Cell adhesion proteins in the cerebrospinal fluid of neonates prenatally exposed to Zika virus: A case-control study.

To compare cell adhesion molecules levels in cerebrospinal fluid (CSF) between Zika virus (ZIKV)-exposed neonates with/without microcephaly (cases) and controls, 16 neonates (cases), 8 (50%) with and 8 (50%) without microcephaly, who underwent lumbar puncture (LP) during the ZIKV epidemic (2015-2016) were included. All mothers reported ZIKV clinical symptoms during gestation, all neonates presented with congenital infection findings, and other congenital infections were ruled out. Fourteen control neonates underwent LP in the same laboratory (2017-2018). Five cell adhesion proteins were measured in the CSF using mass spectrometry. Neurexin-1 (3.50 [2.00-4.00] vs. 7.5 [5.00-10.25], P = 0.001), neurexin-3 (0.00 [0.00-0.00] vs. 3.00 [1.50-4.00], P = 0.001) and neural cell adhesion molecule 2 (NCAM2) (0.00 [0.00-0.75] vs. 1.00 [1.00-2.00], P = 0.001) were significantly lower in microcephalic and non-microcephalic cases than in controls. When these two sub-groups of prenatally ZIKA-exposed children were compared to controls separately, the same results were found. When cases with and without microcephaly were compared, no difference was found. Neurexin-3 (18.8% vs. 78.6%, P = 0.001) and NCAM2 (25.0% vs. 85.7%, P = 0.001) were less frequently found among the cases. A positive correlation was found between cephalic perimeter and levels of these two proteins. Neurexin-2 and neurexin-2b presented no significant differences. Levels of three cell adhesion proteins were significantly lower in CSF of neonates exposed to ZIKV before birth than in controls, irrespective of presence of congenital microcephaly. Moreover, the smaller the cephalic perimeter, the lower CSF cell adhesion protein levels. These findings suggest that low CSF levels of neurexin-1, neurexin-3 and NCAM2 may reflect the effects of ZIKV on foetal brain development.

Zika-exposed microcephalic neonates exhibit higher degree of inflammatory imbalance in cerebrospinal fluid.

Not every neonate with congenital Zika virus (ZIKV) infection (CZI) is born with microcephaly. We compared inflammation mediators in CSF (cerebrospinal fluid obtained from lumbar puncture) between ZIKV-exposed neonates with/without microcephaly (cases) and controls. In Brazil, in the same laboratory, we identified 14 ZIKV-exposed neonates during the ZIKV epidemic (2015-2016), 7(50%) with and 7(50%) without microcephaly, without any other congenital infection, and 14 neonates (2017-2018) eligible to be controls and to match cases. 29 inflammation mediators were measured using Luminex immunoassay and multidimensional analyses were employed. Neonates with ZIKV-associated microcephaly presented substantially higher degree of inflammatory perturbation, associated with uncoupled inflammatory response and decreased correlations between concentrations of inflammatory biomarkers. The groups of microcephalic and non-microcephalic ZIKV-exposed neonates were distinguished from the control group (area under curve [AUC] = 1; P < 0.0001). Between controls and those non-microcephalic exposed to ZIKV, IL-1ß, IL-3, IL-4, IL-7 and EOTAXIN were the top CSF markers. By comparing the microcephalic cases with controls, the top discriminant scores were for IL-1ß, IL-3, EOTAXIN and IL-12p70. The degree of inflammatory imbalance may be associated with microcephaly in CZI and it may aid additional investigations in experimental pre-clinical models testing immune modulators in preventing extensive damage of the Central Nervous System.

Cerebrospinal fluid immunoglobulins are increased in neonates exposed to Zika virus during foetal life.

