David Litt

Role of PCR in the diagnosis of pertussis infection in infants: 5 years' experience of provision of a same-day real-time PCR service in England and Wales from 2002 to 2007.

As part of an enhanced surveillance programme for pertussis in England and Wales, a real-time PCR service for the detection of Bordetella pertussis was introduced for infants aged <or=6 months admitted to a paediatric intensive care unit or paediatric ward with a respiratory illness compatible with pertussis. Two real-time fluorescent resonance energy transfer hybridization probe LightCycler (Roche Diagnostics) PCR assays were used. One (designed in-house) targeted the pertussis toxin S1 promoter (ptxA-pr), and included an internal process control to test for sample inhibition and reagent performance. The other (already published) targeted the insertion element IS481. The analytical sensitivities of the assays were 100 and 10 fg per reaction for the ptxA-pr and IS481 PCRs, respectively. The ptxA-pr assay was specific for B. pertussis, whilst the IS481 PCR also showed some cross-reactivity with Bordetella holmesii and the type strain of Bordetella parapertussis. From April 2002 to March 2007, 848 samples were received from 774 patients and DNA was extracted. Of 824 samples that were suitable for testing, 183 (22.2 %) had evidence of Bordetella infection (18.9 % ptxA-pr and IS481; 3.3 % IS481 only), 621 (75.4 %) were negative and 20 (2.4 %) were inhibitory for the PCR. Within the targeted age group of <or=6 months, most patients (130/138) with evidence of Bordetella spp. by PCR were <or=3 months old. The overall percentage increase in laboratory-confirmed cases due to PCR compared with culture for the 5 year period described ranged from 9 to 26 % per year (mean 19 %). Real-time PCR is an invaluable tool both for enhanced epidemiological surveillance and for the provision of a rapid diagnosis of pertussis where results can affect patient and contact management.

Invasive Haemophilus influenzae Serotype e and f Disease, England and Wales

Incidence of serotype e was 3-fold lower than serotype f, but it caused more severe clinical disease.

Whole genome sequencing of Streptococcus pneumoniae: development, evaluation and verification of targets for serogroup and serotype prediction using an automated pipeline

Streptococcus pneumoniae typically express one of 92 serologically distinct capsule polysaccharide (cps) types (serotypes). Some of these serotypes are closely related to each other; using the commercially available typing antisera, these are assigned to common serogroups containing types that show cross-reactivity. In this serotyping scheme, factor antisera are used to allocate serotypes within a serogroup, based on patterns of reactions. This serotyping method is technically demanding, requires considerable experience and the reading of the results can be subjective. This study describes the analysis of the S. pneumoniae capsular operon genetic sequence to determine serotype distinguishing features and the development, evaluation and verification of an automated whole genome sequence (WGS)-based serotyping bioinformatics tool, PneumoCaT (Pneumococcal Capsule Typing). Initially, WGS data from 871 S. pneumoniae isolates were mapped to reference cps locus sequences for the 92 serotypes. Thirty-two of 92 serotypes could be unambiguously identified based on sequence similarities within the cps operon. The remaining 60 were allocated to one of 20 ‘genogroups’ that broadly correspond to the immunologically defined serogroups. By comparing the cps reference sequences for each genogroup, unique molecular differences were determined for serotypes within 18 of the 20 genogroups and verified using the set of 871 isolates. This information was used to design a decision-tree style algorithm within the PneumoCaT bioinformatics tool to predict to serotype level for 89/94 (92 + 2 molecular types/subtypes) from WGS data and to serogroup level for serogroups 24 and 32, which currently comprise 2.1% of UK referred, invasive isolates submitted to the National Reference Laboratory (NRL), Public Health England (June 2014–July 2015). PneumoCaT was evaluated with an internal validation set of 2065 UK isolates covering 72/92 serotypes, including 19 non-typeable isolates and an external validation set of 2964 isolates from Thailand (n = 2,531), USA (n = 181) and Iceland (n = 252). PneumoCaT was able to predict serotype in 99.1% of the typeable UK isolates and in 99.0% of the non-UK isolates. Concordance was evaluated in UK isolates where further investigation was possible; in 91.5% of the cases the predicted capsular type was concordant with the serologically derived serotype. Following retesting, concordance increased to 99.3% and in most resolved cases (97.8%; 135/138) discordance was shown to be caused by errors in original serotyping. Replicate testing demonstrated that PneumoCaT gave 100% reproducibility of the predicted serotype result. In summary, we have developed a WGS-based serotyping method that can predict capsular type to serotype level for 89/94 serotypes and to serogroup level for the remaining four. This approach could be integrated into routine typing workflows in reference laboratories, reducing the need for phenotypic immunological testing.

