Rapid testing for group B streptococcus during labour: a test accuracy study with evaluation of acceptability and cost-effectiveness

OBJECTIVE: To determine the accuracy, acceptability and cost-effectiveness of polymerase chain reaction (PCR) and optical immunoassay (OIA) rapid tests for maternal group B streptococcal (GBS) colonisation at labour. DESIGN: A test accuracy study was used to determine the accuracy of rapid tests for GBS colonisation of women in labour. Acceptability of testing to participants was evaluated through a questionnaire administered after delivery, and acceptability to staff through focus groups. A decision-analytic model was constructed to assess the cost-effectiveness of various screening strategies. SETTING: Two large obstetric units in the UK. PARTICIPANTS: Women booked for delivery at the participating units other than those electing for a Caesarean delivery. INTERVENTIONS: Vaginal and rectal swabs were obtained at the onset of labour and the results of vaginal and rectal PCR and OIA (index) tests were compared with the reference standard of enriched culture of combined vaginal and rectal swabs. MAIN OUTCOME MEASURES: The accuracy of the index tests, the relative accuracies of tests on vaginal and rectal swabs and whether test accuracy varied according to the presence or absence of maternal risk factors. RESULTS: PCR was significantly more accurate than OIA for the detection of maternal GBS colonisation. Combined vaginal or rectal swab index tests were more sensitive than either test considered individually [combined swab sensitivity for PCR 84% (95% CI 79-88%); vaginal swab 58% (52-64%); rectal swab 71% (66-76%)]. The highest sensitivity for PCR came at the cost of lower specificity [combined specificity 87% (95% CI 85-89%); vaginal swab 92% (90-94%); rectal swab 92% (90-93%)]. The sensitivity and specificity of rapid tests varied according to the presence or absence of maternal risk factors, but not consistently. PCR results were determinants of neonatal GBS colonisation, but maternal risk factors were not. Overall levels of acceptability for rapid testing amongst participants were high. Vaginal swabs were more acceptable than rectal swabs. South Asian women were least likely to have participated in the study and were less happy with the sampling procedure and with the prospect of rapid testing as part of routine care. Midwives were generally positive towards rapid testing but had concerns that it might lead to overtreatment and unnecessary interference in births. Modelling analysis revealed that the most cost-effective strategy was to provide routine intravenous antibiotic prophylaxis (IAP) to all women without screening. Removing this strategy, which is unlikely to be acceptable to most women and midwives, resulted in screening, based on a culture test at 35-37 weeks' gestation, with the provision of antibiotics to all women who screened positive being most cost-effective, assuming that all women in premature labour would receive IAP. The results were sensitive to very small increases in costs and changes in other assumptions. Screening using a rapid test was not cost-effective based on its current sensitivity, specificity and cost. CONCLUSIONS: Neither rapid test was sufficiently accurate to recommend it for routine use in clinical practice. IAP directed by screening with enriched culture at 35-37 weeks' gestation is likely to be the most acceptable cost-effective strategy, although it is premature to suggest the implementation of this strategy at present.

Intrapartum tests for group B streptococcus:accuracy and acceptability of screening

Objective: To assess the accuracy and acceptability of polymerase chain reaction (PCR) and optical immunoassay (OIA) tests for the detection of maternal group B streptococcus (GBS) colonisation during labour, comparing their performance with the current UK policy of risk factor-based screening. Design Diagnostic test accuracy study. Setting and population Fourteen hundred women in labour at two large UK maternity units provided vaginal and rectal swabs for testing. Methods The PCR and OIA index tests were compared with the reference standard of selective enriched culture, assessed blind to index tests. Factors influencing neonatal GBS colonisation were assessed using multiple logistic regression, adjusting for antibiotic use. The acceptability of testing to participants was evaluated through a structured questionnaire administered after delivery. Main outcome measures The sensitivity and specificity of PCR, OIA and risk factor-based screening. Results Maternal GBS colonisation was 21% (19-24%) by combined vaginal and rectal swab enriched culture. PCR test of either vaginal or rectal swabs was more sensitive (84% [79-88%] versus 72% [65-77%]) and specific (87% [85-89%] versus 57% [53-60%]) than OIA (P <0.001), and far more sensitive (84 versus 30% [25-35%]) and specific (87 versus 80% [77-82%]) than risk factor-based screening (P <0.001). Maternal antibiotics (odds ratio, 0.22 [0.07-0.62]; P = 0.004) and a positive PCR test (odds ratio, 29.4 [15.8-54.8]; P <0.001) were strongly related to neonatal GBS colonisation, whereas risk factors were not (odds ratio, 1.44 [0.80-2.62]; P = 0.2). Conclusion Intrapartum PCR screening is a more accurate predictor of maternal and neonatal GBS colonisation than is OIA or risk factor-based screening, and is acceptable to women. © RCOG 2010 BJOG An International Journal of Obstetrics and Gynaecology.

