John C. Hartley

The Likelihood of an Indeterminate Test Result from a Whole-Blood Interferon-γ Release Assay for the Diagnosis of Mycobacterium tuberculosis Infection in Children Correlates With Age and Immune Status

Background: Interferon-&ggr; release assays for the diagnosis of infection with Mycobacterium tuberculosis have been increasingly used in recent years and are endorsed by national guidelines, but experience regarding their use in children is still limited. Methods: We retrospectively evaluated the routine use of the QuantiFERON-TB Gold In-Tube assay (QFT-IT) in a pediatric tertiary care center with a high prevalence of immunocompromising conditions. The relationship between age, immune status, and likelihood of an indeterminate test result was analyzed using logistic regression analysis and fractional polynomials. Results: Two hundred thirty-seven tests from 237 children were included in the analysis. Fifty-nine children (25%) were immunocompromised by our definition. An indeterminate test result was obtained in 83 children (35%). The likelihood of an indeterminate test result was inversely correlated with age (P < 0.001) for children who were not known to be immunocompromised, and decreased by 13% per year of age. Impaired immunity (P < 0.001) was independently associated with a higher probability of an indeterminate QFT-IT. Among 161 children with a documented tuberculin skin test, 89% had a concordant QFT-IT (&kgr; = 0.71). Twelve of 16 patients with culture-proven TB had a positive QFT-IT. Conclusion: These data suggest that QFT-IT may not provide a determinate test result in a substantial proportion of children in a tertiary care setting due to the combination of young age and primary and acquired immune deficiencies.

Infection and sudden unexpected death in infancy: a systematic retrospective case review

BACKGROUND The cause and mechanism of most cases of sudden unexpected death in infancy (SUDI) remain unknown, despite specialist autopsy examination. We reviewed autopsy results to determine whether infection was a cause of SUDI. METHODS We did a systematic retrospective case review of autopsies, done at one specialist centre between 1996 and 2005, of 546 infants (aged 7-365 days) who died suddenly and unexpectedly. Cases of SUDI were categorised as unexplained, explained with histological evidence of bacterial infection, or explained by non-infective causes. Microbial isolates gathered at autopsy were classified as non-pathogens, group 1 pathogens (organisms usually associated with an identifiable focus of infection), or group 2 pathogens (organisms known to cause septicaemia without an obvious focus of infection). FINDINGS Of 546 SUDI cases, 39 autopsies were excluded because of viral or pneumocystis infection or secondary bacterial infection after initial collapse and resuscitation. Bacteriological sampling was done in 470 (93%) of the remaining 507 autopsies. 2079 bacteriological samples were taken, of which 571 (27%) were sterile. Positive cultures yielded 2871 separate isolates, 484 (32%) of which showed pure growth and 1024 (68%) mixed growth. Significantly more isolates from infants whose deaths were explained by bacterial infection (78/322, 24%) and from those whose death was unexplained (440/2306, 19%) contained group 2 pathogens than did those from infants whose death was explained by a non-infective cause (27/243, 11%; difference 13.1%, 95% CI 6.9-19.2, p<0.0001 vs bacterial infection; and 8.0%, 3.2-11.8, p=0.001 vs unexplained). Significantly more cultures from infants whose deaths were unexplained contained Staphylococcus aureus (262/1628, 16%) or Escherichia coli (93/1628; 6%) than did those from infants whose deaths were of non-infective cause (S aureus: 19/211, 9%; difference 7.1%, 95% CI 2.2-10.8, p=0.005; E coli: 3/211, 1%, difference 4.3%, 1.5-5.9, p=0.003). INTERPRETATION Although many post-mortem bacteriological cultures in SUDI yield organisms, most seem to be unrelated to the cause of death. The high rate of detection of group 2 pathogens, particularly S aureus and E coli, in otherwise unexplained cases of SUDI suggests that these bacteria could be associated with this condition.

