Trends in serotype distribution and antimicrobial susceptibility pattern of invasive Haemophilus influenzae isolates from Brazil, 2009-2021.

INTRODUCTION: Invasive Haemophilus influenzae (Hi) disease poses a significant global health challenge. With the relaxation of COVID-19 pandemic measures and declining H. influenzae serotype b (Hib) vaccination coverage, there is concern about a potential increase in Hi cases worldwide. METHODOLOGY: This study analyzed 1437 invasive Hi isolates in Brazil over 13 years, determining capsular serotypes, antimicrobial susceptibility, and genetic relatedness through multilocus sequence typing. RESULTS: The primary source of isolation for these invasive H. influenzae isolates was blood (54.4%), followed by cerebrospinal fluid (37.1%) and lung specimens (8.5%), respectively. Consequently, bacteremia (47%) was the most common clinical presentation, followed by meningitis (39.6%) and pneumonia (13.4%). Non-encapsulated Hi (NTHi) predominated among the isolates (51.4%), along with serotype a (22%) and serotype b (21.5%) among the encapsulated isolates. The majority of the encapsulated isolates were isolated from children under 14 years of age (76.7%), while NTHi isolates were identified in patients older than 15 years, particularly those ≥ 60 years old (40%). Ampicillin resistance was observed in 17.1% of cases, displaying ß-lactamase production as the principal resistance mechanism. MLST revealed a diverse NTHi population, whereas the encapsulated isolates presented a clonal structure. CONCLUSION: This study describes the prevalence of NTHi isolates circulating in Brazil after two decades of the Hib vaccine immunization program. Continuous universal surveillance is crucial for implementing prompt public health measures to prevent and control invasive Hi disease and monitor changes in antibiotic resistance profiles.

Genomic characterization of invasive meningococcal X isolates from Brazil, 1992–2022 / Caracterización genómica de aislamientos invasivos de meningococo X de Brasil, 1992–2022

Introduction: Invasive meningococcal disease (IMD) is a major health problem. Given the post-COVID-19 pandemic scenario with the loosening of the non-pharmacological measures to control the virus transmission and considering the observed global reduction of meningococcal vaccination coverage, an increase in IMD cases can be expected. Methodology: Using whole-genome sequencing, we characterized six Neisseria meningitidis serogroup X (MenX) isolates recovered from IMD cases in Brazil in the last 30 years. Results: The predominance (66.6%, 4/6) of ST2888 presenting fHbp 160, NHBA 129, NadA 21, and PorA 19,15 was found on isolates. Two novel STs, 15458 and 15477, were described. Conclusion: This study describes the circulation of MenX lineage ST2888 in Brazil, previously reported only in Europe. Continuous universal surveillance is crucial to implement prompt public health measures aiming to prevent and control non-vaccine preventable serogroup X IMD cases.(AU)

Emergence of Neisseria meningitidis W South American sublineage strain variant in Brazil: disease and carriage.

Introduction. Invasive meningococcal disease is a major health problem, impacting morbidity and mortality worldwide. Exploratory genomics has revealed insights into adaptation, transmissibility and virulence to elucidate endemic, outbreaks or epidemics caused by Neisseria meningitidis serogroup W (MenW) strains.Gap Statement. Limited information on the genomics of Neisseria meningitis serogroup W ST11/cc11 is available from emerging countries, especially in contemporary isolates.Aim. To (i) describe the antigenic diversity and distribution of genetic lineages of N. meningitidis serogroup W circulating in Brazil; (ii) study the carriage prevalence of hypervirulent clones in adolescents students and (iii) analyse the potential risk factors for meningococcal carriage.Methodology. Using whole-genome sequencing, we analysed the genomic diversity of 92 invasive N. meningitidis serogroup W isolates circulating in Brazil from 2016 to 2019. A cross-sectional survey of meningococcal carriage was conducted in 2019, in the city of Florianópolis, Brazil, among a representative sample of 538 students.Results. A predominance (58.5 %, 41/82) of ST11/cc11 presenting PorB2-144, PorA VR1-5, VR2-2, FetA 1-1, and a novel fHbp peptide 1241 was found on invasive N. meningitidis W isolates, on the other hand, a high diversity of clonal complexes was found among carriage isolates. The overall carriage rate was 7.5 % (40/538). A total of 28 of 538 swab samples collected were culture positive for N. meningitidis, including four serogroup/genogroup B isolates (14.8 %;4/27), 1 serogroup/genogroup Y isolate (3.7 %;1/27), 22 (81.5 %; 22/27) non-groupable isolates. No MenW isolate was identified among carriages isolates.Conclusion. This report describes the emergence of the new MenW ST11/cc11 South America sublineage variant, named here, 2016 strain, carrying a novel fHbp peptide 1241, but its emergence, was not associated with an increased MenW carriage prevalence. Continuous surveillance is necessary to ascertain the role of this sublineage diversification and how its emergence can impact transmission.

