Detecção de DNA microbiano em amostras clínicas e culturas bacterianas

Mestrado em Microbiologia MolecularAs infecções do sistema circulatório constituem um campo prioritário de intervenção, com cerca de 200000 casos de bacterémia e fungémia anualmente nos EUA, com taxas de mortalidade associadas entre 20 e 50%. Os métodos convencionais de detecção de microrganismos a partir do sangue (hemocultura) atingiram um nível de desenvolvimento difícil de melhorar. No entanto, continua a ser desejável conseguir resultados com maior sensibilidade, capazes de contornar o efeito inibitório da antibioterapia empírica muitas vezes instituída previamente à colheita, em tempo clinicamente útil. Foi testado um sistema comercial de extracção, amplificação e detecção por PCR em tempo real, SeptiFast Test, (Roche), capaz de identificar 25 espécies de bactérias e fungos directamente a partir de amostras de sangue, em cerca de 6 horas. Os resultados obtidos, quando comparados com a evidência de infecção, mostraram sensibilidade semelhante à da hemocultura (67 e 68% respectivamente) e igual valor preditivo negativo (88%), no entanto o VPN do SeptiFast é limitado aos 25 microrganismos contemplados no teste molecular. A aplicação prática do teste beneficiaria da automatização do protocolo de extracção de ácidos nucleicos, sendo importante a selecção criteriosa dos pacientes testados de forma a que este seja mais vantajoso numa relação custo-benefício. O sistema SeptiFast representa um método complementar aos métodos convencionais, podendo em fornecer informação importante em tempo clinicamente útil. No que respeita à identificação de agentes bacterianos isolados em cultura, por vezes surgem dificuldades em chegar a uma identificação conclusiva pelos métodos fenotípicos convencionais. Foi implementada uma metodologia de sequenciação automática directa, usando o sistema MicroSeq 500 Bacterial Identification Kit (Applied Biosystems). Durante um ano foram estudadas 54 estirpes com perfis de identificação ambíguos pelos métodos tradicionais. Os resultados obtidos por sequenciação foram compatíveis com a suspeita clínica, ou confirmados pelos resultados do programa de controlo de qualidade externo NEQAS. A identificação por sequenciação tornou-se uma ferramenta importante no apoio à identificação bacteriana, particularmente em bacilos gram positivo, bacilos gram negativo fastidiosos, Streptococcus do grupo viridans e bactérias anaeróbias, organismos de identificação fenotípica problemática.Bloodstream infections remain a priority, with about 200000 cases of bacteremia and fungemia annually in the USA., with mortality rates between 20 and 50%. Blood culture systems have reached such development, that further improvements will be difficult. Molecular methods offer the possibility of higher sensitivity in turnaround time, overcoming the inhibitory effect of the empirical therapy often instituted prior to sample collection. A commercial system for microbial extraction, amplification and detention by real time PCR - SeptiFast Test, (Roche) was tested. This system detects 25 species of bacteria and fungi directly from whole blood, in about 6 hours. Compared with evidence of infection, the test showed a sensitivity value similar to blood culture (67 and 68% respectively) and the same negative predictive value (88%), however for SeptiFast the NPV is limited to the 25 microorganisms contemplated in the test. The molecular system would benefit from an automated nucleic acid extraction method, being of utmost importance the criteria for patient selection in order to improve the cost-benefit relation. SeptiFast Test seems to be a complementary method to conventional assays, able to provide reliable information in turnaround time. Most clinical bacterial isolates are quickly and accurately identified by conventional techniques used in the microbiology laboratory. However there are several situations in which molecular identification can significantly improve both the time to and accuracy of identification. An automatic sequencing method was implemented for bacterial identification - MicroSeq 500 Bacterial Identification Kit (Applied Biosystems). During one year, 54 isolates with ambiguous identification profiles by standard methods were studied. The sequence based identification results were compatible with clinical suspicion or with the results from an external quality control program - NEQAS. Sequence based identification became an important tool in the support of bacterial identification, particularly in gram positive bacilli, fastidious gram negative bacilli, anaerobic bacteria and viridans streptococci, microorganisms for witch phenotypic methods are presented with greater challenges

Tiragem molecular de pseudomonas aeruginosa pelo Sistema DiversiLab

The isolation of Pseudomonas aeruginosa in the blood of a newborn, as well as in the water of two incubators of UCIN of Oporto Hospitalar Centre, triggered the need to clarify the existence of an outbreak. Molecular typing of the three isolates was performed by Diversilab system, bioMérieux, which revealed a high degree of similarity in genomic profiles. The timely response allowed intervention measures to prevent the spread of these infections.  O isolamento de Pseudomonas aeruginosa na hemocultura de um recém-nascido, bem como na água de duas das incubadoras da UCIN do Centro Hospitalar do Porto, despoletou a necessidade de esclarecer a existência de um surto. Foi efectuada a tipagem molecular dos três isolados pelo sistema Diversilab, bioMérieux, que revelou elevado grau de similaridade nos perfis genómicos. A resposta atempada permitiu implementar medidas para evitar a disseminação destas infecções

Dolutegravir twice-daily dosing in children with HIV-associated tuberculosis: a pharmacokinetic and safety study within the open-label, multicentre, randomised, non-inferiority ODYSSEY trial

