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Health Protection Agency, Respiratory and Systemic Infections Laboratory, Central Public Health Laboratory, London
Health Protection Agency, South East Region
South East Public Health Observatory
Health Protection Agency, Meningococcal Reference Unit, North West Regional Laboratory, Manchester, United Kingdom.
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 REPORT
Two 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.
DNA extraction from pneumococcal isolate. The S. pneumoniae isolate from case 2 was grown overnight on Columbia agar base (Oxoid, Basingstoke, United Kingdom) with the addition of 2.5% defibrinated horse blood. Colonies were suspended in saline, and 250 μl was heated at 95°C for 20 min on a QBT2 heating block (Grant Instruments Ltd., Cambridge, United Kingdom). The samples were snap chilled for 5 min at –20°C before centrifugation at 16,058 x g for 10 min in a Biofuge primo R (Kendro Laboratory Products, Hanau, Germany). The supernatant was used for PCR.
DNA extraction from clinical samples. The EDTA blood sample from case 1 was extracted with the QIAGEN DNA blood mini kit (QIAGEN, Crawley, United Kingdom) following the manufacturer's instructions for extraction of a 400-μl sample. The CSF sample from case 1 was extracted using the GenSpin kit (Whatman Biosciences, Maidstone, United Kingdom) following the manufacturer's instructions.
MLST target amplification. For clinical samples, each 50-μl reaction mixture for first round nested touchdown PCR contained 200 μM dATP, dGTP, dCTP, dTTP (Roche Diagnostics Ltd., Lewes, United Kingdom), 0.5 mM each primer (Table 1), 5 μl of 10X buffer (containing 15 mM MgCl2), 10 μl of Q-Solution, 5 μl of extracted sample, 9.5 μl of sterile water, and 0.5 μl (5 U/μl) of HotStarTaq DNA polymerase (QIAGEN, Crawley, United Kingdom). The second round nested PCR used the same reaction mixture but with second round primers (Table 1) and 5 μl of PCR product from the first round reaction. For the cultured isolate, each 50-μl PCR mixture contained 200 μM dATP, dGTP, dCTP, and dTTP (Roche Diagnostics Ltd., Lewes, United Kingdom), 1 mM each primer (Table 1), 1.5 mM MgCl2, 5 μl of 10X buffer, 1 μl of 1% W1, 0.5 μl (5 U/μl) of Taq DNA polymerase (all Invitrogen Life Technologies, Paisley, Scotland), 1 μl of extracted sample, and 21 μl of sterile water. Multiple PCR cycling conditions were used to amplify the seven MLST targets for both clinical sample and cultured isolate protocols (Table 2).
Sequencing analysis. PCR products were purified for sequencing using the MultiScreen PCR clean up plate (Millipore, Bedford, Mass.) in accordance with the manufacturer's instructions. Sequencing reactions were performed in 10-μl volumes with a CEQ Dye Terminator cycle sequencing Quick Start kit (Beckman Coulter, Fullerton, Calif.) utilizing the reduced volume protocol described by Azadan et al. (1). Thermal cycling conditions for the sequencing reaction and ethanol cleanup of sequencing products were carried out as per the manufacturer's instructions (Beckman Coulter, Fullerton, Calif.); the products were then run on a Beckman Coulter CEQ 8000 automated DNA sequencer. The sequences for the seven gene fragments of each sample were assembled from the resultant chromatograms using BioNumerics (Applied Maths, Belgium). Sequences were assigned allele numbers using the MLST website (http://www.mlst.net).
MLST results. MLST was successfully performed upon the two clinical samples from case 1 and the cultured isolate from case 2. The pneumococcal MLST allelic profiles obtained from the two cases were identical (2-5-1-11-16-3-14) and corresponded to sequence type 53 (ST53). Just 8 of the 2,285 strains recorded on the pneumococcal MLST database (at the time of manuscript submission) were ST53. These ST53 isolates were also reported to be serotype 8 and had been isolated from cases of bacteremia, pneumonia, or meningitis. The pneumococcal MLST database is intrinsically biased in that it does not include all strains ever typed or a representative sampling process but rather strains that were typed and voluntarily submitted to the database from a range of carriage and disease isolate collections. However, given that ST53 comprises just 0.35% of recorded strains, it is reasonable to conclude that this ST has been uncommon in the population even allowing for such biases. The presence of this identical and relatively infrequent sequence type in the two patients is strong supporting evidence for both men being infected by the same strain within a single transmission network.