OBJECTIVE: To compare immunoglobulin levels in cerebrospinal fluid (CSF) of neonates exposed to Zika virus (ZIKV) during foetal life (cases) with levels in CSF of control neonates. METHODS: We identified 16 neonates who underwent lumbar puncture (LP), during the ZIKV epidemic (December/2015 to March/2016) whose mothers reported ZIKV clinical symptoms during gestation (cases). Congenital microcephaly was defined as head circumference ≤31.9 cm (boys) and ≤31.5 cm (girls) for term neonates, or ≤2 standard deviations below the mean for premature (<37 weeks) neonates. Subsequently, we identified neonates who underwent LP in the same lab and fulfilled criteria to be controls: age ≤4 days, CSF white blood cell count ≤8/mm3, CSF protein ≤132 mg/dL, CSF red blood cell count ≤1,000/mm3, neither central nervous system illness, nor congenital infection, nor microcephaly. CSF immunoglobulin concentrations were measured by mass spectrometry. RESULTS: 13 controls were included. IgM, IgA, IgG, IgK, and IgL were significantly higher among cases (p < 0.001). Eight (50%) ZIKV exposed infants had congenital microcephaly. These showed the strongest immunoglobulin elevation of the IgM and IgA classes. CONCLUSION: Neonates exposed to ZIKV infection during gestation present with elevated distinct immunoglobulins in CSF, both in cases that developed microcephaly and in cases that did not.

Respiratory viruses among children with non-severe community-acquired pneumonia: A prospective cohort study.

BACKGROUND: Community-acquired pneumonia (CAP) causes a major burden to the health care system among children under-5 years worldwide. Information on respiratory viruses in non-severe CAP cases is scarce. OBJECTIVES: To estimate the frequency of respiratory viruses among non-severe CAP cases. STUDY DESIGN: Prospective study conducted in Salvador, Brazil. Out of 820 children aged 2-59 months with non-severe CAP diagnosed by pediatricians (respiratory complaints and radiographic pulmonary infiltrate/consolidation), recruited in a clinical trial (ClinicalTrials.gov Identifier NCT01200706), nasopharyngeal aspirate samples were obtained from 774 (94.4%) patients and tested for 16 respiratory viruses by PCRs. RESULTS: Viruses were detected in 708 (91.5%; 95%CI: 89.3-93.3) cases, out of which 491 (69.4%; 95%CI: 65.9-72.7) harbored multiple viruses. Rhinovirus (46.1%; 95%CI: 42.6-49.6), adenovirus (38.4%; 95%CI: 35.0-41.8), and enterovirus (26.5%; 95%CI: 23.5-29.7) were the most commonly found viruses. The most frequent combination comprised rhinovirus plus adenovirus. No difference was found in the frequency of RSVA (16.1% vs. 14.6%; P = 0.6), RSVB (10.9% vs. 13.2%; P = 0.4) influenza (Flu) A (6.3% vs. 5.1%; P = 0.5), FluB (4.5% vs. 1.8%; P = 0.09), parainfluenza virus (PIV) 1 (5.1% vs. 2.8%; P = 0.2), or PIV4 (7.7% vs. 4.1%; P = 0.08), when children with multiple or sole virus detection were compared. Conversely, rhinovirus, adenovirus, enterovirus, bocavirus, PIV2, PIV3, metapneumovirus, coronavirus OC43, NL63, 229E were significantly more frequent among cases with multiple virus detection. CONCLUSIONS: Respiratory viruses were detected in over 90% of the cases, out of which 70% had multiple viruses. Several viruses are more commonly found in multiple virus detection whereas other viruses are similarly found in sole and in multiple virus detection.

Infection by Streptococcus pneumoniae in children with or without radiologically confirmed pneumonia / Infecção por Streptococcus pneumoniae em crianças com ou sem pneumonia radiologicamente confirmada

Abstract Objective: Community-acquired pneumonia is an important cause of morbidity in childhood, but the detection of its causative agent remains a diagnostic challenge. The authors aimed to evaluate the role of the chest radiograph to identify cases of community-aquired pneumonia caused by typical bacteria. Methods: The frequency of infection by Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis was compared in non-hospitalized children with clinical diagnosis of community acquired pneumonia aged 2-59 months with or without radiological confirmation (n = 249 and 366, respectively). Infection by S. pneumoniae was diagnosed by the detection of a serological response against at least one of eight pneumococcal proteins (defined as an increase ≥2-fold in the IgG levels against Ply, CbpA, PspA1 and PspA2, PhtD, StkP-C, and PcsB-N, or an increase ≥1.5-fold against PcpA). Infection by H. influenzae and M. catarrhalis was defined as an increase ≥2-fold on the levels of microbe-specific IgG. Results: Children with radiologically confirmed pneumonia had higher rates of infection by S. pneumoniae. The presence of pneumococcal infection increased the odds of having radiologically confirmed pneumonia by 2.8 times (95% CI: 1.8-4.3). The negative predictive value of the normal chest radiograph for infection by S. pneumoniae was 86.3% (95% CI: 82.4-89.7%). There was no difference on the rates of infection by H. influenzae and M. catarrhalis between children with community-acquired pneumonia with and without radiological confirmation. Conclusions: Among children with clinical diagnosis of community-acquired pneumonia submitted to chest radiograph, those with radiologically confirmed pneumonia present a higher rate of infection by S. pneumoniae when compared with those with a normal chest radiograph.

