Etiology of meningitis among adults in three quaternary hospitals in Mozambique, 2016-2017: The role of HIV.

BACKGROUND: Meningitis remains an important cause of morbi-mortality in adults in sub-Saharan Africa. Data on the etiological investigation of meningitis in adults in Mozambique is limited and most studies were conducted in southern Mozambique. Identification of the etiology of meningitis in adults are crucial to guide prevention and treatments strategies. In this study, we determine the burden of fungal and bacterial meningitis among adults at the three largest hospitals in Mozambique. METHOD: We performed analysis of data from the routine sentinel surveillance system for meningitis in Mozambique from January 2016 to December 2017. Cerebrospinal fluid (CSF) samples were collected from eligible adults (≥18 years old) who met World Health Organization (WHO) case definition criteria for Meningitis. All samples were tested by cryptococcal antigen (CrAg) lateral flow assay (LFA), culture and triplex real-time polymerase chain reaction (qPCR) assay and all patients were tested for human immunodeficiency virus (HIV) using the national algorithm for HIV testing. RESULTS: Retrospective analysis of 1501 CSF samples from adults clinically suspected of meningitis revealed that 10.5% (158/1501) were positive for bacterial and fungal meningitis. Of these 158 confirmed cases, the proportion of Cryptococcal meningitis and pneumococcal meningitis was38.6% (95% CI: 31.0% to 46.7%) and 36.7% (95% CI: 29.2% to 44.7%), respectively. The other bacterial agents of meningitis identified include Neisseria meningitidis (8.9%; 14/158), Escherichia coli (6.3%; 10/158), Haemophilus influenzae (5.1%; 8/158) and S. aureus (4.4%; 7/158), which represent (24.7%; 39/158) of the total confirmed cases. CONCLUSION: Altogether, our findings show a high burden of Cryptococcal meningitis among adults in Mozambique, especially in people living with HIV, followed by pneumococcal meningitis. Our findings suggest that rollout of CrAg Lateral Flow Assay in the health system in Mozambique for early detection of cryptococcus neoformans is necessary to improve overall patient care.

The Role of Molecular Testing in Pediatric Meningitis Surveillance in Southern and East African Countries, 2008-2017.

BACKGROUND: As part of the global Invasive Bacterial Vaccine-Preventable Diseases Surveillance Network, 12 African countries referred cerebrospinal fluid (CSF) samples to South Africa's regional reference laboratory. We evaluated the utility of real-time polymerase chain reaction (PCR) in detecting and serotyping/grouping Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae (HNS). METHODS: From 2008 to 2017, CSF samples collected from children <5 years old with suspected meningitis underwent routine microbiology testing in-country, and 11 680 samples were submitted for HNS PCR at the regional reference laboratory. Unconditional logistic regression, with adjustment for geographic location, was performed to identify factors associated with PCR positivity. RESULTS: The overall HNS PCR positivity rate for all countries was 10% (1195 of 11 626 samples). In samples with both PCR and culture results, HNS PCR positivity was 11% (744 of 6747 samples), and HNS culture positivity was 3% (207 of 6747). Molecular serotype/serogroup was assigned in 75% of PCR-positive specimens (762 of 1016). Compared with PCR-negative CSF samples, PCR-positive samples were more often turbid (adjusted odds ratio, 6.80; 95% confidence interval, 5.67-8.17) and xanthochromic (1.72; 1.29-2.28), had elevated white blood cell counts (6.13; 4.71-7.99) and high protein concentrations (5.80; 4.34-7.75), and were more often HNS culture positive (32.70; 23.18-46.12). CONCLUSION: PCR increased detection of vaccine-preventable bacterial meningitis in countries where confirmation of suspected meningitis cases is impeded by limited culture capacity.

Toward Establishing Integrated, Comprehensive, and Sustainable Meningitis Surveillance in Africa to Better Inform Vaccination Strategies.