A comparison of different pre-lysis methods and extraction kits for recovery of Stretococcus agalacticae (Lancefield group B Streptococcus) DNA from whole blood

Sub-optimal recovery of bacterial DNA from whole blood samples can limit the sensitivity of molecular assays to detect pathogenic bacteria. We compared 3 different pre-lysis protocols (none, mechanical pre-lysis and achromopeptidasepre-lysis) and 5 commercially available DNA extraction platforms for direct detection of Group B Streptococcus (GBS) in spiked whole blood samples, without enrichment culture. DNA was extracted using the QIAamp Blood Mini kit (Qiagen), UCP Pathogen Mini kit (Qiagen), QuickGene DNA Whole Blood kit S (Fuji), Speed Xtract Nucleic Acid Kit 200 (Qiagen) and MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche Diagnostics Corp). Mechanical pre-lysis increased yields of bacterial genomic DNA by 51.3 fold (95% confidence interval; 31.6–85.1, p < 0.001) and pre-lysis with achromopeptidase by 6.1 fold (95% CI; 4.2–8.9, p < 0.001), compared with no pre-lysis. Differences in yield dueto pre-lysis were 2–3 fold larger than differences in yield between extraction methods. Including a pre-lysis step can improve the limits of detection of GBS using PCR or other molecular methods without need for culture.

Serotype distribution and antimicrobial susceptibility of group B streptococci in pregnant women: results from a Swiss tertiary centre.

OBJECTIVE To evaluate the rates of penicillin, clindamycin and erythromycin resistance and the serotype distribution among isolates of group B streptococcus (GBS) obtained from pregnant women at the University Hospital of Bern in Switzerland. METHODS We prospectively collected screening samples for GBS colonisation at the University Women's Hospital Bern, Switzerland, between March 2009 and August 2010. We included 364 GBS isolates collected from vaginal, cervical or vaginal-perianal swabs at any gestation time. The minimal inhibitory concentrations for penicillin, clindamycin and erythromycin were established using Etest with 24 hours of incubation, and inducible clindamycin resistance was tested with double disk diffusion tests. Serotyping was done with a rapid latex agglutination test or, if not conclusive, with polymerase chain-reaction (PCR) testing. We looked for significant associations between resistance patterns, age groups, serotype and ethnicity. RESULTS All isolates were susceptible to penicillin. Resistance rates were 14.5% for erythromycin and 8.2% for clindamycin. Of 364 isolates, 5.8% were susceptible to clindamycin but not to erythromycin, although demonstrating inducible clindamycin resistance. Hence, the final reported clindamycin resistance rate was 14%. Serotype III was the most frequent serotype (29%), followed by V (25%) and Ia (19%). Serotype V was associated with erythromycin resistance (p = 0.0007). In comparison with all other ethnicities, patients from Asia showed a higher proportion of erythromycin and clindamycin resistance (p = 0.018). No significant association between resistance patterns and age groups was found. CONCLUSION In pregnant women with GBS colonisation, penicillin is the antibiotic of choice for intrapartum prophylaxis to prevent neonatal early-onset GBS sepsis. In women with penicillin allergy and at high risk for anaphylactic reaction, clindamycin may be an alternative. The resistance rate for clindamycin at our institution was 14%; therefore, susceptibility must be tested before administration.