Risk of Invasive Haemophilus influenzae Infection During Pregnancy and Association With Adverse Fetal Outcomes

IMPORTANCE Unencapsulated Haemophilus influenzae frequently causes noninvasive upper respiratory tract infections in children but can also cause invasive disease, especially in older adults. A number of studies have reported an increased incidence in neonates and suggested that pregnant women may have an increased susceptibility to invasive unencapsulated H. influenzae disease. OBJECTIVE To describe the epidemiology, clinical characteristics, and outcomes of invasive H. influenzae disease in women of reproductive age during a 4-year period. DESIGN, SETTING, AND PARTICIPANTS Public Health England conducts enhanced national surveillance of invasive H. influenzae disease in England and Wales. Clinical questionnaires were sent prospectively to general practitioners caring for all women aged 15 to 44 years with laboratory-confirmed invasive H. influenzae disease during 2009-2012, encompassing 45,215,800 woman-years of follow-up. The final outcome was assessed in June 2013. EXPOSURES Invasive H. influenzae disease confirmed by positive culture from a normally sterile site. MAIN OUTCOMES AND MEASURES The primary outcome was H. influenzae infection and the secondary outcomes were pregnancy-related outcomes. RESULTS In total, 171 women had laboratory-confirmed invasive H. influenzae infection, which included 144 (84.2%; 95% CI, 77.9%-89.3%) with unencapsulated, 11 (6.4%; 95% CI, 3.3%-11.2%) with serotype b, and 16 (9.4%; 95% CI, 5.4%-14.7%) with other encapsulated serotypes. Questionnaire response rate was 100%. Overall, 75 of 171 women (43.9%; 95% CI, 36.3%-51.6%) were pregnant at the time of infection, most of whom were previously healthy and presented with unencapsulated H. influenzae bacteremia. The incidence rate of invasive unencapsulated H. influenzae disease was 17.2 (95% CI, 12.2-24.1; P < .001) times greater among pregnant women (2.98/100,000 woman-years) compared with nonpregnant women (0.17/100,000 woman-years). Unencapsulated H. influenzae infection during the first 24 weeks of pregnancy was associated with fetal loss (44/47; 93.6% [95% CI, 82.5%-98.7%]) and extremely premature birth (3/47; 6.4% [95% CI, 1.3%-17.5%]). Unencapsulated H. influenzae infection during the second half of pregnancy was associated with premature birth in 8 of 28 cases (28.6%; 95% CI, 13.2%-48.7%) and stillbirth in 2 of 28 cases (7.1%; 95% CI, 0.9%-23.5%). The incidence rate ratio for pregnancy loss was 2.91 (95% CI, 2.13-3.88) for all serotypes of H. influenzae and 2.90 (95% CI, 2.11-3.89) for unencapsulated H. influenzae compared with the background rate for pregnant women. CONCLUSIONS AND RELEVANCE Among women in England and Wales, pregnancy was associated with a greater risk of invasive H. influenzae infection. These infections were associated with poor pregnancy outcomes.

Bacterial bronchitis caused by Streptococcus pneumoniae and nontypable Haemophilus influenzae in children: the impact of vaccination.

BACKGROUND Protracted bacterial bronchitis is a major cause of persistent cough in childhood. The organisms most commonly isolated are nontypable Haemophilus influenzae and Streptococcus pneumoniae . There are no studies addressing typing of these organisms when recovered from the lower airways. METHODS Isolates of these two organisms (identified in BAL samples from children undergoing routine investigation of a chronic cough thought to be attributable to a protracted bacterial bronchitis) were subject to typing. Samples were collected in Sheffield, England, and Athens, Greece. The majority of the children from Sheffield had received pneumococcal-conjugate vaccines 7 or 13 (PCV-7 or PCV-13) conjugate vaccine but only a minority of Greek children had received PCV-7. RESULTS All 18 S pneumoniae isolates from Greek BAL samples are serotypes contained in PCV-13 while 10 are contained in PCV-7. In contrast, 28 of the 39 samples from Sheffield contained serotypes that are not included in PCV-13. All 26 of the nontypable H influenzae samples obtained in Sheffield produced distinct multilocus variable-number tandem repeat analysis profiles. There was a significant difference between children from Athens and Sheffield in the distribution of serotypes contained or not contained in the pneumococcal vaccine ( P = .04). More specifically, immunization with pneumococcal vaccine was related with isolation of S pneumoniae serotypes not included in the vaccine (OR, 0.021; CI, 0.003-0.115; P < .001). CONCLUSIONS The data suggest that both vaccine and nonvaccine S pneumoniae serotypes may play a role in protracted bacterial bronchitis and provide some hints that serotype replacement may occur in response to the introduction of conjugate vaccines.