Large populations across sub-Saharan Africa remain at risk of devastating acute bacterial meningitis epidemics and endemic disease. Meningitis surveillance is a cornerstone of disease control, essential for describing temporal changes in disease epidemiology, the rapid detection of outbreaks, guiding vaccine introduction and monitoring vaccine impact. However, meningitis surveillance in most African countries is weak, undermined by parallel surveillance systems with little to no synergy and limited laboratory capacity. African countries need to implement comprehensive meningitis surveillance systems to adapt to the rapidly changing disease trends and vaccine landscapes. The World Health Organization and partners have developed a new investment case to restructure vaccine-preventable disease surveillance. With this new structure, countries will establish comprehensive and sustainable meningitis surveillance systems integrated with greater harmonization between population-based and sentinel surveillance systems. There will also be stronger linkage with existing surveillance systems for vaccine-preventable diseases, such as polio, measles, yellow fever, and rotavirus, as well as with other epidemic-prone diseases to leverage their infrastructure, transport systems, equipment, human resources and funding. The implementation of these concepts is currently being piloted in a few countries in sub-Saharan Africa with support from the World Health Organization and other partners. African countries need to take urgent action to improve synergies and coordination between different surveillance systems to set joint priorities that will inform action to control devastating acute bacterial meningitis effectively.

Characterization of strains of Neisseria meningitidis causing meningococcal meningitis in Mozambique, 2014: Implications for vaccination against meningococcal meningitis.

INTRODUCTION: In sub Saharan Africa, the epidemiology, including the distribution of serogroups of strains of N. meningitidis is poorly investigated in countries outside "the meningitis belt". This study was conducted with the aim to determine the distribution of serogroups of strains of N. meningitidis causing meningococcal meningitis in children and adults in Mozambique. METHODS: A total of 106 PCR confirmed Neisseria meningitidis Cerebrospinal Fluid (CSF) samples or isolates were obtained from the biobank of acute bacterial meningitis (ABM) surveillance being implemented by the National Institute of Health, at three central hospitals in Mozambique, from January to December 2014. Serogroups of N. meningitidis were determined using conventional PCR, targeting siaD gene for Neisseria meningitidis. Outer Membrane Proteins (OMP) Genotyping was performed by amplifying porA gene in nine samples. RESULTS: Of the 106 PCR confirmed Neisseria meningitidis samples, the most frequent serotype was A (50.0%, 53/106), followed by W/Y (18.9%, 20/106), C (8.5%, 9/106), X (7.5%, 8/106) and B (0.9%, 1/106). We found non-groupable strains in a total of 15 (14.2%) samples. PorA genotypes from nine strains showed expected patterns with the exception of two serogroup C strains with P1.19,15,36 and P1.19-36,15 and one serogroup X with P1.19,15,36, variants frequently associated to serogroup B. CONCLUSION: Our data shows that the number of cases of meningococcal meningitis routinely reported in central hospitals in Mozambique is significant and the most dominant serogroup is A. In conclusion, although serogroup A has almost been eliminated from the "meningitis belt", this serogroup remains a major concern in countries outside the belt such as Mozambique.

Surveillance of impact of PCV-10 vaccine on pneumococcal meningitis in Mozambique, 2013 - 2015.

BACKGROUND: Vaccination using the 10-valent conjugate vaccine (PCV-10) was introduced into the Extended Program on Immunization in Mozambique in March 2013, however its impact on pediatric pneumococcal meningitis is unknown. In this study, we assessed for the first time the impact of PCV10 on the burden of pneumococcal meningitis in children less than 5 years of age at the three largest hospitals in Mozambique. METHOD: Between March 2013 and December 2015, a total of 744 cerebrospinal fluid (CSF) samples were collected from eligible children, of which 160 (21.5%) were positive for S. pneumoniae. Of these, only 86 samples met the criteria for serotyping and were subsequently serotyped using sequential multiplex PCR (SM-PCR), but 17 samples were non-typable. RESULTS: The proportion of cases of pneumococcal meningitis decreased from 33.6% (124 of 369) in 2013 to 1.9% (3 of 160) in 2015 (p < 0.001). The relative frequency of PCV10 serotype cases also decreased from 84.2% (48 of 57) in 2013 to 0% (0 of 3) in 2015 (p = 0.006). Between 2013 and 2015, serotype coverage of PCV-10 and PCV13 vaccine formulations was 66.7% and 81.2%, respectively. CONCLUSION: Altogether, our findings shows that introduction of PCV-10 immunization resulted in rapid decline of pneumococcal meningitis children less than 5 years old in Mozambique. This decline was accompanied by substantial changes in the pattern of circulating pneumococcal serotypes.

