Rapid colorimetric polymyxin B microelution directly from positive blood bottles: because patients with serious infections should not have to wait for results of culture-based methodologies.

PURPOSE: To evaluate the performance of the rapid colorimetric polymyxin B microelution (RCPEm) in determining polymyxin B resistance directly from Enterobacterales-positive blood cultures. METHODS: A set volume of positive blood culture bottles (diluted 1:10) was inoculated into a glucose-broth-phenol red solution (NP solution), where a polymyxin B disk was previously eluted (final concentration of 3 µg/mL). Test was read each 1 h for up to 4 h. Color change from red/orange to yellow indicated resistant isolates. Results were compared to the reference method, broth microdilution (BMD), performed from colonies grown on solid media from the same blood culture bottle. RESULTS: One hundred fifty-two Enterobacterales-positive blood cultures were evaluated, 22.4% (34/152) of them resistant to polymyxin B (including 6.6% with borderline MICs). When performing directly from positive blood cultures (RCPEm-BC), specificity and sensitivity were 99.1% and 94.1%, respectively. Of note, 79.4% (27/34) of truly resistant isolates required 3 h of incubation, compared to the 18 ± 2 h incubation that microtiter plates of BMD demand before reading can be performed. CONCLUSIONS: RCPEm directly from blood cultures has great potential to be part of the routine of clinical microbiology laboratories to establish polymyxin B susceptibility, impacting outcome of patients with bloodstream infections caused by carbapenem-resistant Enterobacterales.

Detection of polymyxins resistance among Enterobacterales: evaluation of available methods and proposal of a new rapid and feasible methodology.

BACKGROUND: Fast and accurate detection of polymyxins resistance is necessary as they remain the last resources to treat infections caused by Carbapenem-resistant Enterobacterales in many regions. We evaluated the rapid colorimetric polymyxin B elution (RCPE) and developed its miniaturized version, RCPE microelution (RCPEm), aiming to detect polymyxins resistance among Enterobacterales. METHODS: The methodologies consist of exposing the bacterial population in a solution (NP solution) where polymyxin B disks were previously eluted to obtain a concentration of 2 µg/mL for RCPE and 3 µg/mL for RCPEm. RESULTS: Two hundred sixty-seven Enterobacterales were evaluated, 90 (33.7%) resistant to polymyxin B by broth microdilution. It was observed 0.6% of major error (ME) by RCPE, with a specificity of 99.4%. The miniaturized version (RCPEm) presented the same ME and specificity values, but slightly higher sensitivity (97.8% vs. 95.6%) with 2.2% of very major error (VME). CONCLUSIONS: RCPE and RCPEm proved to be useful alternatives to determine polymyxin B susceptibility in clinical microbiology laboratories, presenting low cost, being easy to perform, and demanding short incubation time.

Otopathogens in the middle ear and nasopharynx of children with recurrent acute otitis media.

OBJECTIVE: This study aimed to describe the microbiology of the middle ear and nasopharynx, determining the prevalence of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis in a group of children vaccinated with pneumococcal conjugate vaccine (PCV) who underwent ventilation tube insertion for recurrent acute otitis media. METHODS: We analyzed 278 middle ear effusion and 139 nasopharyngeal samples obtained from 139 children who underwent myringotomy and ventilation tube insertion for recurrent acute otitis media between June 2017 and June 2021. The children's ages ranged from 9 months to 9 years, 10 months, with a median of 21 months. The patients had no signs of acute otitis media or respiratory tract infection and were not on antibiotic therapy at the time of the procedure. The middle ear effusion and nasopharyngeal samples were collected with an Alden-Senturia aspirator and a swab, respectively. Bacteriological studies and multiplex PCR were performed for the detection of the three pathogens. Direct molecular determination of pneumococcal serotypes was performed by real-time PCR. The chi-square test was used to verify associations between categorical variables and measures of strength of association based on prevalence ratios, considering a 95% confidence interval a 5% significance level. RESULTS: Vaccination coverage was 77.7% with the basic regimen plus booster dose and 22.3% with the basic regimen alone. Middle ear effusion culture identified H. influenzae in 27 children (19.4%), S. pneumoniae in 7 (5.0%), and M. catarrhalis in 7 (5.0%). PCR detected H. influenzae in 95 children (68.3%), S. pneumoniae in 52 (37.4%), and M. catarrhalis in 23 (16.5%), a three-to seven-fold increase compared to culture. In the nasopharynx, culture isolated H. influenzae in 28 children (20.1%), S. pneumoniae in 29 (20.9%), and M. catarrhalis in 12 (8.6%). PCR identified H. influenzae in 84 children (60.4%), S. pneumoniae in 58 (41.7%), and M. catarrhalis in 30 (21.5%), a two-to three-fold increase in detection. The most common pneumococcal serotype was 19A, both in the ears and the nasopharynx. In the ears, of the 52 children who had pneumococcus, 24 (46.2%) had serotype 19A. In the nasopharynx, of the 58 patients who had pneumococcus, 37 (63.8%) had serotype 19A. Of all 139 children, 53 (38.1%) had polymicrobial samples (more than 1 of the 3 otopathogens) in the nasopharynx. Of the 53 children who had polymicrobial samples in the nasopharynx, 47 (88.7%) also had 1 of the 3 otopathogens in the middle ear, mainly H. influenzae (40%-75.5%), especially when it was found in the nasopharynx in conjunction with S. pneumoniae. CONCLUSION: The prevalence of bacteria in a group of Brazilian children immunized with the PCV who required ventilation tube insertion for recurrent acute otitis media was similar to that reported in other parts of the world after the advent of PCV. H. influenzae was the most frequent bacteria, both in the nasopharynx and the middle ear, while S. pneumoniae serotype 19A was the most common pneumococcus in the nasopharynx and middle ear. Polymicrobial colonization of the nasopharynx was strongly associated with detection of H. influenzae in the middle ear.