Differing Prevalence and Diversity of Bacterial Species in Fetal Membranes from Very Preterm and Term Labor

Background Intrauterine infection may play a role in preterm delivery due to spontaneous preterm labor (PTL) and preterm prolonged rupture of membranes (PPROM). Because bacteria previously associated with preterm delivery are often difficult to culture, a molecular biology approach was used to identify bacterial DNA in placenta and fetal membranes. Methodology/Principal findings We used broad-range 16S rDNA PCR and species-specific, real-time assays to amplify bacterial DNA from fetal membranes and placenta. 74 women were recruited to the following groups: PPROM <32 weeks (n = 26; 11 caesarean); PTL with intact membranes <32 weeks (n = 19; all vaginal birth); indicated preterm delivery <32 weeks (n = 8; all caesarean); term (n = 21; 11 caesarean). 50% (5/10) of term vaginal deliveries were positive for bacterial DNA. However, little spread was observed through tissues and species diversity was restricted. Minimal bacteria were detected in term elective section or indicated preterm deliveries. Bacterial prevalence was significantly increased in samples from PTL with intact membranes [89% (17/19) versus 50% (5/10) in term vaginal delivery p = 0.03] and PPROM (CS) [55% (6/11) versus 0% (0/11) in term elective CS, p = 0.01]. In addition, bacterial spread and diversity was greater in the preterm groups with 68% (13/19) PTL group having 3 or more positive samples and over 60% (12/19) showing two or more bacterial species (versus 20% (2/10) in term vaginal deliveries). Blood monocytes from women with PTL with intact membranes and PPROM who were 16S bacterial positive showed greater level of immune paresis (p = 0.03). A positive PCR result was associated with histological chorioamnionitis in preterm deliveries. Conclusion/Significance Bacteria are found in both preterm and term fetal membranes. A greater spread and diversity of bacterial species were found in tissues of women who had very preterm births. It is unclear to what extent the greater bacterial prevalence observed in all vaginal delivery groups reflects bacterial contamination or colonization of membranes during labor. Bacteria positive preterm tissues are associated with histological chorioamnionitis and a pronounced maternal immune paresis.

Comparative Effectiveness of Intravenous vs Oral Antibiotics for Postdischarge Treatment of Acute Osteomyelitis in Children

IMPORTANCE Postdischarge treatment of acute osteomyelitis in children requires weeks of antibiotic therapy, which can be administered orally or intravenously via a peripherally inserted central catheter (PICC). The catheters carry a risk for serious complications, but limited evidence exists on the effectiveness of oral therapy. OBJECTIVE To compare the effectiveness and adverse outcomes of postdischarge antibiotic therapy administered via the PICC or the oral route. DESIGN, SETTING, AND PARTICIPANTS We performed a retrospective cohort study comparing PICC and oral therapy for the treatment of acute osteomyelitis. Among children hospitalized from January 1, 2009, through December 31, 2012, at 36 participating childrens hospitals, we used discharge codes to identify potentially eligible participants. Results of medical record review confirmed eligibility and defined treatment group allocation and study outcomes. We used within- and across-hospital propensity score-based full matching to adjust for confounding by indication. INTERVENTIONS Postdischarge administration of antibiotics via the PICC or the oral route. MAIN OUTCOMES AND MEASURES The primary outcome was treatment failure. Secondary outcomes included adverse drug reaction, PICC line complication, and a composite of all 3 end points. RESULTS Among 2060 children and adolescents (hereinafter referred to as children) with osteomyelitis, 1005 received oral antibiotics at discharge, whereas 1055 received PICC-administered antibiotics. The proportion of children treated via the PICC route varied across hospitals from 0 to 100%. In the across-hospital (risk difference, 0.3% [95% CI, -0.1% to 2.5%]) and within-hospital (risk difference, 0.6% [95% CI, -0.2% to 3.0%]) matched analyses, children treated with antibiotics via the oral route (reference group) did not experience more treatment failures than those treated with antibiotics via the PICC route. Rates of adverse drug reaction were low (<4% in both groups) but slightly greater in the PICC group in across-hospital (risk difference, 1.7% [95% CI, 0.1%-3.3%]) and within-hospital (risk difference, 2.1% [95% CI, 0.3%-3.8%]) matched analyses. Among the children in the PICC group, 158 (15.0%) had a PICC complication that required an emergency department visit (n = 96), a rehospitalization (n = 38), or both (n = 24). As a result, the PICC group had a much higher risk of requiring a return visit to the emergency department or for hospitalization for any adverse outcome in across-hospital (risk difference, 14.6% [95% CI, 11.3%-17.9%]) and within-hospital (risk difference, 14.0% [95% CI, 10.5%-17.6%]) matched analyses. CONCLUSIONS AND RELEVANCE Given the magnitude and seriousness of PICC complications, clinicians should reconsider the practice of treating otherwise healthy children with acute osteomyelitis with prolonged intravenous antibiotics after hospital discharge when an equally effective oral alternative exists.