Background: Children with HIV-associated tuberculosis (TB) have few antiretroviral therapy (ART) options. We aimed to evaluate the safety and pharmacokinetics of dolutegravir twice-daily dosing in children receiving rifampicin for HIV-associated TB. Methods: We nested a two-period, fixed-order pharmacokinetic substudy within the open-label, multicentre, randomised, controlled, non-inferiority ODYSSEY trial at research centres in South Africa, Uganda, and Zimbabwe. Children (aged 4 weeks to <18 years) with HIV-associated TB who were receiving rifampicin and twice-daily dolutegravir were eligible for inclusion. We did a 12-h pharmacokinetic profile on rifampicin and twice-daily dolutegravir and a 24-h profile on once-daily dolutegravir. Geometric mean ratios for trough plasma concentration (Ctrough), area under the plasma concentration time curve from 0 h to 24 h after dosing (AUC0–24 h), and maximum plasma concentration (Cmax) were used to compare dolutegravir concentrations between substudy days. We assessed rifampicin Cmax on the first substudy day. All children within ODYSSEY with HIV-associated TB who received rifampicin and twice-daily dolutegravir were included in the safety analysis. We described adverse events reported from starting twice-daily dolutegravir to 30 days after returning to once-daily dolutegravir. This trial is registered with ClinicalTrials.gov (NCT02259127), EudraCT (2014–002632-14), and the ISRCTN registry (ISRCTN91737921). Findings: Between Sept 20, 2016, and June 28, 2021, 37 children with HIV-associated TB (median age 11·9 years [range 0·4–17·6], 19 [51%] were female and 18 [49%] were male, 36 [97%] in Africa and one [3%] in Thailand) received rifampicin with twice-daily dolutegravir and were included in the safety analysis. 20 (54%) of 37 children enrolled in the pharmacokinetic substudy, 14 of whom contributed at least one evaluable pharmacokinetic curve for dolutegravir, including 12 who had within-participant comparisons. Geometric mean ratios for rifampicin and twice-daily dolutegravir versus once-daily dolutegravir were 1·51 (90% CI 1·08–2·11) for Ctrough, 1·23 (0·99–1·53) for AUC0–24 h, and 0·94 (0·76–1·16) for Cmax. Individual dolutegravir Ctrough concentrations were higher than the 90% effective concentration (ie, 0·32 mg/L) in all children receiving rifampicin and twice-daily dolutegravir. Of 18 children with evaluable rifampicin concentrations, 15 (83%) had a Cmax of less than the optimal target concentration of 8 mg/L. Rifampicin geometric mean Cmax was 5·1 mg/L (coefficient of variation 71%). During a median follow-up of 31 weeks (IQR 30–40), 15 grade 3 or higher adverse events occurred among 11 (30%) of 37 children, ten serious adverse events occurred among eight (22%) children, including two deaths (one tuberculosis-related death, one death due to traumatic injury); no adverse events, including deaths, were considered related to dolutegravir. Interpretation: Twice-daily dolutegravir was shown to be safe and sufficient to overcome the rifampicin enzyme-inducing effect in children, and could provide a practical ART option for children with HIV-associated TB

Neuropsychiatric manifestations and sleep disturbances with dolutegravir-based antiretroviral therapy versus standard of care in children and adolescents: a secondary analysis of the ODYSSEY trial

BACKGROUND: Cohort studies in adults with HIV showed that dolutegravir was associated with neuropsychiatric adverse events and sleep problems, yet data are scarce in children and adolescents. We aimed to evaluate neuropsychiatric manifestations in children and adolescents treated with dolutegravir-based treatment versus alternative antiretroviral therapy. METHODS: This is a secondary analysis of ODYSSEY, an open-label, multicentre, randomised, non-inferiority trial, in which adolescents and children initiating first-line or second-line antiretroviral therapy were randomly assigned 1:1 to dolutegravir-based treatment or standard-of-care treatment. We assessed neuropsychiatric adverse events (reported by clinicians) and responses to the mood and sleep questionnaires (reported by the participant or their carer) in both groups. We compared the proportions of patients with neuropsychiatric adverse events (neurological, psychiatric, and total), time to first neuropsychiatric adverse event, and participant-reported responses to questionnaires capturing issues with mood, suicidal thoughts, and sleep problems. FINDINGS: Between Sept 20, 2016, and June 22, 2018, 707 participants were enrolled, of whom 345 (49%) were female and 362 (51%) were male, and 623 (88%) were Black-African. Of 707 participants, 350 (50%) were randomly assigned to dolutegravir-based antiretroviral therapy and 357 (50%) to non-dolutegravir-based standard-of-care. 311 (44%) of 707 participants started first-line antiretroviral therapy (ODYSSEY-A; 145 [92%] of 157 participants had efavirenz-based therapy in the standard-of-care group), and 396 (56%) of 707 started second-line therapy (ODYSSEY-B; 195 [98%] of 200 had protease inhibitor-based therapy in the standard-of-care group). During follow-up (median 142 weeks, IQR 124–159), 23 participants had 31 neuropsychiatric adverse events (15 in the dolutegravir group and eight in the standard-of-care group; difference in proportion of participants with ≥1 event p=0·13). 11 participants had one or more neurological events (six and five; p=0·74) and 14 participants had one or more psychiatric events (ten and four; p=0·097). Among 14 participants with psychiatric events, eight participants in the dolutegravir group and four in standard-of-care group had suicidal ideation or behaviour. More participants in the dolutegravir group than the standard-of-care group reported symptoms of self-harm (eight vs one; p=0·025), life not worth living (17 vs five; p=0·0091), or suicidal thoughts (13 vs none; p=0·0006) at one or more follow-up visits. Most reports were transient. There were no differences by treatment group in low mood or feeling sad, problems concentrating, feeling worried or feeling angry or aggressive, sleep problems, or sleep quality. INTERPRETATION: The numbers of neuropsychiatric adverse events and reported neuropsychiatric symptoms were low. However, numerically more participants had psychiatric events and reported suicidality ideation in the dolutegravir group than the standard-of-care group. These differences should be interpreted with caution in an open-label trial. Clinicians and policy makers should consider including suicidality screening of children or adolescents receiving dolutegravir

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved