Mid-trimester microbial invasion of the amniotic cavity and the risk of preterm birth.

OBJECTIVE: To evaluate the rate of mid-trimester microbial invasion of the amniotic cavity (MIAC) in asymptomatic women and its association with preterm birth. STUDY DESIGN: This is a prospective cohort study of asymptomatic women undergoing mid-trimester amniocentesis for genetic testing between 14 and 24 weeks of gestation. For each participant, a sample of amniotic fluid was incubated in an aerobic and anaerobic facultative culture media and another sample was tested for the presence of specific Mycoplasma species (Ureaplasma urealyticum, Ureaplasma parvum, and Mycoplasma hominis) using quantitative-PCR. Results were not revealed to the participants or their health care providers. All participants were followed until delivery. MIAC was defined by a positive culture or a positive PCR for Mycoplasma species. The primary outcome was a spontaneous preterm birth or preterm premature rupture of membranes before 35 weeks of gestation. RESULTS: We included 812 women at a median gestational age of 16 5/7 (interquartile: 15 6/7-17 4/7) weeks. Twenty-six (3.2%) had a spontaneous delivery before 35 weeks. We observed no case of positive PCR for Mycoplasma species and 4 (0.5%) cases of positive culture that were all considered to be skin contaminants. None of those four cases was associated with preterm birth. Nulliparity, low family income and history of preterm birth were associated with spontaneous delivery before 35 weeks. CONCLUSION: We found no case of mid-trimester MIAC using a combination of culture and Mycoplasma-specific PCR techniques in a large cohort of low-risk asymptomatic pregnant women. We estimate that mid-trimester MIAC is rare in low-risk population but more sensitive and broad-range microbiologic techniques, such as 16S DNA detection by PCR, could be further evaluated.

Oropharyngeal Carriage of Kingella kingae and Transient Synovitis of the Hip in Young Children: A Case-control Study.

BACKGROUND: Transient synovitis of the hip affects mostly preschool children, and its etiology is unknown. Kingella kingae has been identified recently as a common etiologic agent of osteoarticular infections (OAI) in young children and could potentially be associated to transient synovitis of the hip. The main objective of this study was to evaluate the association between transient synovitis of the hip and oropharyngeal carriage of K. kingae among preschool children. METHODS: This was a prospective case-control study conducted at a tertiary care pediatric emergency department. Cases were children between 6 and 71 months of ages with a diagnosis of transient synovitis of the hip. For each transient synovitis case, an age-matched control was recruited among children presenting for a trauma. A second control group included children with any OAI. The independent variable was the presence of oropharyngeal K. kingae identified by a specific polymerase chain reaction assay. The primary analysis was the association between oropharyngeal K. kingae carriage and final diagnosis. RESULTS: A total of 73 children were included in the study. Among them, 25 had a transient synovitis, 16 an OAI, and 22 controls. Baseline demographics were similar between the groups. There was no difference in oropharyngeal carriage of K. kingae for children with transient synovitis (5/25; 0.20) in comparison to controls (3/22; 0.14), while it was higher for children with OAI (10/16; 0.63). CONCLUSIONS: There is no association between oropharyngeal K. kingae and transient synovitis of the hip among preschool children.

Efficient SARS-CoV-2 detection in unextracted oro-nasopharyngeal specimens by rRT-PCR with the Seegene Allplex™ 2019-nCoV assay.

BACKGROUND: The fight against the COVID-19 pandemic has created an urgent need to rapidly detect infected people. The challenge for clinical laboratories has been finding a high throughput, cost-efficient, and accurate testing method in the context of extraction reagents shortage on a global scale. To answer this need, we studied SARS-CoV-2 detection in oro-nasopharyngeal (ONP) swabs stored in Universal Transport Media (UTM) or in RNase-free water by rRT-PCR with Seegene Allplex™ 2019-nCoV assay without RNA extraction. RESULTS: Optimal results were obtained when swabs stored in UTM were diluted 1/5 and 1/2 in RNase-free water. Thermal lysis before rRT-PCR testing slightly improved detection rate. In addition, proteinase K (PK) treatment allowed for a significant reduction of invalid results and increased sensitivity for detection of low viral load specimens. In a panel of positive samples with all 3 viral genes amplified and N gene Cycle threshold values (Ct values) from 15 to 40, our detection rate was 98.9% with PK and 94.4% without. In a challenging panel of low positive samples with only the N gene being detectable at Ct values > 30, detection rate was increased from 53.3 to 76.7% with the addition of PK, and invalid rate fell off from 18.3 to 0%. Furthermore, we demonstrated that our method reliably detects specimens with Ct values up to 35, whereas false negative samples become frequent above this range. Finally, we show that swabs should be stored at - 70 °C rather than 4 °C when testing cannot be performed within 72 h of collection. CONCLUSION: We successfully optimized the unextracted rRT-PCR process using the Seegene Allplex™ 2019-nCoV assay to detect SARS-CoV-2 RNAs in nasopharyngeal swabs. This improved method offers cost savings and turnaround time advantages compared to automated extraction, with high efficiency of detection that could play an important role in the surveillance of Covid-19.

