EuroPneumo Special Issue / pneumonia 2015 Oct 21;7:I–72

not possible to differentiate between serotypes that that are caused by protein truncation due the testing method (e.g.

9A and 9V). In addition to pure bacterial culture, identification of serotype direct from extracted DNA of nasopharyngeal

and ear swabs as well as serum samples is our next target to ensure rapid diagnosis can be achieved. Within 24 hours of

sample acquirement, up to 96 samples can be tested simultaneously before analysis and interpretation with a software

program. Furthermore, we aim to be able to identify multiple pneumococcal serotypes from a single sample to reflect

multi-serotype pneumococcal carriage. Overall, our system is suitable for high throughput analysis of pneumococcal

samples for both the research and medical communities.


A novel multiplex real-time PCR assay for identifying and typing of

bacterial meningoencephalitis pathogens: development and validation

on clinical samples

Milen Milenkov 1 , Mala Rakoto Andrianarivelo 2 , Mickael Harioly Nirina 2 , Lalaina Rahajamanana 3 ,

Angelot Rakotomalala 2 , Liliane Raboba 3 , Annick Robinson 3 , Mélina Messaoudi 1 , Jean-Noël Telles 1 ,

Hubert Endtz 1 , Bénédicte Contamin 1 , Glaucia Paranhos-Baccalà 1


Fondation Mérieux, Lyon, France; 2 Centre d’Infectiologie Charles Mérieux, Antananarivo, Madagascar; 3 CHU Mère-Enfants Tsaralalàna,

Antananarivo, Madagascar

Bacterial meningoencephalitis is a major public health concern especially in developing countries. Common identification

techniques are time consuming as they require samples culture and often lack sensitivity. Here, we describe a rapid,

two-step multiplex real-time PCR method for identifying and typing Neisseria meningitidis, Streptococcus pneumoniae,

and Haemophilus influenzae type b. The first step of the test is a quadruplex, real-time PCR, targeting highly conserved

and species specific genes for S. pneumoniae, N. meningitidis, H. influenzae type b, and Streptococcus zooepidemicus

(internal control). The second step consists of 2 multiplex PCR assays for the identification of the 42 most prevalent S.

pneumoniae serotypes (1, 2, 3, 4, 5, 6A/B, 6C, 7F, 8, 9N/L, 9V, 10A, 10F, 11A, 12F, 13, 14, 15B/C, 16F, 18A/B/C /F, 19A,

19F, 21, 22F, 23A, 23B, 23F, 24F, 31, 33F, 34, 35A, 35B, 35F, 38, 39) and for meningococcal serogroups A, B, C, W135, X

and Y. The analytical specificity of all primers/probe pairs has been validated on laboratory strains, representative of

each serotype/serogroup targeted by the assay, as well as on closely related members of the genera Streptococcus,

Haemophilus, and Neisseria. The test was then used to analyse 252 samples of cerebrospinal fluid from patients with

suspected meningoencephalitis in Antananarivo, Madagascar. Forty-two (17%) were positive for S. pneumoniae and we

were able to determine the serotype of 35 of them (83%). N. meningitidis serogroup W135 was found in 1 sample and

another was positive for N. meningitidis serogroup C. The major advantage of the proposed molecular assay resides in

the possibility to perform a direct analysis of a clinical sample, thus providing results within a couple of hours. It can be

also used for monitoring epidemiological trends and serotype replacement upon vaccine introduction.


Evolution of Streptococcus pneumoniae serotype 14 in Catalonia,

Spain, after the introduction of 13-valent pneumococcal conjugate

vaccine (PCV13) (2010–2014)

Cristina Esteva 1 , Eva del Amo 1 , Paula Gassiot 2 , Xavier Raga 3 , Carmina Sanjose 4 , Mar O Perez 5 ,

Pilar Ciruela 6 , Marina Cunquero 1 , Irene Latorre 1 , Carmen Muñoz-Almagro 1 , Catalan Study Group

Invasive Pneumococcal Disease 6


Molecular Microbiology Department, Universitary Hospital Sant Joan de Déu, Esplugues de Llobregat, Barcelona, Spain; 2 Microbiology Department,

Figueres Hospital, Figueres, Girona, Spain; 3 Microbiology Department, Sant Pau y Santa Tecla Hospital, Tarragona, Spain; 4 Microbiology Department,

Alt Penedes Hospital, Vilafranca del Penedes, Spain; 5 Microbiology Department, Verge de la Cinta Hospital, Tortosa, Spain, 6 Public Health Agency,

Government of Catalonia, Barcelona, Spain

Serotype 14 (S14) has been considered one of the main serotypes causing invasive pneumococcal disease (IPD). Since

2001, conjugate vaccines are available to prevent IPD caused by different serotypes included S14. Since 2010, PCV13

is the vaccine recommended in children in our region, but it is estimated only 50% are vaccinated. Our aim was to

pneumonia 2015 Volume 7


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