Serotype distribution and antimicrobial resistance of Streptococcus pneumoniae in children with acute bacterial meningitis in Mozambique: implications for a national immunization strategy.

BACKGROUND: S. pneumoniae is the leading cause of acute bacterial meningitis (ABM) in children. Vaccination using the 10-valent conjugate vaccine (PCV-10) was recently introduced into the National Immunization Program in Mozambique, but data on serotype coverage of this vaccine formulation are scarce. In this study, we investigated the serotype distribution and antimicrobial resistance of isolates of S. pneumoniae causing ABM in children < 5 years at the two largest hospitals in Mozambique. METHODS: Between March 2013 and March 2014, a total of 352 cerebrospinal fluid (CSF) samples were collected from eligible children, of which 119 (33.8 %) were positive for S. pneumoniae. Of these, only 50 samples met the criteria for serotyping and were subsequently serotyped using sequential multiplex PCR (SM-PCR), but 15 samples were non-typable. RESULTS: The most common serotypes of S. pneumoniae were 1 (18.2 %), 5 (15.2 %), 14 (12.1 %), 9 V (12.1 %), 23 F (9.1 %), 6A (9.1 %), 4 (9.1 %) and 6B (6.1 %). Serotypes 1, 5, 9 V, 6A and 12 were mostly prevalent in Northern Mozambique, while serotypes 23 F, 4, 6B, 3 and 15B were predominant in Southern. Serotype coverage of PCV-10 and PCV-13 vaccine formulations were 81.8 % and 93.9 %, respectively. Serotypes 1, 3, 4, 6B, 14, 23 F were resistant to penicillin and sensitive to ceftriaxone. CONCLUSIONS: Our findings shows that changing the current in use PCV-10 vaccine formulation to PCV-13 formulation might increase substantially the protection against invasive strains of S. pneumoniae as the PCV-10 vaccine formulation does not cover the serotypes 3 and 6A, which are prevalent in Mozambique.

Frequency of Pathogenic Paediatric Bacterial Meningitis in Mozambique: The Critical Role of Multiplex Real-Time Polymerase Chain Reaction to Estimate the Burden of Disease.

BACKGROUND: In Sub-Saharan Africa, including Mozambique, acute bacterial meningitis (ABM) represents a main cause of childhood mortality. The burden of ABM is seriously underestimated because of the poor performance of culture sampling, the primary method of ABM surveillance in the region. Low quality cerebrospinal fluid (CSF) samples and frequent consumption of antibiotics prior to sample collection lead to a high rate of false-negative results. To our knowledge, this study is the first to determine the frequency of ABM in Mozambique using real-time polymerase chain reaction (qPCR) and to compare results to those of culture sampling. METHOD: Between March 2013 and March 2014, CSF samples were collected at 3 regional hospitals from patients under 5 years of age, who met World Health Organization case definition criteria for ABM. Macroscopic examination, cytochemical study, culture, and qPCR were performed on all samples. RESULTS: A total of 369 CSF samples were collected from children clinically suspected of ABM. qPCR showed a significantly higher detection rate of ABM-causing pathogens when compared to culture (52.3% [193/369] versus 7.3% [27/369], p = 0.000). The frequency of Streptococcus pneumoniae, Haemophilus influenzae, group B Streptococci, and Neisseria meningitidis were 32.8% (121/369), 12.2%, (45/369), 3.0% (16/369) and 4.3% (11/369), respectively, significantly higher compared to that obtained on culture (p < 0.001 for each). CONCLUSION: Our findings demonstrate that culture is less effective for the diagnosis of ABM than qPCR. The common use of culture rather than qPCR to identify ABM results in serious underestimation of the burden of the disease, and our findings strongly suggest that qPCR should be incorporated into surveillance activities for ABM. In addition, our data showed that S. pneumoniae represents the most common cause of ABM in children under 5 years of age.

Frequência e epidemiologia molecular das estirpes causadoras de meningites bacterianas agudas em crianças menores de 5 anos de idade em Moçambique / Frequency and molecular epidemiology of strains causing acute bacterial meningitis in children under 5 years of age in Mozambique

Na maioria dos países da África sub-sahariana, onde a meningite bacteriana aguda (MBA) continua a ser uma das principais causas de mortalidade infantil, a vigilância de MBA é baseada principalmente em cultura. Além disso, as amostras de LCR de baixa qualidade e alta frequência de consumo de antibióticos antes da colheita de amostras, leva a uma alta taxa de resultados falso-negativos. Este estudo foi realizado com o objetivo de descrever a epidemiologia e o perfil de susceptibilidade aos antibióticos dos principais agentes etiológicos da MBA, isolados em amostras de crianças menores de 5 anos de idade em Moçambique.