Impact of the adolescent pertussis booster dose on the incidence of pertussis in British Columbia and Quebec, Canada.

Impact of an adolescent tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis (Tdap) vaccine program was assessed in the provinces of British Columbia and Quebec, Canada. In both provinces, the Tdap booster has been in place since 2004, targeting Grade 9 students (14-15-years-of-age). Incidence rate ratios (IRRs) standardizing notification rates among teens 15-19-years-old to infants <1-year-old decreased following introduction of the Tdap program and were significantly halved during the 2009-2012 post-Tdap versus 2000-2003 pre-Tdap period. This program impact, however, is tempered by the observation that pertussis incidence among 15-19-year-olds was already lower than any other pediatric age group, following gradual decline from pre-teen rates even before the Tdap program. The risk of hospitalization among adolescents 15-19-years-old was also low throughout at <1/100,000. Furthermore, IRRs increased in 2013-2017 when an increasing proportion of 15-19-year-olds were primed with acellular pertussis vaccine only, suggesting short-lived Tdap booster-dose effectiveness that warrants further monitoring.

Multiplex Polymerase Chain Reaction Panel for Suspected Pertussis: What About a Positive Mycoplasma pneumoniae Result?

BACKGROUND: The use of bacterial multiplex polymerase chain reaction (PCR) in children with suspected pertussis sometimes yields unexpected positive results for Mycoplasma pneumoniae. We aimed to evaluate the clinical significance of positive M. pneumoniae results in this population. METHODS: Retrospective cohort of consecutive patients with suspected pertussis tested with a bacterial multiplex PCR (including Bordetella pertussis and M. pneumoniae) between June 2015 and March 2017. Medical records were reviewed to compare demographics, clinical presentations and outcomes of patients positive for M. pneumoniae with those positive for B. pertussis and those with negative results, using multivariable logistic regression. RESULTS: A total of 1244 patients were included as follows: 56 (4.5%) with M. pneumoniae, 116 (9.3%) with B. pertussis and 1029 (82.7%) with negative results. Mean age was respectively 4.8 years, 6.5 years and 2.8 years (P < 0.05). Children with M. pneumoniae were less likely to present with cardinal symptoms of pertussis such as paroxysmal cough [adjusted odds ratio (OR): 0.19, 95% confidence interval (CI): 0.08-0.40) but were more likely to have fever (adjusted OR: 10.53, 95% CI: 3.54-39.49) and other nonspecific respiratory symptoms compared with children with B. pertussis. Children with M. pneumoniae had very similar clinical presentations to those with a negative PCR, but were more likely to have radiologically confirmed pneumonia (adjusted OR: 5.48, 95% CI: 2.96-9.99) and were less likely to be diagnosed with a concomitant viral infection (adjusted OR: 0.32, 95% CI: 0.07-0.99). CONCLUSIONS: In children with suspected pertussis, the detection of M. pneumoniae is clinically relevant. However, the impact of this finding on patients' outcome is still unclear.

Clinical and Microbiological Characteristics of Invasive Group A Streptococcal Infections Before and After Implementation of a Universal Varicella Vaccine Program.

Since the introduction of the varicella vaccine to the routine immunization schedule, we have observed a 70% reduction in the rate of varicella-associated invasive group A streptococcal infections (IGASI). In the mean time, the clinical presentation of IGASI and microbiological characteristics of GAS strains have changed significantly.

First documented case of human infection with ovine Shiga-toxin-producing Escherichia coli serotype O52:H45.