Next-Generation Whole Genome Sequencing Identifies the Direction of Norovirus Transmission in Linked Patients

BACKGROUND Noroviruses are a highly transmissible and major cause of nosocomial gastroenteritis resulting in bed and hospital-ward closures. Where hospital outbreaks are suspected, it is important to determine the routes of spread so that appropriate infection-control procedures can be implemented. To investigate a cluster of norovirus cases occurring in children undergoing bone marrow transplant, we undertook norovirus genome sequencing by next-generation methods. Detailed comparison of sequence data from 2 linked cases enabled us to identify the likely direction of spread. METHODS Norovirus complementary DNA was amplified by overlapping polymerase chain reaction (PCR) from 13 stool samples from 5 diagnostic real-time PCR-positive patients. The amplicons were sequenced by Roche 454, the genomes assembled by de novo assembly, and the data analyzed phylogenetically. RESULTS Phylogenetic analysis indicated that patients were infected by viruses similar to 4 distinct GII.4 subtypes and 2 patients were linked by the same virus. Of the 14 sites at which there were differences between the consensus sequences of the 2 linked viral genomes, 9 had minor variants present within one or the other patient. Further analysis confirmed that minor variants at all 9 sites in patient B w ere present as the consensus sequence in patient A. CONCLUSIONS Phylogenetic analysis excluded a common source of infection in this apparent outbreak. Two of 3 patients on the same ward had closely related viruses, raising the possibility of cross-infection despite protective isolation. Analysis of deep sequencing data enabled us to establish the likely direction of nosocomial transmission.

Molecular Fingerprinting of Mycobacterium abscessus Strains in a Cohort of Pediatric Cystic Fibrosis Patients

ABSTRACT Forty-one Mycobacterium abscessus complex isolates from 17 pediatric cystic fibrosis (CF) patients were typed using a novel variable-number tandem repeat (VNTR) scheme and an automated repetitive-PCR (rep-PCR) system. Both VNTR and rep-PCR typing methods differentiate between members of the M. abscessus complex. The isolates from individual patients are indistinguishable, and the data strongly suggest that individual CF patients are persistently infected with one strain and also suggests that different CF patients can harbor the same strain.

Duplex Real-Time PCR Assay for Detection of Streptococcus pneumoniae in Clinical Samples and Determination of Penicillin Susceptibility

ABSTRACT We have developed a duplex real-time PCR for the rapid diagnosis of Streptococcus pneumoniae infection from culture-negative clinical samples with the simultaneous determination of penicillin susceptibility. The assay amplifies a lytA gene target and a penicillin binding protein 2b (pbp2b) gene target in penicillin-susceptible organisms. The assay was shown to be sensitive (detects 0.5 CFU per PCR) and specific for the detection of S. pneumoniae DNA. The assay was validated by comparing pbp2b PCR results with MIC data for 27 S. pneumoniae isolates. All 5 isolates with penicillin MICs of >1.0 mg/liter were pbp2b real-time PCR negative, as were 9 of the 10 isolates with penicillin MICs of 0.12 to 1.0 mg/liter. One isolate with a penicillin MIC of 0.12 to 1.0 mg/liter gave an equivocal pbp2b real-time PCR result. Twelve isolates were penicillin susceptible (MICs of ≤0.06 mg/liter) and pbp2b real-time PCR positive. These data were used to establish an algorithm for the interpretation of penicillin susceptibility from the duplex PCR result. pbp2b real-time PCR results were also compared to an established PCR-restriction fragment length polymorphism (RFLP) method previously applied to these 27 isolates and 46 culture-negative clinical samples (containing S. pneumoniae DNA by broad-range 16S rRNA gene PCR). Discordant results were seen for four isolates and six culture-negative clinical samples, as PCR-RFLP could not reliably detect penicillin MICs of 0.12 to 1.0 mg/liter. We report prospective application of the duplex PCR assay to the diagnosis of S. pneumoniae infection from 200 culture-negative clinical specimens sent to the laboratory for diagnostic broad-range 16S rRNA gene PCR. One hundred six were negative in the duplex PCR. Ninety-four were lytA PCR positive, and 70 of these were also pbp2b PCR positive and interpreted as penicillin susceptible. Fourteen were pbp2b PCR negative and interpreted as having reduced susceptibility to penicillin. For the remaining 10 samples, susceptibility to penicillin was not determined.