In Sub-Saharan Africa, including Mozambique, acute bacterial meningitis (ABM) represents a main cause of childhood mortality. The burden of ABM is seriously underestimated because of the poor performance of culture sampling, the primary method of ABM surveillance in the region. Low quality cerebrospinal fluid (CSF) samples and frequent consumption of antibiotics prior to sample collection lead to a high rate of false-negative results. To our knowledge, this study is the first to determine the frequency of ABM in Mozambique using real-time polymerase chain reaction (qPCR) and to compare results to those of culture sampling.

Frequência e epidemiologia molecular das estirpes causadoras de meningites bacterianas agudas em crianças menores de 5 anos de idade em Moçambique / Frequency and molecular epidemiology of strains causing acute bacterial meningitis in children under 5 years of age in Mozambique

Introdução: Na maioria dos países da África sub-sahariana, onde a meningite bacteriana aguda (MBA) continua a ser uma das principais causas de mortalidade infantil, a vigilância de MBA é baseada principalmente em cultura. Além disso, as amostras de LCR de baixa qualidade e alta frequência de consumo de antibióticos antes da colheita de amostras, leva a uma alta taxa de resultados falso-negativos. Este estudo foi realizado com o objetivo de descrever a epidemiologia e o perfil de susceptibilidade aos antibióticos dos principais agentes etiológicos da MBA, isolados em amostras de crianças menores de 5 anos de idade em Moçambique. Metodologia: Durante período de março de 2013 a março de 2014, foram colhidas amostras de LCR de crianças menores de 5 anos de idade, internadas, e que reuniram os critérios de definição de caso da OMS para MBA, em três hospitais (quaternários) regionais. Os exames macroscópicos e citoquímico, cultura e qPCR foram realizadas em todas as amostras. Resultados: Das 369 amostras de LCR colhidas em crianças com suspeita clínica da MBA. Portanto, a taxa de detecção dos patógenos bacterianos foi significativamente elevada quando usada a qPCR em relação à cultura [52.3% (193/369) para M-qPCR contra 7.3% (27/399) para a cultura]. S. pneumonia e foi mais frequente (62.7%, 121/369), seguido por H. influenza e (23.3%, 45/369), estreptococos do grupo B (S. agalactiae) (8,3%, 16/369) e N. meningites (5,7%, 11/ 369). Idade inferior a 12 meses, (aOR) = 3.9, p= 0,002), contagem de leucócitosde10 a 100 células/mm3 (aOR = 8,7; IC 95% = 3,6-21,1; p= 0,000) e > 100 células/mm3(aOR = 26.7, p= 0,000) e níveis de glicose menor que 40 mg/dL (aOR = 18.6, p= 0,001) foram estatisticamente associadas a MBA. Todos os isolados de pneumococos (n = 17) foram sensíveis à ceftriaxona, e 11,8% (2/17) foram sensíveis à penicilina. Todos os isolados de Hib(n = 5) foram sensíveis à ampicilina e a ceftriaxna e80% (4/5) foram sensíveis à cloranfenicol. Entre os 3isolados de meningococos, apenas um foi resistente à ampicilina, e cloranfenicol (33,3%para cada) mas todos foram sensíveis àceftriaxona para cada um. Todos os 2 isolados de S. agalactiaeforam sensíveis à eritromicina, levofloxacina e ceftriaxona e todos eram resistentes à vancomicina e à ampicilina. Os serotipos mais prevalentes foram 1 (18,2), 5 (15,2), 14 (12,1), 9V (12.1), 23F (9,1), 6A (9,1), 4 (9,1) e 6B (6,1).A proporção de isolados pertencentes a serotipos incluídos nas vacinas PCV-7, PCV-10, e PCV-13 foram 45,5%, 63,6% e 81,8%, respectivamente. Os serotipos 1, 3, 4, 6B, 14 e 23F foram todos resistentes à penicilina, enquanto para a ceftriaxona, todos foram sensíveis. Conclusão: Nossos achados mostram que a MBAé altamente prevalente em crianças com meningitee que S. pneumonia e foi a causa mais comum de meningite bacteriana aguda em crianças <5 anos de idade. Além disso, nossos dados demonstram que a cultura apresenta um rendimento baixo no diagnóstico da MBA, e sugerem fortemente que a qPCR deve ser incorporada nas actividades de vigilância de MBA. A introdução daPCV-13 pode ser vantajoso devido a emergência dos sero tipo 3 e 6A em Moçambique.