Original ResearchChest InfectionsBacterial Bronchitis Caused by Streptococcus pneumoniae and Nontypable Haemophilus influenzae in Children: The Impact of Vaccination

BACKGROUND Protracted bacterial bronchitis is a major cause of persistent cough in childhood. The organisms most commonly isolated are nontypable Haemophilus influenzae and Streptococcus pneumoniae . There are no studies addressing typing of these organisms when recovered from the lower airways. METHODS Isolates of these two organisms (identified in BAL samples from children undergoing routine investigation of a chronic cough thought to be attributable to a protracted bacterial bronchitis) were subject to typing. Samples were collected in Sheffield, England, and Athens, Greece. The majority of the children from Sheffield had received pneumococcal-conjugate vaccines 7 or 13 (PCV-7 or PCV-13) conjugate vaccine but only a minority of Greek children had received PCV-7. RESULTS All 18 S pneumoniae isolates from Greek BAL samples are serotypes contained in PCV-13 while 10 are contained in PCV-7. In contrast, 28 of the 39 samples from Sheffield contained serotypes that are not included in PCV-13. All 26 of the nontypable H influenzae samples obtained in Sheffield produced distinct multilocus variable-number tandem repeat analysis profiles. There was a significant difference between children from Athens and Sheffield in the distribution of serotypes contained or not contained in the pneumococcal vaccine ( P = .04). More specifically, immunization with pneumococcal vaccine was related with isolation of S pneumoniae serotypes not included in the vaccine (OR, 0.021; CI, 0.003-0.115; P < .001). CONCLUSIONS The data suggest that both vaccine and nonvaccine S pneumoniae serotypes may play a role in protracted bacterial bronchitis and provide some hints that serotype replacement may occur in response to the introduction of conjugate vaccines.

Childhood Bacterial Meningitis in Ulaanbaatar, Mongolia, 2002–2004

BACKGROUND Childhood bacterial meningitis is severe and largely preventable by vaccination. Few data on childhood bacterial meningitis in Northeast and Central Asia exist. Our aim was to determine the incidence and etiology of childhood bacterial meningitis in Ulaanbaatar, Mongolia. METHODS We conducted prospective, population-based, active hospital surveillance for clinical meningitis in children 2 months to 5 years of age. Clinical data, blood, and cerebrospinal fluid were collected according to a standard protocol. Laboratory testing was performed at 2 reference laboratories in Ulaanbaatar. RESULTS From February 2002 to January 2005, 201 suspected meningitis cases were identified in residents of Ulaanbaatar. The average annual incidence rate for confirmed and probable bacterial meningitis (cases with culture-negative, purulent cerebrospinal fluid) was 68 cases per 100,000 children aged 2 months to 5 years. The average annual incidence rate of confirmed cases was 28 cases per 100,000 children for Haemophilus influenzae type b meningitis, 11 cases per 100,000 children for pneumococcal meningitis, and 13 cases per 100,000 children for meningococcal meningitis. Adjusting for cases without complete cerebrospinal fluid information and culture-negative, probable bacterial cases, the estimated incidence rate was 40 cases per 100,000 children for H. influenzae type b meningitis, 15 cases per 100,000 children for pneumococcal meningitis, and 17 cases per 100,000 children for meningococcal meningitis. CONCLUSION H. influenzae type b is the leading cause of childhood bacterial meningitis in Ulaanbaatar, and the incidence rate is higher than that reported from other Asian countries. These data supported the recent introduction of H. influenzae type b conjugate vaccine in Mongolia. Ongoing surveillance will monitor the impact of the vaccine.