Streptococcus pneumoniae serotype 19A from carriers and invasive disease: virulence gene profile and pathogenicity in a Galleria mellonella model.

PURPOSE: This study aimed to evaluate and compare the presence of genes related to surface proteins between isolates of Streptococcus pneumoniae from healthy carriers (HC) and invasive pneumococcal disease (IPD) with a particular focus on serotype 19A. METHODS: The presence of these genes was identified by real-time PCR. Subsequently, we employed the Galleria mellonella larval infection model to study their effect on pathogenicity in vivo. RESULTS: The percentage of selected virulence genes was similar between the HC and IPD groups (p > 0.05), and the genes lytA, nanB, pavA, pcpA, phtA, phtB, phtE, rrgA, and sipA were all present in both groups. However, the virulence profile of the isolates differed individually between HC and IPD groups. The highest lethality in G. mellonella was for IPD isolates (p < 0.01), even when the virulence profile was the same as compared to the HC isolates or when the nanA, pspA, pspA-fam1, and pspC genes were not present. CONCLUSIONS: The occurrence of the investigated virulence genes was similar between HC and IPD S. pneumoniae serotype 19A groups. However, the IPD isolates showed a higher lethality in the alternative G. mellonella model than the HC isolates, regardless of the virulence gene composition, indicating that other virulence factors may play a decisive role in virulence. Currently, this is the first report using the in vivo G. mellonella model to study the virulence of clinical isolates of S. pneumoniae.

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.

Polymyxin NP tests (from colonies and directly from blood cultures): accurate and rapid methodologies to detect polymyxin B susceptibility among Enterobacterales.

Detection of polymyxins susceptibility is challenging. We aimed to evaluate Rapid Polymyxin NP from colonies (NP-colony) and directly from positive blood bottles (NP-bottle), using polymyxin B instead of colistin among Enterobacterales. Both had similar and acceptable accuracy. This is the first study performing NP-bottle using polymyxin B instead of colistin.

Polymyxin B broth disk elution: a feasible and accurate methodology to determine polymyxin B susceptibility in Enterobacterales.

Determination of polymyxins susceptibility by clinical laboratories is a nightmare, mainly because of physicochemical properties of the drug. Elution tests have already been proposed for colistin, but not for polymyxin B. We aimed to evaluate accuracy of Polymyxin B broth disk elution (PBDE) to determine the susceptibility to this drug. We evaluated 196 Enterobacterales (45.9% polymyxin B-resistant). PBDE was done in 15-mL cation-adjusted Mueller-Hinton broth where one polymyxin B disk (300 U) was eluted (2 µg/mL). BMD was performed as reference method. Categorical Agreement (CA), Major Error (ME) and Very Major Error (VME) were 99.5%, 0% and 1.11% (one false-negative K. pneumoniae MIC 4 µg/mL), respectively. As some institutions preferably use polymyxin B over colistin and in some countries colistin are not commercially available, to specifically evaluate polymyxin B is important. PBDE proved to be a cheap and easy to perform methodology to evaluate susceptibility to polymyxin B among Enterobacterales.

