Amoxicillin/clavulanate in combination with rifampicin/clarithromycin is bactericidal against Mycobacterium ulcerans.

BACKGROUND: Buruli ulcer (BU) is a skin neglected tropical disease (NTD) caused by Mycobacterium ulcerans. WHO-recommended treatment requires 8-weeks of daily rifampicin (RIF) and clarithromycin (CLA) with wound care. Treatment compliance may be challenging due to socioeconomic determinants. Previous minimum Inhibitory Concentration and checkerboard assays showed that amoxicillin/clavulanate (AMX/CLV) combined with RIF+CLA were synergistic against M. ulcerans. However, in vitro time kill assays (TKA) are a better approach to understand the antimicrobial activity of a drug over time. Colony forming units (CFU) enumeration is the in vitro reference method to measure bacterial load, although this is a time-consuming method due to the slow growth of M. ulcerans. The aim of this study was to assess the in vitro activity of RIF, CLA and AMX/CLV combinations against M. ulcerans clinical isolates by TKA, while comparing four methodologies: CFU enumeration, luminescence by relative light unit (RLU) and optical density (at 600 nm) measurements, and 16S rRNA/IS2404 genes quantification. METHODOLOGY/PRINCIPAL FINDINGS: TKA of RIF, CLA and AMX/CLV alone and in combination were performed against different M. ulcerans clinical isolates. Bacterial loads were quantified with different methodologies after 1, 3, 7, 10, 14, 21 and 28 days of treatment. RIF+AMX/CLV and the triple RIF+CLA+AMX/CLV combinations were bactericidal and more effective in vitro than the currently used RIF+CLA combination to treat BU. All methodologies except IS2404 quantitative PCR provided similar results with a good correlation with CFU enumeration. Measuring luminescence (RLU) was the most cost-effective methodology to quantify M. ulcerans bacterial loads in in vitro TKA. CONCLUSIONS/SIGNIFICANCE: Our study suggests that alternative and faster TKA methodologies can be used in BU research instead of the cumbersome CFU quantification method. These results provide an in vitro microbiological support to of the BLMs4BU clinical trial (NCT05169554, PACTR202209521256638) to shorten BU treatment.

Comparison of 8 weeks standard treatment (rifampicin plus clarithromycin) vs. 4 weeks standard plus amoxicillin/clavulanate treatment [RC8 vs. RCA4] to shorten Buruli ulcer disease therapy (the BLMs4BU trial): study protocol for a randomized controlled multi-centre trial in Benin.

BACKGROUND: Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans that affects skin, soft tissues, and bones, causing long-term morbidity, stigma, and disability. The recommended treatment for BU requires 8 weeks of daily rifampicin and clarithromycin together with wound care, physiotherapy, and sometimes tissue grafting and surgery. Recovery can take up to 1 year, and it may pose an unbearable financial burden to the household. Recent in vitro studies demonstrated that beta-lactams combined with rifampicin and clarithromycin are synergistic against M. ulcerans. Consequently, inclusion of amoxicillin/clavulanate in a triple oral therapy may potentially improve and shorten the healing process. The BLMs4BU trial aims to assess whether co-administration of amoxicillin/clavulanate with rifampicin and clarithromycin could reduce BU treatment from 8 to 4 weeks. METHODS: We propose a randomized, controlled, open-label, parallel-group, non-inferiority phase II, multi-centre trial in Benin with participants stratified according to BU category lesions and randomized to two oral regimens: (i) Standard: rifampicin plus clarithromycin therapy for 8 weeks; and (ii) Investigational: standard plus amoxicillin/clavulanate for 4 weeks. The primary efficacy outcome will be lesion healing without recurrence and without excision surgery 12 months after start of treatment (i.e. cure rate). Seventy clinically diagnosed BU patients will be recruited per arm. Patients will be followed up over 12 months and managed according to standard clinical care procedures. Decision for excision surgery will be delayed to 14 weeks after start of treatment. Two sub-studies will also be performed: a pharmacokinetic and a microbiology study. DISCUSSION: If successful, this study will create a new paradigm for BU treatment, which could inform World Health Organization policy and practice. A shortened, highly effective, all-oral regimen will improve care of BU patients and will lead to a decrease in hospitalization-related expenses and indirect and social costs and improve treatment adherence. This trial may also provide information on treatment shortening strategies for other mycobacterial infections (tuberculosis, leprosy, or non-tuberculous mycobacteria infections). TRIAL REGISTRATION: ClinicalTrials.gov NCT05169554 . Registered on 27 December 2021.