Resumo Objetivo: Avaliar o papel do raios X de tórax na identificação de casos de pneumonia adquirida na comunidade (PAC) causada por agentes bacterianos. Métodos: A frequência de infecção por Streptococcus pneumoniae, Haemophilus influenzae e Moraxella catarrhalis em crianças com PAC não hospitalizadas foi comparada com a presença de confirmação radiológica da pneumonia (n = 249 crianças com pneumonia radiologicamente confirmada e 366 crianças com raios X de tórax normal). Infecção por S. pneumoniae foi diagnosticada com base na resposta sorológica a pelo menos uma dentre oito proteínas pneumocócicas investigadas (aumento ≥ 2 vezes nos níveis de IgG em relação a Ply, CbpA, PspA1 e 2, PhtD, StkP-C e PcsB-N ou aumento≥ 1,5 vez em relação aPcpA). Infecção por H. influenzae e M. catarrhalis foi definida por aumento ≥ 2 vezes nos níveis de IgG específica a antígenos de cada agente. Resultados: Crianças com pneumonia radiologicamente confirmada apresentaram maior taxa de infecção pelo pneumococo. Além disso, a presença de infecção pneumocócica foi um fator preditor de pneumonia radiologicamente confirmada, o que aumenta sua chance de detecção em 2,8 vezes (IC 95%: 1,8-4,3). O valor preditivo negativo do raios X normal para a infecção por S. pneumoniae foi 86,3% (IC95%: 82,4%-89,7%). Não houve diferença nas frequências de infecção por H. influenzae e M. catarrhalis entre crianças com PAC com ou sem confirmação radiológica. Conclusão: Crianças com diagnóstico clínico de PAC submetidas a um raios X de tórax que apresentam confirmação radiológica têm maior taxa de infecção por S. pneumoniae comparadas com as crianças com raios X normal.

Serologically diagnosed acute human bocavirus 1 infection in childhood community-acquired pneumonia.

AIM: To assess the role of human bocavirus 1 (HBoV1) as a causative agent of non-severe community-acquired pneumonia (CAP) in children. METHODS: Patients aged 2-59 months with non-severe CAP (respiratory complaints and radiographic pulmonary infiltrate/consolidation) attending a University Hospital in Salvador, Brazil were enrolled in a prospective cohort. From 820 recruited children in a clinical trial (ClinicalTrials.gov NCT01200706), nasopharyngeal aspirate (NPA), and acute and convalescent serum samples were obtained from 759 (92.6%) patients. NPAs were tested for 16 respiratory viruses by PCR. Acute HBoV1 infection was confirmed by measuring specific IgM and IgG responses in paired serum samples. RESULTS: Respiratory viruses were detected in 693 (91.3%; 95%CI: 89.1-93.2) CAP cases by PCR. HBoV1-DNA was detected in 159 (20.9%; 95%CI: 18.2-24.0) cases. Of these 159 PCR positive cases, acute HBoV1 infection was confirmed serologically in 38 cases (23.9%; 95%CI: 17.8-31.0). Overall, acute HBoV1 infection was confirmed in 5.0% (38/759) of non-severe CAP patients. HBoV1 was detected in 151 cases with at least one other virus making 31.7% of all multiple virus (n = 477) detections. Among all 759 cases, 216 had one respiratory virus detected, and sole HBoV1 was detected in only 8 (3.7%). Acute HBoV1 infection was serologically diagnosed in 34 (22.5%) HBoV1-DNA-positive cases with another virus, compared to 4 (50.0%) cases with sole virus detection (p = 0.09). CONCLUSION: HBoV1 was detected by PCR in one fifth of the children with non-severe CAP and acute HBoV1 infection was serologically confirmed in one quarter of these cases.