Phenotypic and molecular characterization of hyperpigmented group B Streptococci.

Group B Streptococcus (GBS) causes invasive infections in neonates, older adults and patients with comorbidities. β-hemolysin/cytolysin is an important GBS virulence factor. It is encoded by the cyl operon and confers GBS hemolytic activity. Isolates displaying hyperpigmentation are typically hyperhemolytic. Comparison of clonally identical isolates displaying different levels of pigmentation has shown transcriptional dysregulation due to mutations in components of the control of the virulence S/R (CovS/R) regulatory system. In addition, hyperpigmented isolates show decreased CAMP factor and decreased capsule thickness. In analogy to findings in group A Streptococcus, a pivotal role of CovS/R has been proposed in the host-pathogen interaction of invasive GBS infection. However, corresponding investigations on multiple clinical GBS isolates have not been performed. We prospectively collected hyperpigmented isolates found in a diagnostic laboratory and performed phenotypic, molecular and transcriptional analyses. In the period from 2008 to 2012, we found 10 isolates obtained from 10 patients. The isolates reflected both invasive pathogens and colonizers. In three cases, clonally identical but phenotypically different variants were also found. Hence, the analyses included 13 isolates. No capsular serotype was found to be significantly more frequent. Bacterial pigments were analyzed via spectrophotometry and for their hemolytic activity. Data obtained for typical absorbance spectra peaks correlated significantly with hemolytic activity. Molecular analysis of the cyl operon showed that it was conserved in all isolates. The covR sequence displayed mutations in five isolates; in one isolate, the CovR binding site to cylX was abrogated. Our results on clinical isolates support previous findings on CovR-deficient isogenic mutants, but suggest that - at least in some clinical isolates - for β-hemolysin/cytolysin and CAMP factor production, other molecular pathways may be involved.

Etude du développement de la réponse humorale dirigée contre la capsule polysaccharidique de Streptococcus suis et Streptococcus du groupe B

Streptococcus suis et Streptococcus du groupe B (GBS) sont deux bactéries encapsulées qui induisent des pathologies similaires chez l’homme et/ou l’animal, incluant septicémies et méningites. La capsule polysaccharidique (CPS) est un facteur de virulence clé de ces deux pathogènes et les anticorps (Ac) anti-CPS présentent un bon potentiel protecteur. Néanmoins, ces molécules sont faiblement immunogéniques et les mécanismes de la génération de la réponse humorale anti-CPS demeurent méconnus. L’objectif principal de cette thèse était d’évaluer les caractéristiques et les mécanismes du développement de la réponse Ac dirigée spécifiquement contre les CPS de S. suis et GBS, ainsi que l’effet de la biochimie de la CPS dans cette réponse. Nous avons étudié S. suis types 2 et 14 et GBS types III et V, dont les CPS présentent plusieurs similarités dans leurs compositions et leurs structures, incluant la présence d’acide sialique, un sucre potentiellement immunosuppresseur, tout en possédant une antigénicité propre. Nous avons tout d’abord analysé la nature de la réponse Ac anti-CPS sérique face à la bactérie entière. Les souris infectées par S. suis développent une réponse très faible (S. suis type 2) voire insignifiante (S. suis type 14) de profil isotypique restreint à l’IgM et sont incapables de monter une réponse mémoire efficace face à une seconde infection. Un profil similaire est obtenu chez le porc infecté par S. suis type 2. On détecte des titres d’IgM anti-CPS significatifs chez les souris infectées par GBS (type III ou V). Toutefois, la magnitude de la réponse reste globalement faible et aucune commutation de classe n’est observée. Nous avons ensuite examiné l’influence de la biochimie de la CPS sur ces profils de réponse en conduisant des expériences avec la CPS hautement purifiée de ces pathogènes. Tandis que la CPS de GBS type III administrée aux souris conserve des propriétés immunogéniques similaires à celles observées durant l’infection par la bactérie intacte, les CPS de S. suis type 2 et GBS type V perdent toute capacité à induire une réponse Ac spécifique. L’analyse de l’interaction in vitro des CPS avec les cellules dendritiques (DC) murines, des acteurs clés dans la détection des pathogènes et l’orchestration des réponses immunitaires subséquentes, révèle que ces molécules stimulent la production de niveaux conséquents de chémokines via différents récepteurs. Néanmoins, les CPS sont inaptes à induire la sécrétion de cytokines et elles interfèrent avec la capacité des DC à exprimer BAFF, une cytokine clé dans la différenciation des lymphocytes B en plasmocytes. L’utilisation de CPS chimiquement désialylées démontre que l’acide sialique ne joue aucun rôle immunosuppresseur majeur dans le développement de la réponse Ac dirigée contre les CPS purifiées de S. suis ou GBS, ni sur l’interaction des CPS avec les DC in vitro, ni sur profil de la réponse in vivo. D’autres propriétés biochimiques intrinsèques à ces CPS seraient responsables de l’inaptitude de l’hôte infecté à monter une réponse Ac adéquate et les identifier constituera un outil précieux pour une meilleure compréhension de l’immunopathogénèse de S. suis et GBS ainsi que pour développer des moyens de lutte efficaces contre ces bactéries.

