Evaluation of Haemophilus influenzae type b carrier status among children 10 years after the introduction of Hib vaccine in Brazil

The aim of the present study was to assess the prevalence of Haemophilus influenzaetype b (Hib) nasopharyngeal (NP) colonisation among healthy children where Hib vaccination using a 3p+0 dosing schedule has been routinely administered for 10 years with sustained coverage (> 90%). NP swabs were collected from 2,558 children who had received the Hib vaccine, of whom 1,379 were 12-< 24 months (m) old and 1,179 were 48-< 60 m old. Hi strains were identified by molecular methods. Hi carriage prevalence was 45.1% (1,153/2,558) and the prevalence in the 12-< 24 m and 48-< 60 m age groups were 37.5% (517/1,379) and 53.9% (636/1,179), respectively. Hib was identified in 0.6% (16/2,558) of all children in the study, being 0.8% (11/1,379) and 0.4% (5/1,179) among the 12-< 24 m and 48-< 60 m age groups, respectively. The nonencapsulate Hi colonisation was 43% (n = 1,099) and was significantly more frequent at 48-< 60 m of age (51.6%, n = 608) compared with that at 12-< 24 m of age (35.6%, n = 491). The overall resistance rates to ampicillin and chloramphenicol were 16.5% and 3.7%, respectively; the co-resistance was detected in 2.6%. Our findings showed that the Hib carrier rate in healthy children under five years was very low after 10 years of the introduction of the Hib vaccine.

Avaliação da PCR-TR na detecção de Haemophilus influenzae em amostras de secreção de nasofaringe de crianças saudáveis / Evaluation of Real-Time PCR assay for Haemophilus influenzae detection in Nasopharyngeal Samples from Healthy Children

Haemophilus influenzae (Hi) é um microrganismo que faz parte da microbiota transitória da nasofaringe humana e, eventualmente, pode causar doenças em indivíduos suscetíveis. A vacina Hib conjugada além de proporcionar uma proteção direta contra a doença nos vacinados, também reduz a colonização da nasofaringe pelas cepas vacinais. Os estudos de portador de Hi na população permitem a caracterização das cepas circulantes, portanto a identificação correta do microrganismo é de fundamental importância para estimar com acurácia o efeito da vacina. A técnica padrão-ouro para detectar Hi em material clínico é a cultura, um método específico, de sensibilidade variável e que demanda maior tempo para a completa identificação do microrganismo. O uso de técnicas moleculares tem auxiliado na diferenciação de Hi de outras espécies do gênero Haemophilus, devido à alta sensibilidade e especificidade para detectar o agente etiológico. Diferentes ensaios de PCR-TR e PCR foram desenvolvidos para o diagnóstico de Hi utilizando diferentes genes alvo específicos como o gene hpd e o gene fucK. Estes genes são altamente conservados e permite a detecção de Hi capsulado e não capsulado. O principal objetivo deste estudo foi avaliar a acurácia da PCR-TR utilizando o marcador molecular hpd#3 para detectar o Hi na secreção de nasofaringe de crianças saudáveis comparando-a com a cultura. Um total de 410 amostras de secreção de nasofaringe estocadas a -70ºC em meio STGG foi selecionado aleatoriamente para esta avaliação. Pela PCR-TR, 161 (39,2%) amostras foram positivas para o gene hpd e 249 (60,8%) negativas. Pela cultura, 166 (40,5%) amostras foram positivas para Hi e 244 (59,5%) culturas negativas. Sendo assim, a PCR-TR apresentou uma sensibilidade de 90,9% (95% IC: 85,3-94,7) e especificidade de 95,9% (95% IC: 92,4-97,9) quando comparada à cultura para detectar Hi...

Haemophilus influenzae (Hi) colonizes the human nasopharynx and eventually can cause diseases in susceptible individuals. The Hib conjugate vaccine provides direct protection against the diseases and also reduces nasopharyngeal colonization by vaccine strain. Hi carriage studies allow the knowledge of circulating strains and the accurate identification of the microorganism is important to estimate the effect of the vaccine. Culture is the gold standard method to detect Hi in clinical samples, this is a specific method, with variable sensitivity and demand more time for the complete identification of the microorganism. The use of molecular techniques has helped in differentiating Hi from other species of the Haemophilus spp. due to the high sensitivity and specificity to detect the etiologic agent. Different RT-PCR and PCR assays were developed for the diagnosis of Hi using several biomarker genes such as hpd and fucK gene. These genes are highly conserved and are present in encapsulated and non-encapsulated Hi strains, allowing the detection of both variants. The aim of this study was to evaluate the accuracy of RT-PCR to detect Hi in nasopharyngeal samples of healthy children vaccinated against Hib comparing it with the culture. A total of 410 nasopharyngeal swabs stored at -70°C in STGG medium were randomly selected to evaluate RT-PCR assay targeting protein D (hpd#3). Considering the 410 nasopharyngeal swabs, 161 (39.2%) samples were positive for Hi and 249 (60.8%) had negative RT-PCR. By culture, 166 (40.5%) samples were positive for Hi and 244 (59.5%) were negative. Thus, the hpd#3 RT-PCR showed a sensitivity of 90.9% (95% CI: 85.3 - 94.7) and a specificity of 95.9% (95% CI: 92.4 - 97.9) when compared to culture to detect Hi in nasopharyngeal swabs. The results showed no significant difference (McNemar's chi-square = 0.64 p>0.5) between RT-PCR and culture and the Kappa coefficient showed an excellent agreement (0.873) between the two techniques...