Emergence of Neisseria meningitidis W South American sublineage strain variant in Brazil: disease and carriage

Introduction. Invasive meningococcal disease is a major health problem, impacting morbidity and mortality worldwide. Exploratory genomics has revealed insights into adaptation, transmissibility and virulence to elucidate endemic, outbreaks or epidemics caused by Neisseria meningitidis serogroup W (MenW) strains.Gap Statement. Limited information on the genomics of Neisseria meningitis serogroup W ST11/cc11 is available from emerging countries, especially in contemporary isolates.Aim. To (i) describe the antigenic diversity and distribution of genetic lineages of N. meningitidis serogroup W circulating in Brazil; (ii) study the carriage prevalence of hypervirulent clones in adolescents students and (iii) analyse the potential risk factors for meningococcal carriage.Methodology. Using whole-genome sequencing, we analysed the genomic diversity of 92 invasive N. meningitidis serogroup W isolates circulating in Brazil from 2016 to 2019. A cross-sectional survey of meningococcal carriage was conducted in 2019, in the city of Florianópolis, Brazil, among a representative sample of 538 students.Results. A predominance (58.5 %, 41/82) of ST11/cc11 presenting PorB2-144, PorA VR1-5, VR2-2, FetA 1-1, and a novel fHbp peptide 1241 was found on invasive N. meningitidis W isolates, on the other hand, a high diversity of clonal complexes was found among carriage isolates. The overall carriage rate was 7.5 % (40/538). A total of 28 of 538 swab samples collected were culture positive for N. meningitidis, including four serogroup/genogroup B isolates (14.8 %;4/27), 1 serogroup/genogroup Y isolate (3.7 %;1/27), 22 (81.5 %; 22/27) non-groupable isolates. No MenW isolate was identified among carriages isolates.Conclusion. This report describes the emergence of the new MenW ST11/cc11 South America sublineage variant, named here, 2016 strain, carrying a novel fHbp peptide 1241, but its emergence, was not associated with an increased MenW carriage prevalence. Continuous surveillance is necessary to ascertain the role of this sublineage diversification and how its emergence can impact transmission.

Comparative performances of seven quantitative Reverse-Transcription Polymerase Chain Reaction assays (RT-qPCR) for detecting SARS-CoV-2 infection in samples from individuals suspected of COVID-19 in São Paulo, Brazil.