Seroprevalence of meningococcal serogroup C bactericidal antibodies in the Portuguese population, a decade after vaccine introduction in the National Immunisation Programme.

BACKGROUND: The incidence of invasive meningococcal disease due to serogroup C (MenC) decreased in Portugal since the introduction of the conjugate vaccine (MCC) in the free market in 2001 and in the National Immunisation Plan in 2006. Considering the potential waning of the antibody response reported in the literature, the different vaccination schemes that were used in our country over the past decade, and that Neisseria meningitidis serogroup C continues to circulate, the Portuguese population may currently be at increased risk of infection. In the absence of national data, we evaluated the seroprotection level of the Portuguese population against MenC, in order to identify the protected fraction of the population and ponder on the necessity of a booster dose of the MCC vaccine. METHODS: We measured serum bactericidal antibody levels against MenC in a representative sample of the population (n = 1500) aged 2-64 years who participated in the 2015/2016 National Serological Survey. RESULTS: A total of 31.1% (466/1500, 95%CI: 29-33%) of the individuals studied were protected against MenC. The geometric mean titre was 6.5. The proportion of seroprotected was particularly low in children aged 2-4 years (<16%) who received a single dose of the vaccine at 12 months of age (vaccination strategy since 2012). The proportion of seroprotected was higher (44.7% to 53.5%) in adolescent and young adults (15-24 years of age), resulting from vaccination during the catch-up campaign at 5-15 years of age. The highest protection rates were observed when the vaccine was administered during adolescence. CONCLUSION: The small fraction of population seroprotected, combined with the already known waning effect of the antibody response over time, may indicate that the Portuguese population will become progressively more exposed to the risk of infection. Taking in consideration our results, we recommend to change the current vaccination strategy and introduce a booster dose of the MCC vaccine during adolescence.

Epidemiology and genetic variability of respiratory syncytial virus in Portugal, 2014-2018.

INTRODUCTION: Respiratory syncytial virus (RSV) is associated with substantial morbidity and mortality since it is a predominant viral agent causing respiratory tract infections in infants, young children and the elderly. Considering the availability of the RSV vaccines in the coming years, molecular understanding in RSV is necessary. OBJECTIVE: The objective of the present study was to describe RSV epidemiology and genotype variability in Portugal during the 2014/15-2017/18 period. MATERIAL AND METHODS: Epidemiological data and RSV-positive samples from patients with a respiratory infection were collected through the non-sentinel and sentinel influenza surveillance system (ISS). RSV detection, subtyping in A and B, and sequencing of the second hypervariable region (HVR2) of G gene were performed by molecular methods. Phylogenetic trees were generated using the Neighbor-Joining method and p-distance model on MEGA 7.0. RESULTS: RSV prevalence varied between the sentinel (2.5%, 97/3891) and the non-sentinel ISS (20.7%, 3138/16779), being higher (P < 0.0001) among children aged <5 years. Bronchiolitis (62.9%, 183/291) and influenza-like illness (24.6%, 14/57) were associated (P < 0.0001) with RSV laboratory confirmation among children aged <6 months and adults ≥65 years, respectively. The HVR2 was sequenced for 562 samples. RSV-A (46.4%, 261/562) and RSV-B (53.6%, 301/562) strains clustered mainly to ON1 (89.2%, 233/261) and BA9 (92%, 277/301) genotypes, respectively, although NA1 and BA10 were also present until 2015/2016. CONCLUSION: The sequence and phylogenetic analysis reflected the relatively high diversity of Portuguese RSV strains. BA9 and ON1 genotypes, which have been circulating in Portugal since 2010/2011 and 2011/2012 respectively, predominated during the whole study period.