Infection by Streptococcus pneumoniae in children with or without radiologically confirmed pneumonia.

OBJECTIVE: Community-acquired pneumonia is an important cause of morbidity in childhood, but the detection of its causative agent remains a diagnostic challenge. The authors aimed to evaluate the role of the chest radiograph to identify cases of community-aquired pneumonia caused by typical bacteria. METHODS: The frequency of infection by Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis was compared in non-hospitalized children with clinical diagnosis of community acquired pneumonia aged 2-59 months with or without radiological confirmation (n=249 and 366, respectively). Infection by S. pneumoniae was diagnosed by the detection of a serological response against at least one of eight pneumococcal proteins (defined as an increase ≥2-fold in the IgG levels against Ply, CbpA, PspA1 and PspA2, PhtD, StkP-C, and PcsB-N, or an increase ≥1.5-fold against PcpA). Infection by H. influenzae and M. catarrhalis was defined as an increase ≥2-fold on the levels of microbe-specific IgG. RESULTS: Children with radiologically confirmed pneumonia had higher rates of infection by S. pneumoniae. The presence of pneumococcal infection increased the odds of having radiologically confirmed pneumonia by 2.8 times (95% CI: 1.8-4.3). The negative predictive value of the normal chest radiograph for infection by S. pneumoniae was 86.3% (95% CI: 82.4-89.7%). There was no difference on the rates of infection by H. influenzae and M. catarrhalis between children with community-acquired pneumonia with and without radiological confirmation. CONCLUSIONS: Among children with clinical diagnosis of community-acquired pneumonia submitted to chest radiograph, those with radiologically confirmed pneumonia present a higher rate of infection by S. pneumoniae when compared with those with a normal chest radiograph.

Evolution of acute infection with atypical bacteria in a prospective cohort of children with community-acquired pneumonia receiving amoxicillin.

Background: Atypical bacteria are treatable causative agents of community-acquired pneumonia (CAP). However, there is no conclusive evidence that a child with CAP should receive empirical treatment against such agents. Objectives: We assessed the possibility of association between clinical failure and acute infection by these bacteria among children with CAP treated with amoxicillin. Patients and methods: Patients aged 2-59 months with non-severe CAP received amoxicillin during prospective follow-up. Acute and convalescent blood samples were collected. Probable acute infection by Mycoplasma pneumoniae (specific IgM antibodies), by Chlamydia pneumoniae or Chlamydia trachomatis (specific IgM antibodies and/or IgG/IgA titre change) was investigated. Outcomes were assessed during follow-up at 2, 5 and 14-28 days. Treatment failure included development of danger signs, persistent fever, tachypnoea or death. ClinicalTrials.gov: NCT01200706. Results: Of 787 children, 86 (10.9%; 95% CI = 8.9%-13.3%) had acute M. pneumoniae infection. C. pneumoniae acute infection was found in 79 of 733 (10.8%; 95% CI = 8.7%-13.2%) and C. trachomatis was found in 3 of 28 (10.7%; 95% CI = 2.8%-26.5%) <6 months old. Among patients with or without treatment failure at 2 days, acute M. pneumoniae infection (11.7% versus 10.7%; P = 0.7), acute C. pneumoniae infection (8.5% versus 11.3%; P = 0.3) and acute C. trachomatis infection (16.7% versus 9.1%; P = 0.5) were found. No significant differences were found with regard to treatment failure at the 5 day evaluation. Overall, amoxicillin was substituted in 3.5% versus 2.7% among patients with or without acute infection by one of these bacteria ( P = 0.6). Conclusions: The overall substitution rate of amoxicillin was very low. It is not necessary to give an empirical non-ß-lactam antibiotic as a first-line option to treat every child between 2 and 59 months old with non-severe CAP.

An update on antimicrobial options for childhood community-acquired pneumonia: a critical appraisal of available evidence.