Introduction: Brazil is the second largest country with COVID-19 positive cases worldwide. Due to the potent spread of the virus and the scarcity of kits and supplies, the Brazilian Ministry of Health has granted authorization for the use of kits available during this emergency, without an accurate evaluation of their performance. This study compared the performance and cost-effectiveness of seven molecular assays/kits available in São Paulo, Brazil, for SARS-CoV-2 diagnosis. Materials and methods: A total of 205 nasopharyngeal/oropharyngeal samples from suspected cases of COVID-19, were tested using the following assays: (i) GeneFinder COVID-19 plus RealAmp kit; (ii) 2019-nCoV RNA PCR-Fluorescence Probing, Da An Gene Co.; (iii) in-house RT-qPCR SARS-CoV-2 IAL; (iv) 2019-nCoV kit, IDT; (v) molecular SARS-CoV-2 (E) kit, Bio-Manguinhos; (vi) Allplex 2019-nCoV modified Assay, Seegene Inc, and (vii) Biomol one-step COVID-19 kit, IBMP. The criteria for determining a SARS-CoV-2 true positive result included the cycle threshold cut-off values, the characteristics of exponential/linear curves, the gene target diversity, and a positive result in at least two assays. Results: The overall sensitivity of the assays listed were GeneFinder 83.6%, Da An Gene 100.0%, IAL 90.4%, IDT 94.6%, Bio-Manguinhos 87.7%, Allplex 97.3%, and IBMP 87.7%. The minor sensitive gene target was RdRP. Although all assays had a Cohen's Kappa index ≥0.893, the best tests used multiplex assays identifying N-gene and/or E-gene targets. Conclusion: All assays tested accurate for diagnosis, but considering cost-effectiveness (cost, time consumption, number of samples tested, and performance), the in-house IAL assay was ideal for COVID-19 diagnosis in São Paulo, Brazil.

Comparative performances of seven quantitative Reverse - Transcription Polymerase Chain Reaction assays (RT-qPCR) for detecting SARS-CoV-2 infection in samples from individuals suspected of COVID-19 in São Paulo, Brazil

Introduction Brazil is the second largest country with COVID-19 positive cases worldwide. Due to the potent spread of the virus and the scarcity of kits and supplies, the Brazilian Ministry of Health has granted authorization for the use of kits available during this emergency, without an accurate evaluation of their performance. This study compared the performance and cost-effectiveness of seven molecular assays/kits available in São Paulo, Brazil, for SARS-CoV-2 diagnosis Materials and methods A total of 205 nasopharyngeal/oropharyngeal samples from suspected cases of COVID-19, were tested using the following assays: (i) GeneFinder COVID-19 plus RealAmp kit; (ii) 2019-nCoV RNA PCR-Fluorescence Probing, Da An Gene Co.; (iii) in-house RT-qPCR SARS-CoV-2 IAL; (iv) 2019-nCoV kit, IDT; (v) molecular SARS-CoV-2 (E) kit, Bio-Manguinhos; (vi) Allplex 2019-nCoV modified Assay, Seegene Inc, and (vii) Biomol one-step COVID-19 kit, IBMP. The criteria for determining a SARS-CoV-2 true positive result included the cycle threshold cut-off values, the characteristics of exponential/linear curves, the gene target diversity, and a positive result in at least two assays Results The overall sensitivity of the assays listed were GeneFinder 83.6%, Da An Gene 100.0%, IAL 90.4%, IDT 94.6%, Bio-Manguinhos 87.7%, Allplex 97.3%, and IBMP 87.7%. The minor sensitive gene target was RdRP. Although all assays had a Cohen's Kappa index ≥0.893, the best tests used multiplex assays identifying N-gene and/or E-gene targets Conclusion All assays tested accurate for diagnosis, but considering cost-effectiveness (cost, time consumption, number of samples tested, and performance), the in-house IAL assay was ideal for COVID-19 diagnosis in São Paulo, Brazil.

Genomic surveillance of Neisseria meningitidis serogroup B invasive strains: Diversity of vaccine antigen types, Brazil, 2016-2018.