MLST offers an unambiguous, discriminatory, and portable approach to bacterial characterization and typing based on the PCR amplification and sequencing of housekeeping gene fragments. This approach allows direct comparison of results between laboratories and enables allelic profiles to be easily examined for relationships to known clones using databases available on the internet (7, 21). A large outbreak of pneumococcal conjunctivitis with many culture-positive cases was also investigated with MLST (14). Our report is at the opposite extreme, with just two cases and an isolate on only one. It demonstrates the capacity of MLST to add to epidemiological understanding of infectious disease transmission at all levels of outbreak and emphasizes the possibility of definitive typing even in culture-negative patients. A full MLST profile was obtained from both blood and CSF samples of case 1, neither of which yielded an organism on culture. MLST of S. pneumoniae from samples in a culture-negative patient has not, to the best of our knowledge, been reported. The lack of culture-positive results from case 1 may be due to the fact that the blood and CSF samples were taken after administration of an antibiotic which would prevent a viable culture being obtained. However, bacterial DNA would still be present, allowing detection via PCR-based methodologies. The successful culturing of S. pneumoniae from a blood sample but not from a CSF sample in case 2 is harder to explain but may simply be due to sample size. It is relatively straightforward to obtain the 5 ml of blood from an adult required by most modern culturing systems; however, the amount of CSF obtained from a lumbar puncture can vary widely from approximately 10 μl to 1 ml. So it may be possible that in this instance only a very small amount of CSF, which didn't contain any viable organisms, was available for culturing purposes.
Chronic alcohol excess is a major risk factor for invasive pneumococcal disease, possibly the most important one among young and middle-aged adults (3, 15, 19). Altered risk associated with homelessness or living in shelters is less well established, although reports of outbreaks among these populations suggest that risk might be increased (6, 17). A range of social and biological factors, particularly a high smoking prevalence, among alcoholics and the homeless may further increase their risk (17-19). Improved investigation of sporadic and linked cases among these groups should increase understanding of the pneumococcal disease burden that they bear. Alcoholism is not considered an indication for immunization in the United Kingdom unless there is evidence of liver disease (20). This contrasts with other countries such as the United States, where these men would have met indications for pneumococcal immunization (4). There is no U.K. policy on pneumococcal immunization in shelters for the homeless.
The protective efficacy of pneumococcal vaccine may be decreased by chronic alcoholism (and by many other conditions that predispose to pneumococcal infection) (20). Since conjugate vaccines might prove more immunogenic in alcoholic and other individuals with increased risk (9, 16, 22), the questions will become more complicated, incorporating which vaccine to use as well as whether immunization is indicated. Serotype distributions affect the relative benefits of either approach. While MLST does not definitively determine the serotype of S. pneumoniae from a culture-negative individual, it is possible to predict potential serotypes by examining which serotypes correspond to which STs on the MLST website (8). There are molecularly based methods which directly determine the serotype/group of S. pneumoniae isolates (2, 11, 12), and it may be possible to adapt these for use on PCR-positive clinical samples where no organism has been cultured, increasing the information available to researchers and policy makers.
In conclusion, application of MLST directly on clinical samples as well as a cultured isolate supported epidemiological information that these cases were linked. The culture-negative case was therefore also likely to be serotype 8, which is covered by the currently available 23-valent plain polysaccharide vaccine. This report highlights the need for investment in an evidence base to support rational immunization policy for pneumococcal infection in population groups with increased risk of disease and indicates a role for MLST in addition to serotyping in contributing to this evidence base. It also describes the first reported application of pneumococcal MLST in an individual with no positive culture and highlights the possibilities for wider application of this approach in the public health management of pneumococcal disease.
ACKNOWLEDGMENTS
The authors thank Andrew Stacey, Microbiology Department, Royal Berkshire and Battle Hospital, Reading, United Kingdom, for facilitation of this work.
This publication made use of the multilocus sequence typing website (http://www.mlst.net) developed by Man-Suen Chan and David Aanensen and funded by the Wellcome Trust.
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