Modelling anti-pertussis toxin IgG antibody decay following primary and preschool vaccination with an acellular pertussis vaccine in UK subjects using a modified oral fluid assay

Recent vaccination with pertussis vaccine can confound serological and oral fluid (OF) assays targeting anti-pertussis toxin (anti-PT) IgG antibodies as a marker of recent infection. This study sought to establish the minimum potentially confounding time period based on experimental data to assist interpretation from such samples submitted from UK subjects for pertussis diagnosis. Anti-PT IgG antibody response and decay were measured post-vaccination using a modified OF IgG antibody-capture ELISA (GACELISA). Data were obtained from 72 infants after the third acellular pertussis vaccine dose in the primary schedule (4 months of age) and from 119 children after the single dose at preschool age (3 years 4 months to 5 years 8 months of age). Specimens were taken at approximately 1 month intervals for 9 months post-primary immunization (third dose) and 13 months post-preschool booster (PSB). The modified GACELISA demonstrated a sensitivity of 52/56 (92.9 %: 95 % CI 82.7-98.0) and a specificity of 120/128 (93.8 %: 95 % CI 88.0-97.3) and showed good agreement with the National Reference Laboratory standard anti-PT IgG serum ELISA (rank correlation = 0.80) and the original OF assay (rank correlation = 0.79). Modelling of the decline in antibody titres showed a reduction of 54 % and 34 % for each doubling of time after day 14 for the post-third primary dose and post-PSB subjects, respectively. These data suggest that the minimum confounding time period is approximately 300 days for samples obtained post-primary immunization and at least 3 years for samples submitted from UK children following immunization with the PSB. These data will greatly assist the interpretation of single high diagnostic anti-PT IgG titres by allowing an estimate of the positive predictive value, when the number of days post-immunization and prevalence are known or assumed.

Direct molecular typing of Bordetella pertussis from clinical specimens submitted for diagnostic quantitative (real-time) PCR

Molecular typing of Bordetella pertussis is routinely performed on bacterial isolates, but not on DNA extracted from nasopharyngeal aspirates or pernasal swabs submitted for diagnostic real-time PCR (qPCR). We investigated whether these DNA extracts were suitable for multilocus variable-number tandem repeat analysis (MLVA) and DNA sequence-based typing. We analysed all the available qPCR-positive samples received by our laboratory from patients <1 year of age between January 2008 and August 2010. Eighty-one per cent (106/131) of these generated a complete MLVA profile. This rose to 92 % (105/114) if only samples positive for both of the two targets used for the B. pertussis PCR (insertion element IS481 and pertussis toxin promoter ptxP) were analysed. Sequence-based typing of the pertactin, pertussis toxin S1 subunit and pertussis promoter regions (prn, ptxA and ptxP) was attempted on 89 of the DNA extracts that had generated a full MLVA profile. Eighty-three (93 %) of these produced complete sequences for all three targets. Comparison of molecular typing data from the 89 extracts with those from 111 contemporary bacterial isolates showed that the two sources yielded the same picture of the B. pertussis population [dominated by the MLVA-27 prn(2) ptxA(1) ptxP(3) clonal type]. There was no significant difference in MLVA type distribution or diversity between the two sample sets. This suggests that clinical extracts can be used in place of, or to complement, bacterial cultures for typing purposes (at least, in this age group). With small modifications to methodology, generating MLVA and sequence-based typing data from qPCR-positive clinical DNA extracts is likely to generate a complete dataset in the majority of samples from the <1 year age group. Its success with samples from older subjects remains to be seen. However, our data suggest that it is suitable for inclusion in molecular epidemiological studies of the B. pertussis population or as a tool in outbreak investigations.

Oral fluid testing for pertussis, England and wales, june 2007-august 2009.

Existing pertussis surveillance systems tend to underidentify less severe cases among older children and adults. For routine follow-up of notified, nonconfirmed, clinically diagnosed pertussis cases, use of an oral fluid test was pilot tested in England and Wales during June 2007–August 2009. During that period, 1,852 cases of pertussis were confirmed by established laboratory methods and another 591 by oral fluid testing only. Although introduction of serologic testing in 2002 led to the greatest increase in ascertainment of pertussis, oral fluid testing increased laboratory ascertainment by 32% overall; maximal increase (124%) occurred among children 5–9 years of age. Patients whose pertussis was confirmed by oral fluid testing were least likely to be hospitalized, suggesting that milder community cases were being confirmed by this method. Oral fluid testing is an easily administered, noninvasive surveillance tool that could further our understanding of pertussis epidemiology and thereby contribute to decisions on vaccination strategies.