Unique Case of Helicobacter sp. Osteomyelitis in an Immunocompetent Child Diagnosed by Broad-Range 16S PCR

ABSTRACT We report the first case of Helicobacter sp. osteomyelitis in an immunocompetent child. The infection was diagnosed by broad-range 16S PCR followed by sequencing of the resulting amplicon. All other microbiological investigations proved negative. This case highlights the importance of molecular methods in the diagnosis of unsuspected etiological agents and the potential role of Helicobacter sp. in human infection.

The use of a two-gene sequencing approach to accurately distinguish between the species within the Mycobacterium abscessus complex and Mycobacterium chelonae

Mycobacterium abscessus [M. abscessus (sensu lato) or M. abscessus complex] comprises three closely related species: M. abscessus (sensu stricto), hereafter referred to as M. abscessus, M. bolletii and M. massiliense. We describe here an accurate and robust method for distinguishing M. chelonae from M. abscessus, M. bolletii and M. massiliense, using polymerase chain reaction (PCR) and the sequencing of house-keeping gene targets (hsp65 and rpoB). Sequencing of the sodA gene is of little additional value in discriminating between species, but M. massiliense can be rapidly identified by amplification of the truncated erm(41) gene without the need for amplicon sequencing. We have applied the method to 81 isolates from 40 patients from two hospitals, the majority of whom were cystic fibrosis (CF) patients. Of these patients, 21 had previously been identified as M. chelonae and 59 as M. abscessus complex using commercial line probe assays. We identified these as 46 M. abscessus isolates, 20 M. massiliense isolates, five M. bolletii isolates and nine M. chelonae isolates and confirmed the one M. fortuitum isolate. This is the first study that has identified the individual members of the M. abscessus complex in a UK cohort of mainly CF patients.

The British antibiotic and silver-impregnated catheters for ventriculoperitoneal shunts multi-centre randomised controlled trial (the BASICS trial): study protocol

BackgroundInsertion of a ventriculoperitoneal shunt (VPS) for the treatment of hydrocephalus is one of the most common neurosurgical procedures in the UK, but failures caused by infection occur in approximately 8% of primary cases. VPS infection is associated with considerable morbidity and mortality and its management results in substantial cost to the health service. Antibiotic-impregnated (rifampicin and clindamycin) and silver-impregnated VPS have been developed to reduce infection rates. Whilst there is some evidence showing that such devices may lead to a reduction in VPS infection, there are no randomised controlled trials (RCTs) to support their routine use.Methods/designOverall, 1,200 patients will be recruited from 17 regional neurosurgical units in the UK and Ireland. Patients of any age undergoing insertion of their first VPS are eligible. Patients with previous indwelling VPS, active and on-going cerebrospinal fluid (CSF) or peritoneal infection, multiloculated hydrocephalus requiring multiple VPS or neuroendoscopy, and ventriculoatrial or ventriculopleural shunt planned will be excluded. Patients will be randomised 1:1:1 to either standard silicone (comparator), antibiotic-impregnated, or silver-impregnated VPS. The primary outcome measure is time to VPS infection. Secondary outcome measures include time to VPS failure of any cause, reason for VPS failure (infection, mechanical failure, or patient failure), types of bacterial VPS infection (organism type and antibiotic resistance), and incremental cost per VPS failure averted.DiscussionThe British antibiotic and silver-impregnated catheters for ventriculoperitoneal shunts multi-centre randomised controlled trial (the BASICS trial) is the first multi-centre RCT designed to determine whether antibiotic or silver-impregnated VPS reduce early shunt infection compared to standard silicone VPS. The results of this study will be used to inform current neurosurgical practice and may potentially benefit patients undergoing shunt surgery in the future.Trial registrationInternational Standard Randomised Controlled Trial Number:ISRCTN49474281.