Suscetibilidade antimicrobiana de Streptococcus agalactiae isolados de gestantes em um hospital materno infantil de Porto Alegre, Rio Grande do Sul / Antimicrobial susceptibility of Streptococcus agalactiae isolated from pregnant women at a maternity hospital in Porto Alegre, Rio Grande do Sul, Brazil

OBJETIVOS: Caracterizar o perfil de suscetibilidade antimicrobiana de Streptococcus agalactiae isolados de gestantes atendidas em um hospital público. MÉTODOS: O estudo foi realizado em um hospital materno-infantil público de Porto Alegre, RS, no qual a pesquisa de S. agalactiae em gestantes faz parte da rotina obstétrica. Foram incluídas no estudo as pesquisas por swab anal/vaginal realizadas no período de julho de 2015 a fevereiro de 2016. Os isolados bacterianos foram identificados por testes fenotípicos e foi determinada a suscetibilidade aos antimicrobianos ampicilina, clindamicina, eritromicina e ofloxacino. Foram investigados também os genes de resistência à eritromicina ermB e mefA. RESULTADOS: No total, 294 coletas foram incluídas e destas, 26 (8%) foram positivas para S. agalactiae. Todos os isolados avaliados foram sensíveis à ampicilina e foram observadas resistências à eritromicina (21,4%), clindamicina (14,3%) e ofloxacino (7,1%), sendo que 66% dos isolados resistentes à eritromicina apresentaram o genótipo mefA. CONCLUSÕES: Os resultados deste estudo corroboram com o consenso de que em gestantes colonizadas com S. agalactiae é aconselhada a antibioticoprofilaxia intraparto com penicilina G ou ampicilina. A expressiva proporção de isolados resistentes à eritromicina e clindamicina, indicados para a antibioticoprofilaxia intraparto em caso de alergia aos antibióticos beta-lactâmicos, enfatiza a importância da determinação do perfil de suscetibilidade antimicrobiana prévia desses isolados, medida que ainda não faz parte da rotina de exames pré-natais em muitas instituições.

AIMS: To characterize the antimicrobial susceptibility profile of Streptococcus agalactiae isolated from pregnant women attended at a public hospital. METHODS: The study was carried out in a public maternal and child hospital in Porto Alegre, RS, Brazil, in which the screening for S. agalactiae in pregnant women is part of the obstetrics routine. The study was carried out on anal/vaginal swab tests performed from July 2015 to February 2016. Bacterial isolates were identified by phenotypic tests, and the susceptibility to ampicillin, clindamycin, erythromycin and ofloxacin was determined. The erythromycin resistance genes ermB and mefA were also investigated. RESULTS: A total of 294 samples were included, and of these, 26 (8%) were positive for S. agalactiae. All isolates were susceptible to ampicillin, and resistance to erythromycin (21.4%), clindamycin (14.3%) and ofloxacin (7.1%) were observed. The mefA genotype was observed in 66% of the erythromycin resistant isolates. CONCLUSIONS: Results of this study corroborate the consensus that in pregnant women colonized with S. agalactiae, intrapartum antibiotic prophylaxis with penicillin G or ampicillin is indicated. The relevant proportion of isolates resistant to erythromycin and clindamycin, indicated for intrapartum antibiotic prophylaxis in case of allergy to beta-lactam antibiotics, emphasizes the importance of determining the profile of antimicrobial susceptibility of these isolates, a measure that is not yet part of routine prenatal tests in many institutions.

Characterisation of Streptococcus pneumoniae isolates from invasive disease in adults following the introduction of PCV10 in Brazil.

Purpose. Invasive pneumococcal disease (IPD) in the elderly is an important public health issue due to the increased proportion of this population in many countries including Brazil. We aimed to characterise pneumococci isolates in adults >50 years with IPD, following the introduction of the 10-valent pneumococcal conjugate vaccine (PCV10) as part of the National Childhood Immunisation Program for children ≤2 years in March 2010.Methodology. Between 2013 and 2015, pneumococcal isolates were collected and serotypes were determined using multiplex PCR and/or Quellung reaction. Antimicrobial susceptibility was defined by E-test (bioMérieux); genetic diversity was determined using Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) and, in selected isolates, Multi Locus Sequence Typing (MLST) was performed.Results/Key findings. Among 102 pneumococcal isolates, the most frequent serotypes were 19A, 13 of 102 (12.7 %) and 22F, 10 of 102 (9.8 %). Ninety-eight isolates were tested for antimicrobial susceptibility. Intermediate resistance to penicillin was present in 2/98 (2.0 %), ceftriaxone in 7/98 (7.1 %) and meropenem in 7/95 (7.4 %) of the isolates (non-meningitis breakpoint: 4 µg ml-1/2 µg ml-1/0.5 µg ml-1, respectively). Resistance to penicillin (meningitis breakpoint ≥0.12 µg ml-1) was observed in 31/98 (31.6 %) of the isolates. Genetic analysis presented two relevant clonal groups, belonging to non-PCV10 serotypes: 19A (ST320, linked to non-susceptibility) and 22F (ST6403).Conclusion. Our data suggest a predominance of non-PCV10 serotypes among IPD in the elderly population in circulating strains ca. 3 to 5 years after the introduction of PCV10 in Brazil.

