39 Jefferies et al found a pneumolysin allele 5 in ST53 that could facilitate clonal expansion of serotype 8, which would be of particular concern.…”

The clinical and public health value of non-culture methods in the investigation of a cluster of unexplained pneumonia cases

“…The samples were tested for the S. pneumoniae autolysin and pneumolysin genes using the method previously described for dual target testing of clinical samples [3]. Non-culture MLST was attempted on any dual-target PCR-positive samples following the method recently described by Birtles et al [4].…”