Performance of surveillance case definitions for respiratory syncytial virus infections through the sentinel influenza surveillance system, Portugal, 2010 to 2018.

BackgroundWell-established influenza surveillance systems (ISS) can be used for respiratory syncytial virus (RSV) surveillance. In Portugal, RSV cases are detected through the ISS using the European Union (EU) influenza-like illness (ILI) case definition.AimTo investigate clinical predictors for RSV infection and how three case definitions (EU ILI, a modified EU acute respiratory infection, and one respiratory symptom) performed in detecting RSV infections in Portugal.MethodsThis observational retrospective study used epidemiological and laboratory surveillance data (October 2010-May 2018). Associations between clinical characteristics and RSV detection were analysed using logistic regression. Accuracy of case definitions was assessed through sensitivity, specificity, and area under the receiver operating characteristic curve (AUC). A 0.05 significance level was accepted.ResultsThe study involved 6,523 persons, including 190 (2.9%) RSV cases. Among 183 cases with age information, RSV infection was significantly more frequent among individuals < 5 years (n = 23; 12.6%) and ≥ 65 years (n = 45; 24.6%) compared with other age groups (p < 0.0001). Cough (odds ratio (OR): 2.4; 95% confidence interval (CI): 1.2-6.5) was the best RSV-infection predictor considering all age groups, while shortness of breath was particularly associated with RSV-positivity among ≤ 14 year olds (OR: 6.7; 95% CI: 2.6-17.4 for 0-4 year olds and OR: 6.7; 95% CI: 1.5-28.8 for 5-14 year olds). Systemic symptoms were significantly associated with RSV-negative and influenza-positive cases. None of the case definitions were suitable to detect RSV infections (AUC = 0.51).ConclusionTo avoid underestimating the RSV disease burden, RSV surveillance within the Portuguese sentinel ISS would require a more sensitive case definition than ILI and, even a different case definition according to age.

Lessons learned from a prolonged norovirus GII.P16-GII.4 Sydney 2012 variant outbreak in a long-term care facility in Portugal, 2017.

OBJECTIVE: To investigate an outbreak of acute gastroenteritis caused by norovirus (NoV) in a long-term care facility (LTCF) in Portugal to describe and estimate its extent, and we implemented control measures. DESIGN: Outbreak investigation. METHODS: Probable cases were residents or staff members in the LTCF with at least 1 of the following symptoms: (1) diarrhea, (2) vomiting, (3) nausea, and/or (4) abdominal pain between October 31 and December 8, 2017. Confirmed cases were probable cases with positive NoV infection detected by real-time polymerase chain reaction (RT-PCR) and the same genotype in stool specimens. RESULTS: The outbreak was caused by NoV GII.P16-GII.4 Sydney 2012 variant and affected 146 people. The highest illness rates were observed in residents (97 of 335, 29%) and nurses (16 of 83, 19%). All 11 resident wards were affected. Data on cases and their working or living areas suggest that movement between wards facilitated the transmission of NoV, likely from person to person. CONCLUSIONS: The delay in the identification of the causative agent, a lack of restrictions of resident and staff movement between wards, and ineffective initial deep-cleaning procedures resulted an outbreak that continued for >1 month. The outbreak ended only after implementation of strict control measures. Recommendations for controlling future NoV outbreaks in LTCFs include emphasizing the need to control resident's movements and to restrict visitors, timely and effective environmental cleaning and disinfection, leave of absence for ill staff, and encouraging effective hand hygiene.