Characteristics of serogroup 20 S.pneumoniae isolates from Brazil.

BACKGROUND: Although serogroup 20 is not part of any conjugate pneumococcal vaccine, its serotype 20A, but not 20B, belongs to the polysaccharide 23-valent formula. Little is known about its clinical, laboratorial and epidemiological characteristics. METHODS: The purpose of this study was to evaluate the bacterial genotypes (by PFGE and MLST), clinical characteristics of patients (from review of medical records) and antimicrobial susceptibility of serogroup 20 isolates which were recovered from patients with invasive pneumococcal disease (IPD) from 2007 to 2012. Subtyping to determine 20A and 20B types was also performed by sequencing the genes of the cps locus. RESULTS: Sixteen isolates were genotyped and were highly related. All pneumococci were resistant to tetracycline and 31 % were non-susceptible to trimethoprim/sulfamethoxazole. Penicillin MIC ranged from 0.004 to 1 µg/mL and non-susceptibility (MIC ≥ 0.12 µg/mL) was observed in 5/16 isolates (31 %). All isolates belonged to subtype 20B. Most patients were male with a median age of 62 years and presented at least one underlying disease (mostly respiratory conditions). All isolates belonged to ST8889 and to a unique PFGE clone. CONCLUSIONS: A high clonal occurrence of serotype 20B pneumococci recovered from patients with IPD in Brazil was observed. As a non-PCV10 serotype, selective pressure may be responsible for this unusual occurrence of serogroup 20. However, temporal variation effect should not be underestimated; therefore it is an issue that warrants continued monitoring.

Streptococcus pneumoniae appendicitis in an adult patient.

Streptococcus pneumoniae is a rare cause of appendicitis.We report apneumococcal appendicitis with secondary peritonitis in a human immunodeficiency virus­positive adult, with favorable outcome after surgery and antibiotic therapy. Secondary peritonitis is frequently complication of S pneumoniae appendicitis in the few reported cases,and no specific risk factor has been identified so far.

Susceptibility profiles and correlation with pneumococcal serotypes soon after implementation of the 10-valent pneumococcal conjugate vaccine in Brazil.

OBJECTIVES: To evaluate the susceptibility patterns among Streptococcus pneumoniae recovered during the years 2010-2012 and to correlate these with serotypes. METHODS: Pneumococci from invasive sites were serotyped by sequential multiplex PCR and/or Quellung reaction. Etest strips were used to determine the minimal inhibitory concentrations, and the Clinical and Laboratory Standards Institute (CLSI) guidelines were used for interpretation. Genetic determinants of macrolide resistance were assessed by PCR, and the occurrence of the D phenotype was analyzed following the recommendations of the CLSI. RESULTS: One hundred fifty-nine S. pneumoniae were studied; most were recovered from blood and were associated with serotypes 14, 3, 4, 23F, 20, 7F, 12F, 19A, and 19F. Pneumococcal conjugate vaccine PCV7, PCV10, and PCV13 and 23-valent polysaccharide vaccine serotypes represented 38.2%, 48.7%, 64.5%, and 85.5%, respectively. ß-Lactam non-susceptibility (non-meningitis) was basically related to serotype 19A. For meningitis, it was observed in 21.4% (serotypes 14, 3, 9V, 23F, and 24F). Resistance to erythromycin occurred in 8.2% and mefA was the most common macrolide genetic determinant. One isolate was resistant to levofloxacin. Non-susceptibility to trimethoprim-sulfamethoxazole was 37.7% and to tetracycline was 22.0%. CONCLUSIONS: Our population of pneumococci represents a transition era, soon after the introduction of PCV10. Non-susceptible patterns were found to be associated with classical PCV serotypes (especially serotype 14), which is still highly prevalent, and non-PCV10 ones (19A), which may disseminate, occupying the biological niche left by the vaccine serotypes.