BACKGROUND: Neisseria meningitidis serogroup B remains a prominent cause of invasive meningococcal disease (IMD) in Brazil. Because two novel protein-based vaccines against serogroup B are available, the main purpose of this study was to provide data on the diversity and distribution of meningococcal vaccine antigen types circulating in Brazil. METHODOLOGY: Genetic lineages, vaccine antigen types, and allele types of antimicrobial-associated resistance genes based on whole-genome sequencing of a collection of 145 Neisseria meningitidis serogroup B invasive strains recovered in Brazil from 2016 to 2018 were collected. RESULTS: A total of 11 clonal complexes (ccs) were identified among the 145 isolates, four of which were predominant, namely, cc461, cc35, cc32, and cc213, accounting for 72.0% of isolates. The most prevalent fHbp peptides were 24 (subfamily A/variant 2), 47 (subfamily A/variant 3), 1 (subfamily B/variant 1) and 45 (subfamily A/variant 3), which were predominantly associated with cc35, cc461, cc32, and cc213, respectively. The NadA peptide was detected in only 26.2% of the isolates. The most frequent NadA peptide 1 was found almost exclusively in cc32. We found seven NHBA peptides that accounted for 74.5% of isolates, and the newly described peptide 1390 was the most prevalent peptide exclusively associated with cc461. Mutated penA alleles were detected in 56.5% of the isolates, whereas no rpoB and gyrA mutant alleles were found. CONCLUSION: During the study period, changes in the clonal structure of circulating strains were observed, without a predominance of a single hyperinvasive lineage, indicating that an epidemiologic shift has occurred that led to a diversity of vaccine antigen types in recent years in Brazil.

Nasopharyngeal carriage of Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus in a Brazilian elderly cohort.

We aimed to investigate the nasopharyngeal colonization (NPC) by Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus in the elderly population and to assess the demographic factors associated with NPC. This was an observational cohort study in which outpatients aged ≥60 years were enrolled from April to August 2017, with a follow-up visit from September through December 2017. Nasopharyngeal (NP) swabs were collected, bacteria were detected and isolated, and isolates were subjected to phenotypic and molecular characterization using standard microbiological techniques. At enrolment, the rates of S. aureus, methicillin-resistant S. aureus (MRSA), H. influenzae, and S. pneumoniae among 776 elderly outpatients were 15.9%, 2.3%, 2.5%, and 2.2%, respectively. Toxin production was detected in 21.1% of methicillin-susceptible S. aureus, and three SCCmec types were identified: II/IIb, IVa, and VI. At the follow-up visit, all carriage rates were similar (p > 0.05) to the rates at enrolment. Most of S. pneumoniae serotypes were not included in pneumococcal conjugate vaccines (PCVs), except for 7F, 3, and 19A. All strains of H. influenzae were non-typeable. Previous use of antibiotics and 23-valent pneumococcal polysaccharide vaccination (p < 0.05) were risk factors for S. aureus and MRSA carriage; S. aureus colonization was also associated with chronic kidney disease (p = 0.021). S. pneumoniae carriage was associated with male gender (p = 0.032) and an absence of diabetes (p = 0.034), while not receiving an influenza vaccine (p = 0.049) and chronic obstructive pulmonary disease (p = 0.031) were risk factors for H. influenzae colonization. The frailty of study participants was not associated with colonization status. We found a higher S. aureus carriage rate compared with the S. pneumoniae- and H. influenzae-carriage rates in a well-attended population in a geriatric outpatient clinic. This is one of the few studies conducted in Brazil that can support future colonization studies among elderly individuals.

Meningococcal Carriage among Adolescents after Mass Meningococcal C Conjugate Vaccination Campaigns in Salvador, Brazil.