Challenging measles case definition: three measles outbreaks in three Health Regions of Portugal, February to April 2018.

We report three simultaneous measles outbreaks with 112 confirmed cases in three Health Regions of Portugal, from February to April 2018. The mean age of cases was 30 years, 79% worked in a healthcare setting and 87% were vaccinated. Genotype B3 was identified in 84 cases from the three outbreaks. Primary cases in each outbreak were imported. Several cases presented with modified measles, highlighting the importance of rethinking the measles case definition for vaccinated cases.

Outbreak Caused by Escherichia coli O18: K1: H7 Sequence Type 95 in a Neonatal Intensive Care Unit in Barcelona, Spain.

BACKGROUND: Escherichia coli is one of the most frequent causes of late-onset neonatal sepsis. The aim of this study was to characterize an outbreak of neonatal sepsis occurring in the neonatal intensive care unit of the Hospital Clinic of Barcelona from April to August 2013. METHODS: After presentation of the index case, all E. coli isolates from previously hospitalized neonates, health-care workers and neonates admitted to the neonatal intensive care unit from April to October 2013 were tested for K1 antigen positivity and epidemiologically compared by pulse-field gel electrophoresis. Furthermore, the E. coli K1 strains collected from neonates during this period were analyzed by different methods (serotyping, phylotyping, polymerase chain reaction of virulence factors, antimicrobial resistance and "in vitro" assays in Human Brain Microvascular Endothelial Cells (HBMEC)). RESULTS: An E. coli O18:K1:H7 sequence type 95 and phylogenetic group B2 strain was the cause of the outbreak involving 6 preterm neonates: 1 with late septicemia because of a urinary focus and 5 with late-onset septicemia and meningitis, 3 of whom died. All showed the same pulsotype, full resistance to ampicillin and intermediate resistance to gentamicin. The outbreak strain carried the pathogenicity island (PAI) IIJ96-like domain that could explain the high-grade bacteremia necessary to develop meningitis. CONCLUSIONS: All the E. coli isolates responsible for this outbreak belonged to a single clone suggesting a common source of infection, and it was categorized as O18:K1:H7. Despite the bacteria's pathogenicity has an important role in the severity of infection, the host-associated factors were crucial for the fatal outcomes.

Characterization of Vaginal Escherichia coli Isolated from Pregnant Women in Two Different African Sites.

The relevance of vaginal colonization of pregnant women by Escherichia coli is poorly understood, despite these strains sharing a similar virulence profile with other extraintestinal pathogenic E. coli producing severe obstetric and neonatal infections. We characterized the epidemiology, antimicrobial susceptibility and virulence profiles of 84 vaginal E. coli isolates from pregnant women from Rabat (Morocco) and Manhiça (Mozambique), two very distinct epidemiological settings. Low levels of antimicrobial resistance were observed to all drugs tested, except for trimethoprim-sulfamethoxazole in Manhiça, where this drug is extensively used as prophylaxis for opportunistic HIV infections. The most prevalent virulence factors were related to iron acquisition systems. Phylogroup A was the most common in Rabat, while phylogroups E and non-typeable were the most frequent in Manhiça. Regardless of the apparently "low virulence" of these isolates, the frequency of infections is higher and the outcomes more devastating in constrained-resources conditions, especially among pregnant women and newborns.

Vaginal versus Obstetric Infection Escherichia coli Isolates among Pregnant Women: Antimicrobial Resistance and Genetic Virulence Profile.