Cost-effectiveness of universal prophylaxis in pregnancy with prior group B streptococci colonization.

OBJECTIVE: To estimate the costs and outcomes of rescreening for group B streptococci (GBS) compared to universal treatment of term women with history of GBS colonization in a previous pregnancy. STUDY DESIGN: A decision analysis model was used to compare costs and outcomes. Total cost included the costs of screening, intrapartum antibiotic prophylaxis (IAP), treatment for maternal anaphylaxis and death, evaluation of well infants whose mothers received IAP, and total costs for treatment of term neonatal early onset GBS sepsis. RESULTS: When compared to screening and treating, universal treatment results in more women treated per GBS case prevented (155 versus 67) and prevents more cases of early onset GBS (1732 versus 1700) and neonatal deaths (52 versus 51) at a lower cost per case prevented ($8,805 versus $12,710). CONCLUSION: Universal treatment of term pregnancies with a history of previous GBS colonization is more cost-effective than the strategy of screening and treating based on positive culture results.

Sépsis neonatal com meningite a streptococcus do grupo B na era do rastreio e da quimioprofilaxia : a propósito de um caso clínico

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Genome-wide mapping of cystitis due to Streptococcus agalactiae and Escherichia coli in mice identifies a unique bladder transcriptome that signifies pathogen-specific antimicrobial defense against urinary tract infection

The most common causes of urinary tract infections (UTIs) are Gram-negative pathogens such as Escherichia coli; however, Gram-positive organisms including Streptococcus agalactiae, or group B streptococcus (GBS), also cause UTI. In GBS infection, UTI progresses to cystitis once the bacteria colonize bladder, but the host responses triggered in the bladder immediately following infection are largely unknown. Here, we used genome-wide expression profiling to map the bladder transcriptome of GBS UTI in mice infected transurethrally with uropathogenic GBS that was cultured from a 35 year-old women with cystitis. RNA from bladders was applied to Affymetrix Gene-1.0ST microarrays; qRT-PCR was used to analyze selected gene responses identified in array datasets. A surprisingly small significant gene list of 172 genes was identified at 24h; this compared to 2507 genes identified in a side-by-side comparison with uropathogenic E. coli (UPEC). No genes exhibited significantly altered expression at 2h in GBS-infected mice according to arrays despite high bladder bacterial loads at this early time point. The absence of a marked early host response to GBS juxtaposed with broad-based bladder responses activated by UPEC at 2h. Bioinformatics analyses including integrative systems-level network mapping revealed multiple activated biological pathways in the GBS cystitis transcriptome that regulate leukocyte activation, inflammation, apoptosis, and cytokine-chemokine biosynthesis. These findings define a novel, minimalistic type of bladder host response triggered by GBS UTI, which comprises collective antimicrobial pathways that differ dramatically from those activated by UPEC. Overall, this study emphasizes the unique nature of bladder immune activation mechanisms triggered by distinct uropathogens.