We report a concurrent case of infection with non-O157 Shiga-toxin-producing Escherichia coli (STEC) strain in an 8-month-old child. Laboratory and epidemiological investigations indicated child exposure to contaminated sheep meat following the Muslim feast of sacrifice (Eid al-Adha). Microbiological and molecular typing confirmed that the ovine strain O52:H45 (stx1+, eae-, hlyA-) was the causal agent. This is the first documented case of human infection to this STEC serotype.

Invasive group A Streptococcus disease in French-Canadian children is not associated with a defect in MyD88/IRAK4-pathway.

BACKGROUND: Beta-hemolytic Group A Streptococcus invasive disease (iGASd) has been subject to intense research since its re-emergence in the late 1980s. In Quebec, an increase in the number of severe iGASd cases has recently been observed. Because of the inter-individual variability in the severity of iGASd, a hereditary predisposition to invasive disease can be suspected. Given that iGASd occurs in MyD88- and IRAK4-deficient patients, although rarely, the increasing frequency of iGASd in the population of French-Canadian children may be associated with a deficiency in the host's innate immune response. METHODS: In this report, we assessed the influence of: (i) bacterial genotype and virulence factors, (ii) immune-cellular features, and (iii) Myd88/IRAK4-dependent response to GAS in vitro on the susceptibility to iGASd in a paediatric cohort of 16 children: 11 French-Canadian and 5 from diverse origin. FINDINGS: GAS virulence factors and genotype are not implicated in the susceptibility toward iGASd, and cellular and MyD88/IRAK4 deficiencies are excluded in our patients. CONCLUSIONS: Although it has been shown that the MyD88/IRAK4-dependent signal is involved in the response to invasive GAS, our data indicates that a MyD88/IRAK4-mediated signalling defect is not the main factor responsible for the susceptibility to severe iGASd in a paediatric population from the province of Quebec.

Comparison of three different methods for detection of Shiga toxin-producing Escherichia coli in a tertiary pediatric care center.

Shiga toxin-producing Escherichia coli (STEC) is a well-known cause of sporadic and epidemic food-borne gastroenteritis. A low infectious dose, approximately 10 microorganisms, is sufficient to cause disease that may lead to hemolytic-uremic syndrome. The objective of this study was to compare the performances of an in-house real-time PCR, a commercial enzyme immunoassay (EIA) (Premier EHEC; Meridian Bioscience), and culture on sorbitol MacConkey agar for the detection of STEC in a tertiary care pediatric hospital. Of 632 stool samples tested, 21 were positive for STEC. All were detected by PCR, 6 were detected by EIA, and only 5 O157 STEC isolates were identified by culture. Among the 15 specimens falsely negative by EIA, there were 9 Stx1, 2 Stx2, and 4 Stx1 and Stx2 STEC isolates. The latter group included 2 O157 STEC isolates that would have been missed if only EIA had been performed. To our knowledge, this is the first prospective study performed in a pediatric hospital which demonstrates the superiority of PCR over EIA for the detection of STEC. We conclude that PCR is specific and more sensitive than EIA. PCR should be considered for routine use in clinical settings where molecular detection facilities are available. Its lower limit of detection, equivalent to the infectious dose, is an obvious advantage for patient care and public health surveillance.

The origin of Fusobacterium nucleatum involved in intra-amniotic infection and preterm birth.

OBJECTIVE: To evaluate the potential oral origin of Fusobacterium nucleatum found in amniotic fluid of women at high risk of preterm birth. METHODS: A transversal study nested into a cohort study of women with preterm labor and/or preterm premature rupture of membranes was undergone. Women with the presence of F. nucleatum in the amniotic fluid and their respective partners were invited to be examined for their periodontal health after delivery, and samples of saliva and subgingival plaque were collected. For each couple, specific PCR detection of Fusobacterium species was performed on each oral sample, and the DNA sequences were compared with the one obtained from amniotic fluid. RESULTS: Three women, all in preterm labor with intact membranes, were included. Intra-amniotic sludge was observed in all of them. A strain of F. nucleatum with 100% sequence identity with the strain detected in the amniotic fluid was found in the oral samples of one of them and of two partners. CONCLUSION: This study suggests that intra-amniotic F. nucleatum could originate from the patient's or the partner's oral microflora.

Invasive pneumococcal disease after implementation of a reduced three-dose pneumococcal conjugate vaccine program: a pediatric tertiary care center experience.