INTRODUCTION: Community-acquired pneumonia (CAP) is a leading cause of death and a major cause of morbidity in children under the age of 5. Appropriate antimicrobial use is one crucial tool in controlling childhood CAP mortality and suffering. AREAS COVERED: Structured search of current literature. PubMed was consulted for published trials conducted in children with CAP. We aimed to provide a comprehensive evaluation of antimicrobials used to treat childhood CAP, including a critical appraisal of the methodological aspects of these clinical trials. EXPERT OPINION: Amoxicillin is the preferred option to treat non-severe non-complicated CAP among children aged ≥2 months. Amoxicillin may be used to treat children in this age group with severe CAP if they do not require hospital assistance. If the patient warrants hospitalization, intravenous penicillin is the chosen option. Heterogeneity was high in the included trials, in regard to clinical inclusion criteria, use of radiological inclusion criteria, placebo use and masking. Higher quality evidence was found in the studies which included amoxicillin. There is a clear dearth of randomized, placebo-controlled, well-performed clinical trials evaluating children with CAP aged under 2 months, or aged 2 months and above with very severe or complicated CAP, or in specific age groups like teenagers.

Epidemiological aspects of influenza A related to climatic conditions during and after a pandemic period in the city of Salvador, Northeastern Brazil.

During the influenza pandemic of 2009, the A(H1N1)pdm09, A/H3N2 seasonal and influenza B viruses were observed to be co-circulating with other respiratory viruses. To observe the epidemiological pattern of the influenza virus between May 2009-August 2011, 467 nasopharyngeal aspirates were collected from children less than five years of age in the city of Salvador. In addition, data on weather conditions were obtained. Indirect immunofluorescence, real-time transcription reverse polymerase chain reaction (RT-PCR), and sequencing assays were performed for influenza virus detection. Of all 467 samples, 34 (7%) specimens were positive for influenza A and of these, viral characterisation identified Flu A/H3N2 in 25/34 (74%) and A(H1N1)pdm09 in 9/34 (26%). Influenza B accounted for a small proportion (0.8%) and the other respiratory viruses for 27.2% (127/467). No deaths were registered and no pattern of seasonality or expected climatic conditions could be established. These observations are important for predicting the evolution of epidemics and in implementing future anti-pandemic measures.

Epidemiological aspects of influenza A related to climatic conditions during and after a pandemic period in the city of Salvador, northeastern Brazil

During the influenza pandemic of 2009, the A(H1N1)pdm09, A/H3N2 seasonal and influenza B viruses were observed to be co-circulating with other respiratory viruses. To observe the epidemiological pattern of the influenza virus between May 2009-August 2011, 467 nasopharyngeal aspirates were collected from children less than five years of age in the city of Salvador. In addition, data on weather conditions were obtained. Indirect immunofluorescence, real-time transcription reverse polymerase chain reaction (RT-PCR), and sequencing assays were performed for influenza virus detection. Of all 467 samples, 34 (7%) specimens were positive for influenza A and of these, viral characterisation identified Flu A/H3N2 in 25/34 (74%) and A(H1N1)pdm09 in 9/34 (26%). Influenza B accounted for a small proportion (0.8%) and the other respiratory viruses for 27.2% (127/467). No deaths were registered and no pattern of seasonality or expected climatic conditions could be established. These observations are important for predicting the evolution of epidemics and in implementing future anti-pandemic measures.

Comparison of oral amoxicillin given thrice or twice daily to children between 2 and 59 months old with non-severe pneumonia: a randomized controlled trial.

OBJECTIVES: Oral amoxicillin (50 mg/kg/day) thrice daily is the first-line therapy for non-severe childhood pneumonia. Compliance could be enhanced if two daily doses are employed. We assessed the equivalence of oral amoxicillin (50 mg/kg/day) thrice or twice daily in those patients. PATIENTS AND METHODS: This randomized (1 : 1), controlled, triple-blinded investigation conducted at one centre in Brazil included children aged 2-59 months with non-severe pneumonia diagnosed by trained paediatricians based on respiratory complaints and radiographic pulmonary infiltrate/consolidation. Participants were randomly assigned to receive one bottle (Amoxicillin 1) at 6 am, 2 pm and 10 pm and the other bottle (Amoxicillin 2) at 8 am and 8 pm: one bottle contained amoxicillin and the other placebo and vice versa. Only the pharmacist knew patients' allocation. Follow-up assessments were done at 2, 5 and 14 days after enrolment. Chest radiographs were read by three independent radiologists. Primary outcome was treatment failure (development of danger signs, persistence of fever, tachypnoea, development of serious adverse reactions, death and withdrawal from the trial) at 48 h. ClinicalTrials.gov: identifier NCT01200706. RESULTS: Four hundred and twelve and 408 participants received amoxicillin thrice or twice daily, respectively. Treatment failure was detected in 94 (22.8%) and 94 (23.0%) patients in intention-to-treat analysis (risk difference 0.2%; 95% CI: -5.5%-6.0%) and in 80 (20.1%) and 85 (21.3%) patients in per-protocol analysis (risk difference 1.2%; 95% CI: -4.4%-6.8%). Pneumonia was radiologically confirmed by concordant reading in 277 (33.8%) cases, among whom treatment failure was registered in 25/133 (18.8%) and 27/144 (18.8%) participants from the thrice and twice daily doses subgroups, respectively (risk difference -0.05%; 95% CI: -9.3%-9.2%). CONCLUSIONS: Oral amoxicillin (50 mg/kg/day) twice daily is as efficacious as thrice daily.