Infection and pathology in Queensland grouper, Epinephelus lanceolatus, (Bloch), caused by exposure to Streptococcus agalactiae via different routes

Since 2007, 96 wild Queensland groupers, Epinephelus lanceolatus, (Bloch), have been found dead in NE Australia. In some cases, Streptococcus agalactiae (Group B Streptococcus, GBS) was isolated. At present, a GBS isolate from a wild grouper case was employed in experimental challenge trials in hatchery-reared Queensland grouper by different routes of exposure. Injection resulted in rapid development of clinical signs including bilateral exophthalmia, hyperaemic skin or fins and abnormal swimming. Death occurred in, and GBS was re-isolated from, 98% fish injected and was detected by PCR in brain, head kidney and spleen from all fish, regardless of challenge dose. Challenge by immersion resulted in lower morbidity with a clear dose response. Whilst infection was established via oral challenge by admixture with feed, no mortality occurred. Histology showed pathology consistent with GBS infection in organs examined from all injected fish, from fish challenged with medium and high doses by immersion, and from high-dose oral challenge. These experimental challenges demonstrated that GBS isolated from wild Queensland grouper reproduced disease in experimentally challenged fish and resulted in pathology that was consistent with that seen in wild Queensland grouper infected with S. agalactiae.

Infection and pathology in Queensland grouper, Epinephelus lanceolatus, (Bloch), caused by exposure to Streptococcus agalactiae via different routes

Since 2007, 96 wild Queensland groupers, Epinephelus lanceolatus, (Bloch), have been found dead in NE Australia. In some cases, Streptococcus agalactiae (Group B Streptococcus, GBS) was isolated. At present, a GBS isolate from a wild grouper case was employed in experimental challenge trials in hatchery-reared Queensland grouper by different routes of exposure. Injection resulted in rapid development of clinical signs including bilateral exophthalmia, hyperaemic skin or fins and abnormal swimming. Death occurred in, and GBS was re-isolated from, 98% fish injected and was detected by PCR in brain, head kidney and spleen from all fish, regardless of challenge dose. Challenge by immersion resulted in lower morbidity with a clear dose response. Whilst infection was established via oral challenge by admixture with feed, no mortality occurred. Histology showed pathology consistent with GBS infection in organs examined from all injected fish, from fish challenged with medium and high doses by immersion, and from high-dose oral challenge. These experimental challenges demonstrated that GBS isolated from wild Queensland grouper reproduced disease in experimentally challenged fish and resulted in pathology that was consistent with that seen in wild Queensland grouper infected with S. agalactiae.

Promotion of fibronectin independent invasion by C5a peptidase into epithelial cells in group A Streptococcus

Background & objectives: Group A Streptococcus, causative agent of several clinical manifestations codes for multiple protein invasins which help the bacterium to enter non-phagocytic cells. C5a peptidase (SCPA) is a surface protein conserved among different serotypes of M1 strain. The present study was taken up to study SCPA promoted fibronectin independent entry of GAS into epithelial cells. Methods: An isogenic 90226 emm1DeltaAB (M1(-)) mutant was constructed, with thermosensitive pGhost vector. This isogenic M1(-) mutant expressed SCPA on the surface as determined by Western blotting and immunofluorescence. Results: On preincubation with anti-SCPA serum, the isogenic M1(-) strain exhibited 54 per cent decreased invasion as compared to the bacteria incubated with control serum. Also, purified recombinant SCPA proteins blocked internalization of M1(-) streptococci into HEp-2 cells. The M1(-) strain invaded at the same efficiency in the presence or absence of fibronectin. Interpretation & conclusion: These results suggested that SCPA acted as a potential invasin of group A streptococcus and promoted invasion independent of fibronectin.