Background: In Sub-Saharan Africa, including Mozambique, acute bacterial meningitis (ABM) represents a main cause of childhood mortality. The burden of ABM is seriously underestimated because of the poor performance of culture sampling, the primary method of ABM surveillance in the region. Low quality cerebrospinal fluid (CSF) samples and frequent consumption of antibiotics prior to sample collection lead to a high rate of false-negative results. To our knowledge, this study is the first to determine the frequency of ABM in Mozambique using real-time polymerase chain reaction (qPCR) and to compare results to those of culture sampling. Method: Between March 2013 and March 2014, CSF samples were collected at 3 regional hospitals from patients under 5 years of age, who met World Health Organization case definition criteria for ABM. Macroscopic examination, cytochemical study, culture, and qPCR were performed on all samples. Results: Out of 369 CSF samples were collected from children clinically suspected of ABM. Detection rate for bacterial pathogen was significantly higher when using qPCR compared to culture [52.3% (193/369) for M-qPCR versus 7.3% (27/399) for culture]. S. pneumonia was more frequent (62.7%, 121/369),followed by H. influenza e (23.3%, 45/369),group B streptococci (8.3%, 16/369)and N. meningitides (5.7%, 11/369).Age below 12 months, (aOR) = 3.9, p= 0.002), leucocyte count10 a 100 cells/mm3(aOR = 8,7; IC 95% = 3,6-21,1; p= 0,000)>100 cells/mm3(aOR = 26.7, p= 0.000)and Glucose lower than 40mg/dL (aOR = 18.6, p= 0.001) were statistically associated with ABM. All pneumococci isolates (n=17) were susceptible to ceftriaxone, and 11.8% (2/17) were susceptible to penicillin. All Hib isolates (n=5) were susceptible to ampicillin and ceftriaxone and 80% (4/5) were susceptible to chloramphenicol. Among the 3 meningococci isolates, only one was resistant to ampicillin, and chloramphenicol (33.3%for each) but all were susceptible to ceftriaxone. All the 2 isolates of group B streptococci were susceptible to erythromycin, levofloxacin and ceftriaxone and all were resistant to vancomycin and ampicillin. The most prevailing serotypes were 1 (18.2), 5 (15.2), 14 (12.1), 9V (12.1), 23F (9.1), 6A (9.1), 4 (9.1) and 6B (6.1). The proportion of isolates belonging to serotypes contained in the vaccine PCV-7, PCV-10, and PCV-13 were 45.5%, 63.6% and 81.8%, respectively. Serotypes 1, 3, 4, 6B, 14 and 23F were all resistant to penicillin while for ceftriaxone, all were susceptible. Conclusion: Our findings showed that ABM is highly prevalent among children with meningitis and S. pneumoniae was the most common cause of acute bacterial meningitis in children < 5 years. Furthermore, our dates demonstrate that culture has a lower performance for ABM diagnostics, and strongly suggest that qPCR should be incorporated into surveillance activities for ABM. Introduction of PCV-13 would be advantageous because of emergence of news serotypes 3 and 6A in Mozambique.

Frequência e epidemiologia molecular das estirpes causadoras de meningites bacterianas agudas em crianças menores de 5 anos de idade em Moçambique

Introdução: Na maioria dos países da África sub-sahariana, onde a meningite bacteriana aguda (MBA) continua a ser uma das principais causas de mortalidade infantil, a vigilância de MBA é baseada principalmente em cultura. Além disso, as amostras de LCR de baixa qualidade e alta frequência de consumo de antibióticos antes da colheita de amostras, leva a uma alta taxa de resultados falso-negativos. Este estudo foi realizado com o objetivo de descrever a epidemiologia e o perfil de susceptibilidade aos antibióticos dos principais agentes etiológicos da MBA, isolados em amostras de crianças menores de 5 anos de idade em Moçambique.