Neisseria meningitidis is a commensal bacterium of the human nasopharynx. In rare cases, it penetrates the mucosa, entering the blood stream and causing various forms of disease. Meningococcal conjugate vaccines can prevent invasive disease not only by direct effect in vaccinated individuals but also by herd protection, preventing acquisition of carriage, which interrupts transmission and leads to protection of unvaccinated persons. In 2010 in Salvador, Brazil, an outbreak of group C meningococcal disease led to a mass meningococcal serogroup C conjugate vaccination drive, targeting those <5 and 10-24 years of age. The present study aimed to estimate the prevalence of and identify factors associated with N. meningitidis carriage among adolescents from Salvador, Brazil, in the post-vaccination period. In spring 2014, we performed a cross-sectional study involving 1,200 public school students aged 11-19 years old. Oropharyngeal swabs were collected to identify N. meningitidis. Of the 59 colonized participants, 36 (61.0%) carried non-groupable N. meningitidis, while genogroup B (11.9%), Y (8.5%), E (6.8%), Z (5.1%), C (3.4%), and W (3.4%) were also detected. The overall prevalence of N. meningitidis carriage was 4.9% (95% confidence interval [CI], 3.6-6.1%); the prevalence of N. meningitidis genogroup C was 0.17% (95% CI, 0.0-0.40%). There was no difference by age. Factors associated with carriage were having only one, shared, bedroom in the household (PR, 2.02; 95% CI, 0.99-4.12, p = 0.05); the mother being the only smoker in the home (PR, 2.48; 95% CI, 1.16-5.29; p = 0.01); and going to pubs/parties more than 5 times/month (PR, 2.61; 95% CI, 1.38-4.92; p = 0.02). Our findings show that the N. meningitidis carriage rate in adolescents from Salvador, Bahia, is low and is potentially influenced by the low prevalence of N. meningitidis genogroup C. However, continued surveillance is important to identify changes in the dynamics of N. meningitidis, including the emergence of diseases due to a non-C serogroup.

Gonococcal endocarditis: an ever-present threat.

The incidence of severe complications of the Neisseria gonorrhoeae infection has presented variations over recent decades since the advent of penicillin. Gonococcal endocarditis (GE) still remains an ever-present threat afflicting the society's poor and sexually active young population. This entity frequently requires surgical intervention and usually exhibits a poor outcome. The interval between the onset of symptoms and the diagnosis does not usually exceed 4 weeks. One of the characteristics of GE is a proclivity for aortic valve involvement with large vegetation and valve ring abscess formation. The authors report the case of a young man with a 2-week history of fever, malaise, weakness, and progressive heart failure symptoms, who had no previous history of genital complaints or cardiopathy. The physical examination was consistent with acute aortic insufficiency, which was most probably of an infectious origin. The echocardiogram showed thickened aortic cusps and valve insufficiency. After hospital admission, the patient's clinical status worsened rapidly and he died on the second day. The autopsy findings disclosed aortic valve destruction with vegetation and a ring abscess besides signs of septic shock, such as diffuse alveolar damage, acute tubular necrosis, and zone 3 hepatocellular necrosis. The blood culture isolated N. gonorrhoeae resistant to penicillin and ciprofloxacin. The authors call attention to the pathogen of this particular infectious endocarditis, and the need for early diagnosis and evaluation by a cardiac surgery team.

Prevalence, risk factors and molecular characteristics of meningococcal carriage among brazilian adolescents

BACKGROUND: In 2010, introduction of the meningococcal C conjugate vaccine in Brazil for children <2 years provided an immediate reduction in the incidence rates of disease among the age groups targeted for the vaccine, but no early impact was observed in unvaccinated age groups. Knowledge about meningococcal carriage is crucial for improving our understanding of the disease epidemiology and for designing effective vaccination programs. Taking in account the very limited published data currently available describing meningococcal carriage in Brazil, we performed a study to evaluate the prevalence of Neisseria meningitidis carriage among adolescent students. METHODS: A cross-sectional study was conducted in 2012 to assess the prevalence of meningococcal carriage among a representative sample of 1208 students 11­19 years of age in Campinas, Brazil. Genotypic and phenotypic characterization of isolated carriage strains and the effect of potential risk factors for carriage were also analyzed. Results: The overall carriage prevalence was 9.9% (95% confidence interval, 8.3­11.8%), with dominance of serogroup C (1.32%), followed by serogroups B (0.99%), E (0.74%), Y (0.49%) and W (0.25%). A lower level of education of the parents was independently associated with a higher risk of carriage. A high diversity of genotypes was found among carriage strains. CONCLUSIONS: The evidence gathered during this study provides estimates of carriage prevalence in Brazilian adolescents, showing an unusually high dominance of serogroup C. These results have important implications in future strategies to optimize the impact of the current meningococcal C vaccination program in Brazil

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 influenzae type 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.