Following the implementation of a government-sponsored reduced three-dose (2 + 1) heptavalent conjugate pneumococcal vaccine (PCV7) program, we report a 61.4% decrease in the number of cases of invasive pneumococcal diseases (IPD) treated at our institution. Four years after the implementation of the three-dose reduced vaccine program, only 7.4% of IPD were caused by PCV7 serotypes, and there was an increase in the proportion of IPD caused by nonPCV7 serotypes; serotype 19A represented 40.7% of the strains isolated during the last year of the study. These results, similar to those previously observed with a regular four-dose (3 + 1) PCV7 schedule, are reassuring as to the effectiveness of a reduced three-dose (2 + 1) PCV7 program. Increasing numbers of IPD caused by nonPCV7 serotypes warrant the use of a new conjugate pneumococcal vaccine that contains serotype 19A.

Clinical and microbiologic characteristics of group A streptococcal necrotizing fasciitis in children.

An increase in the incidence of Group A streptococcal necrotizing fasciitis has recently been observed in Montréal, Canada. Clinical features of children hospitalized for invasive Group A streptococcal infections and various virulence factor genes of the bacteria were concomitantly analyzed. It was determined that varicella and presence of speC gene in group A streptococcal strains were associated with necrotizing fasciitis.

Bacteriology of amniotic fluid in women with suspected cervical insufficiency.

OBJECTIVE: To determine the prevalence of mid-trimester microbial invasion of the amniotic cavity (MIAC) in women with suspected cervical insufficiency. METHODS: A prospective observational cohort study was performed in women with suspected cervical insufficiency and visible fetal membranes who were undergoing amniocentesis to rule out MIAC between 16 and 26 weeks of gestation. Women with preterm premature rupture of membranes, regular uterine contractions, or who had a cervical cerclage were excluded. Gram staining of amniotic fluid, glucose and lactate dehydrogenase (LDH) levels in amniotic fluid, and aerobic and anaerobic amniotic fluid cultures were performed, along with polymerase chain reaction (PCR) for the detection of Ureaplasma and Mycoplasma species. RESULTS: Fifteen women with a mean gestational age of 22.6 +/- 2.3 weeks were included in the study. The diagnosis of MIAC was confirmed in 47% (7/15), of whom 20% (3/15) were infected with more than one bacterial strain and 33% (5/15) with Ureaplasma species. According to receiver-operator curve analyses, amniotic fluid levels of glucose were associated with MIAC (P = 0.02), but not amniotic fluid LDH (P = 0.25). CONCLUSION: MIAC is present in approximately one half of women with suspected cervical insufficiency and visible fetal membranes at speculum examination.

Epidemiological, biochemical and antimicrobial susceptibility characteristics of Streptococcus pseudoporcinus isolated in Quebec, Canada, from 1997 to 2006.

From 1997 to 2006, in the province of Quebec, Canada, 15 isolates of Streptococcus pseudoporcinus from 1 urine and 14 vaginorectal cultures were recovered from the genitourinary tract of pregnant women. All these women originated from the Caribbean or sub-Saharan Africa (P=0.00045 compared with a suitable control group). The S. pseudoporcinus isolates were compared to eight isolates of Streptococcus porcinus identified in Quebec from 1995 to 2006, all from animals, of which five were swine. 16S rRNA gene sequencing was required to differentiate between S. pseudoporcinus and S. porcinus animal isolates.

Eradication of intra-amniotic Streptococcus mutans in a woman with a short cervix.

BACKGROUND: Microbial invasion of the amniotic cavity (MIAC) remains an important pathogenetic factor in preterm births. Although most MIACs seem to originate in vaginal microflora, other reservoirs such as the oral microflora have been suggested. CASE: We report a case of successful eradication of Streptococcus mutans (a bacterium generally found in dental plaque) from the amniotic cavity, using ampicillin, gentamycin, and azythromycin in a woman who presented with threatened preterm labour and a short cervix at 25 weeks' gestation. She delivered at 34 weeks' gestation and was treated for tooth decay 12 weeks after delivery. CONCLUSION: Intra-amniotic infection should be suspected in the presence of a short cervix during the second trimester. Appropriate parenteral antibiotics can eradicate intra-amniotic infection and potentially prolong pregnancy.

Sensitivities of antigen detection and PCR assays greatly increased compared to that of the standard culture method for screening for group B streptococcus carriage in pregnant women.