Aderência, invasão e persistência intracelular de estreptococos do grupo B em células epiteliais respiratórias A549

Estreptococos do grupo B (EGB) comumente colonizam adultos saudáveis, sem sintomas, mas sob certas circunstâncias possui a capacidade de invadir tecidos do hospedeiro, evadir da detecção imunológica e causar doenças invasivas graves. Por conseguinte, os EGB continuam sendo uma das principais causas de mortalidade neonatal, pneumonia, sepse e meningite. Contudo, a patogênese desta infecção ainda está pouco elucidada. O sorotipo V é freqüentemente associado à doença invasiva em mulheres adultas não gestantes e o segundo mais prevalente em mulheres grávidas. O principal objetivo deste trabalho foi estudar a aderência, invasão e persistência intracelular de amostras pertencentes ao sorotipo V (88641-vagina/portador e 90186-sangue/paciente) usando as células epiteliais respiratórias A549. As amostras de EGB demonstraram capacidade de aderir e invadir as células epiteliais A549, mas somente a amostra 90186-sangue apresentou maior invasão quando comparada com a de vagina (P <0.001). Ambas as amostras demonstraram persistência intracelular sem replicação no interior das células A549. Apenas o isolado 90186-sangue sobreviveu dentro das células epiteliais até 24h de incubação (P <0,05). A fusão dos lisossomas das células epiteliais com vacúolos contendo bactérias foi observada em células A549 tratadas com Lyso Tracker Grenn DND-26 para todas as amostras testadas. Nossos dados indicam pela primeira vez que as amostras viáveis do sorotipo V permanecem dentro de vacúolos ácidos epiteliais. Curiosamente, a amostra 90186- sangue induziu vacuolização celular e a amostra 88641-vagina promoveu a morte celular após 7h de incubação. Finalmente, nossos resultados aumentam o nosso conhecimento sobre eventos celulares da fagocitose e da patogênese das doenças invasivas promovidas pelos EGB.

Papel de metaloproteases de Estreptococos do grupo B na interação,viabilidade celular e indução de apoptose e necrose em células endoteliais e epiteliais humanas

Estreptococos do grupo B (EGB) é a principal causa de sepse e meningite neonatal e tem sido recentemente reconhecido como patógeno responsável por infecções invasivas em adultos imunocomprometidos (idosos ou portadores de doenças crônicas). Os EGB produzem inúmeras enzimas extracelulares, várias das quais interagem com o sistema imune do hospedeiro e são importantes durante a interação EGB-hospedeiro, bem como para o desenvolvimento da doença. Estudos anteriores mostraram que metaloproteases estão envolvidas em várias vias metabólicas em diferentes tipos celulares. Por esta razão, nós decidimos investigar o possível envolvimento de metaloproteases de EGB durante a interação celular e apoptose/necrose induzida pelo micro-organismo em células endoteliais da veia umbilical humana (HUVEC) e da linhagem de epitélio respiratório (A549). Tratamento de EGB com inibidores de metaloproteases (EDTA, EGTA e FEN) não induziu alterações no crescimento bacteriano, mas promoveu alterações na expressão de proteínas de superfície, capacidade adesiva e perfil de sobrevivência intracelular do patógeno. O EGB e o sobrenadante do crescimento bacteriano (meio condicionado; MC) promoveram a morte das células HUVEC e A549. Contudo, o tratamento com inibidores de metaloproteases restauraram a viabilidade celular induzida pelos EGB e o MC, sugerindo que metaloproteases bacteriana estão envolvidas no rompimento da barreira celular, promovendo a disseminação bacteriana. Este trabalho descreve pela primeira vez apoptose e necrose induzidas pelo EGB e MC em HUVEC e células A549 após 24h de incubação, respectivamente. Nós também observamos redução da pró-caspase-3 após infecção das HUVEC com EGB e MC, sugerindo ativação da caspase-3. Além disso, o aumento da expressão da proteína pró-apoptótica Bax e diminuição dos níveis da proteína anti-apoptótica Bcl-2 em HUVEC, demonstram o envolvimento do mecanismo apoptótico mitocondrial (via intrínseca). A melhor compreensão das bases moleculares da patogênese do EGB contribui para identificar novas moléculas bacterianas e hospedeiras que podem representar novos alvos terapêuticos ou imunoprofiláticos contra a doença causada por esse patógeno neonatal.