Vaginal Escherichia coli colonization is related to obstetric infections and the consequent development of infections in newborns. Ampicillin resistance among E. coli strains is increasing, which is the main choice for treating empirically many obstetric and neonatal infections. Vaginal E. coli strains are very similar to extraintestinal pathogenic E. coli with regards to the virulence factors and the belonging to phylogroup B2. We studied the antimicrobial resistance and the genetic virulence profile of 82 E. coli isolates from 638 vaginal samples and 63 isolated from endometrial aspirate, placental and amniotic fluid samples from pregnant women with obstetric infections. The prevalence of E. coli in the vaginal samples was 13%, which was significant among women with associated risk factors during pregnancy, especially premature preterm rupture of membranes (p<0.0001). Sixty-five percent of the strains were ampicillin-resistant. The E. coli isolates causing obstetric infections showed higher resistance levels than vaginal isolates, particularly for gentamicin (p = 0.001). The most prevalent virulence factor genes were those related to the iron uptake systems revealing clear targets for interventions. More than 50% of the isolates belonged to the virulent B2 group possessing the highest number of virulence factor genes. The ampicillin-resistant isolates had high number of virulence factors primarily related to pathogenicity islands, and the remarkable gentamicin resistance in E. coli isolates from women presenting obstetric infections, the choice of the most appropriate empiric treatment and clinical management of pregnant women and neonates should be carefully made. Taking into account host-susceptibility, the heterogeneity of E. coli due to evolution over time and the geographical area, characterization of E. coli isolates colonizing the vagina and causing obstetric infections in different regions may help to develop interventions and avoid the aetiological link between maternal carriage and obstetric and subsequent puerperal infections.

Effects of a Mutation in the gyrA Gene on the Virulence of Uropathogenic Escherichia coli.

Fluoroquinolones are among the drugs most extensively used for the treatment of bacterial infections in human and veterinary medicine. Resistance to quinolones can be chromosome or plasmid mediated. The chromosomal mechanism of resistance is associated with mutations in the DNA gyrase- and topoisomerase IV-encoding genes and mutations in regulatory genes affecting different efflux systems, among others. We studied the role of the acquisition of a mutation in the gyrA gene in the virulence and protein expression of uropathogenic Escherichia coli (UPEC). The HC14366M strain carrying a mutation in the gyrA gene (S83L) was found to lose the capacity to cause cystitis and pyelonephritis mainly due to a decrease in the expression of the fimA, papA, papB, and ompA genes. The levels of expression of the fimA, papB, and ompA genes were recovered on complementing the strain with a plasmid containing the gyrA wild-type gene. However, only a slight recovery was observed in the colonization of the bladder in the GyrA complement strain compared to the mutant strain in a murine model of ascending urinary tract infection. In conclusion, a mutation in the gyrA gene of uropathogenic E. coli reduced the virulence of the bacteria, likely in association with the effect of DNA supercoiling on the expression of several virulence factors and proteins, thereby decreasing their capacity to cause cystitis and pyelonephritis.

Antimicrobial resistance and virulence characterization among Escherichia coli clinical isolates causing severe obstetric infections in pregnant women.

The virulence markers and the antimicrobial resistance profiles of 78 Escherichia coli isolates causing obstetric infections accompanied by sepsis or not were studied. Adhesion-related virulence factors were the most prevalent markers. Low rates of resistance to the antimicrobial agents used as first-line therapy suggest their correct implementation in stewardship guidelines.

Characterization of CTX-M-14 and CTX-M-15 producing Escherichia coli strains causing neonatal sepsis.

Neonatal sepsis is a disease affecting newborns ≤1 month of age with clinical symptoms and positive blood cultures. The number of Escherichia coli strains causing neonatal sepsis resistant to the antibiotics used in the treatment is increasing. In this study, two E. coli strains causing sepsis in neonates of mothers infected with an E. coli strain harboring extended spectrum beta-lactamases were characterized. The bla(CTX-M-15) and the bla(CTX-M-14) genes were found in an IncFIA and nontypeable transferable plasmids, respectively. In addition, these highly virulent strains belonged to ST705 and ST156 clonal groups, respectively. The presence of strains, which are highly virulent and resistant to ampicillin, gentamicin, and cephalosporins, makes a change in empirical treatment necessary as well as an increase in the surveillance of these infections.