The inter-observer variation of chest radiograph reading in acute lower respiratory tract infection among children.

This study assessed the inter-observer agreement in the interpretation of several radiographic features in the chest radiographs (CXR) of 803 children aged 2-59 months with non-severe acute lower respiratory tract infection (ALRI). Inclusion criteria comprised: report of respiratory complaints, detection of lower respiratory findings, and presence of pulmonary infiltrate on the CXR taken on admission and read by the pediatrician on duty. Data on demographic and clinical findings on admission were collected from children included in a clinical trial on the use of amoxicillin (ClinicalTrials.gov Identifier NCT01200706). CXR was later read by two independent pediatric radiologists blinded to clinical information and pneumonia was finally diagnosed if there was agreement on the presence of pulmonary infiltrate or pleural effusion. The kappa index (κ) of agreement was calculated. The radiologists agreed that 774 (96.4%) and 3 (0.4%) CXR were appropriate or inappropriate for reading, respectively, and that 222 (28.7%) and 459 (59.3%) CXR presented or did not present pneumonia. In intent to treat analysis, that is, considering the 803 enrolled patients, κ for the presence of pneumonia was 0.725 (95% CI: 0.675-0.775). The overall agreement was 78.7% (normal CXR [n = 385, 60.9%], pneumonia [n = 222, 35.1%], other radiological diagnosis [n = 22, 3.5%], inappropriate for reading [n = 3, 0.5%]). The most frequent radiological findings were alveolar infiltrate (33.2%) and consolidation (32.9%) by radiologist 1 and consolidation (28.3%) and alveolar infiltrate (19.3%) by radiologist 2. Concordance for consolidation was 86.7% (k = 0.683, 95%CI: 0.631-0.741). Agreement was good between two pediatric radiologists when diagnosis of pneumonia among children with non-severe ALRI was compared.

Community acquired Pneumonia among pediatric outpatients in Salvador, northeast Brazil, with emphasis on the role of pneumococcus

Pneumonia is one of the leading causes of hospitalization and death among children in developing counties,, and mortality due to pneumoniae has been associated with S. pneumoniae infection. This investigation was designed to describe the antimicrobial susceptibility and serotype patterns of pneumococcal strains recovered from the blood of children with community-acquired pneumonia (CAP) and to acess the clinical findings of pneumococcal bacteremic patients with pneumonia. In a 26 month prospective study, blood cultures were obtained as often as possible from children(<16 years of age) diagnosed with CAP in two emergency rooms. Antimicrobial drug susceptibility tests and serotyping were performed when pneumococcus was identified. We studied 3,431 cases and cultured blood samples from 65.5 percent of those. Pneumococcus was recovered from 0.8 percent of the blood samples. The differences in age, somnolence, wheezing and hospitalization among children with and without pneumococcal bacteremia were statistically significant. Pneumococcal bacteremia was age-related (mean 1.63 +1.55; median 0.92) and associated with somnolence and hospitalization among children with CAP. One strain was recovered from pleural fluid. Penicillin resistance was detected in 21.0 percent(4/19) of the strains at an intermediate level, whereas 63.0 percent of the strains were resistant to trimethoprim-sulfamethoxazole. The most common serotypes were 14 and 6B, and these serotypes included the resistant strains. Eight of our 18 isolates from blood were of types included in the heptavalent conjugate pneumococcal vaccine, recently licensed in the USA.