Group B streptococcus (GBS) is a major cause of serious infections in neonates. The 2002 revised guidelines of the Centers for Disease Control and Prevention (CDC) for the prevention of perinatal GBS disease recommend that all pregnant women be screened for GBS carriage at between 35 and 37 weeks of gestation and that intrapartum antibiotic prophylaxis be given to carriers. We studied the performances of four different GBS detection assays in the context of antenatal screening. Between May and August 2004, the 605 vaginorectal swab specimens received at our bacteriology laboratory for GBS antenatal detection were tested by the four assays. The standard culture method was done according to the CDC recommendations. The three experimental assays performed with the growth from the selective enrichment (LIM) broth (Todd-Hewitt broth with 15 mug/ml nalidixic acid and 10 mug/ml colistin) after overnight incubation were a GBS antigen detection assay (PathoDx) and two PCR assays (for cfb and scpB). The most accurate assay was the scpB PCR (sensitivity, 99.6%; specificity, 100%), followed by the cfb PCR (sensitivity, 75.3%; specificity, 100%), GBS antigen detection (sensitivity, 57.3%; specificity, 99.5%), and standard culture (sensitivity, 42.3%; specificity, 100%). The GBS antigen detection assay was found to be more sensitive than the standard culture method, and moreover, the assay has a low cost and is easy to perform in all obstetrical centers which have access to the most basic of diagnostic microbiology services. We believe that antigen detection on incubated LIM broth should replace the standard culture method for screening for GBS carriage at 35 to 37 weeks of gestation. The impact of the greater sensitivities of PCR assays on the diminution of neonatal GBS infections remains to be demonstrated.

RNase HI overproduction is required for efficient full-length RNA synthesis in the absence of topoisomerase I in Escherichia coli.

It has long been known that Escherichia coli cells deprived of topoisomerase I (topA null mutants) do not grow. Because mutations reducing DNA gyrase activity and, as a consequence, negative supercoiling, occur to compensate for the loss of topA function, it has been assumed that excessive negative supercoiling is somehow involved in the growth inhibition of topA null mutants. However, how excess negative supercoiling inhibits growth is still unknown. We have previously shown that the overproduction of RNase HI, an enzyme that degrades the RNA portion of an R-loop, can partially compensate for the growth defects because of the absence of topoisomerase I. In this article, we have studied the effects of gyrase reactivation on the physiology of actively growing topA null cells. We found that growth immediately and almost completely ceases upon gyrase reactivation, unless RNase HI is overproduced. Northern blot analysis shows that the cells have a significantly reduced ability to accumulate full-length mRNAs when RNase HI is not overproduced. Interestingly, similar phenotypes, although less severe, are also seen when bacterial cells lacking RNase HI activity are grown and treated in the same way. All together, our results suggest that excess negative supercoiling promotes the formation of R-loops, which, in turn, inhibit RNA synthesis.

Effects of RNA polymerase modifications on transcription-induced negative supercoiling and associated R-loop formation.

Transcription in the absence of topoisomerase I, but in the presence of DNA gyrase, can result in the formation of hypernegatively supercoiled DNA and associated R-loops. In this paper, we have used several strategies to study the effects of elongation/termination properties of RNA polymerase on such transcription-induced supercoiling. Effects on R-loop formation were exacerbated when cells were exposed to translation inhibitors, a condition that stimulated the accumulation of R-loop-dependent hypernegative supercoiling. Translation inhibitors were not acting by decreasing (p)ppGpp levels as the absence of (p)ppGpp in spoT relA mutant strains had little effect on hypernegative supercoiling. However, an rpoB mutation leading to the accumulation of truncated RNAs considerably reduced R-loop-dependent hypernegative supercoiling. Transcription of an rrnB fragment preceded by a mutated and inactive boxA sequence to abolish the rrnB antitermination system also considerably reduced R-loop-dependent supercoiling. Taken together, our results indicate that RNA polymerase elongation/termination properties can have a major impact on R-loop-dependent supercoiling. We discuss different possibilities by which RNA polymerase directly or indirectly participates in R-loop formation in Escherichia coli. Finally, our results also indicate that what determines the steady-state level of hypernegatively supercoiled DNA in topA null mutants is likely to be complex and involves a multitude of factors, including the status of RNA polymerase, transcription-translation coupling, the cellular level of RNase HI, the status of DNA gyrase and the rate of relaxation of supercoiled DNA.