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.

Prevalence, Risk Factors and Molecular Characteristics of Meningococcal Carriage Among Brazilian Adolescents.

BACKGROUND: In 2010, introduction of the meningococcal C conjugate vaccine in Brazil for children <2 years provided an immediate reduction in the incidence rates of disease among the age groups targeted for the vaccine, but no early impact was observed in unvaccinated age groups. Knowledge about meningococcal carriage is crucial for improving our understanding of the disease epidemiology and for designing effective vaccination programs. Taking in account the very limited published data currently available describing meningococcal carriage in Brazil, we performed a study to evaluate the prevalence of Neisseria meningitidis carriage among adolescent students. METHODS: A cross-sectional study was conducted in 2012 to assess the prevalence of meningococcal carriage among a representative sample of 1208 students 11-19 years of age in Campinas, Brazil. Genotypic and phenotypic characterization of isolated carriage strains and the effect of potential risk factors for carriage were also analyzed. RESULTS: The overall carriage prevalence was 9.9% (95% confidence interval, 8.3-11.8%), with dominance of serogroup C (1.32%), followed by serogroups B (0.99%), E (0.74%), Y (0.49%) and W (0.25%). A lower level of education of the parents was independently associated with a higher risk of carriage. A high diversity of genotypes was found among carriage strains. CONCLUSIONS: The evidence gathered during this study provides estimates of carriage prevalence in Brazilian adolescents, showing an unusually high dominance of serogroup C. These results have important implications in future strategies to optimize the impact of the current meningococcal C vaccination program in Brazil.

Use of sodC versus ctrA for real-time polymerase chain reaction-based detection of Neisseria meningitidis in sterile body fluids.

We evaluated the use of a newly described sodC-based real-time-polymerase chain reaction (RT-PCR) assay for detecting Neisseria meningitidis in normally sterile sites, such as cerebrospinal fluid and serum. The sodC-based RT-PCR assay has an advantage over ctrA for detecting nongroupable N. meningitidis isolates, which are commonly present in asymptomatic pharyngeal carriage. However, in our study, sodC-based RT-PCR was 7.5% less sensitive than ctrA. Given the public health impact of possible false-negative results due to the use of the sodC target gene alone, sodC-based RT-PCR for the diagnosis of meningococcal meningitis should be used with caution.

Ocorrência de resultados falso-negativos na reação de PCR em Tempo Real (PCR-TR) no diagnóstico da doença meningocócica / False-negative results in Real-Time PCR (RT-PCR) formeningococcal disease diagnosis

Factors responsible for false-negative results (F-N) in RT-PCR assay for detecting N. meningitidis in serumand CSF samples were investigated. As the meningococcal disease should be rapidly treated because ofits high mortality and epidemic potential, the F-N in diagnostic testing may cause treatment failuresand/or on disease restraint in community. Thus, it is crucial to learn the factors which cause F-N in RTPCRassays. The F-N were induced by inhibition, low quantity of target DNA in extracted samples, andinadequate temperature employed at PCR annealing procedure. As bacterial DNA concentration in samplesmight be highly variable, the ideal sample volume to be extracted could not be defined. As previouslyrecommended for N. meningitidis gene-grouping by RT-PCR assay, the annealing temperature at 60 °Cwas not suitable for B and W135 genogroups. Altogether, these factors induced F-N in 31 samples; therefore,30 % of N. meningitidis detected by RT-PCR were classified as non-genogrouped. The inhibitors and/orthe low amount of target DNA induced F-N on RT-PCR, independently of the specimen volume used forextracting DNA. However, adjustments on the PCR annealing temperature and amount of extracted DNAadded into the reaction might avoid the occurrence of the majority of F-N.