James P. McKenna

Development and clinical validation of a loop-mediated isothermal amplification method for the rapid detection of Neisseria meningitidis

Loop-mediated isothermal amplification (LAMP) is an innovative technique that allows the rapid detection of target nucleic acid sequences under isothermal conditions without the need for complex instrumentation. The development, optimization, and clinical validation of a LAMP assay targeting the ctrA gene for the rapid detection of capsular Neisseria meningitidis were described. Highly specific detection of capsular N. meningitidis type strains and clinical isolates was demonstrated, with no cross-reactivity with other Neisseria spp. or with a comprehensive panel of other common human pathogens. The lower limit of detection was 6 ctrA gene copies detectable in 48 min, with positive reactions readily identifiable visually via a simple color change. Higher copy numbers could be detected in as little as 16 min. When applied to a total of 394 clinical specimens, the LAMP assay in comparison to a conventional TaqMan® based real-time polymerase chain reaction system demonstrated a sensitivity of 100% and a specificity of 98.9% with a κ coefficient of 0.942. The LAMP method represents a rapid, sensitive, and highly specific technique for the detection of N. meningitidis and has the potential to be used as a point-of-care molecular test and in resource-poor settings.

Interlaboratory Comparison of Real-Time Polymerase Chain Reaction Methods to Detect Coxiella Burnetii, The Causative Agent of Q Fever

The bacterium Coxiella burnetii, which has a wide host range, causes Q fever. Infection with C. burnetii can cause abortions, stillbirth, and the delivery of weak offspring in ruminants. Coxiella burnetii infection is zoonotic, and in human beings it can cause chronic, potentially fatal disease. Real-time polymerase chain reaction (PCR) is increasingly being used to detect the organism and to aid in diagnosis both in human and animal cases. Many different real-time PCR methods, which target different genes, have been described. To assess the comparability of the C. burnetii real-time PCR assays in use in different European laboratories, a panel of nucleic acid extracts was dispatched to 7 separate testing centers. The testing centers included laboratories from both human and animal health agencies. Each laboratory tested the samples using their in-house real-time PCR methods. The results of this comparison show that the most common target gene for real-time PCR assays is the IS1111 repeat element that is present in multiple copies in the C. burnetii genome. Many laboratories also use additional real-time PCR tests that target single-copy genes. The results of the current study demonstrate that the assays in use in the different laboratories are comparable, with general agreement of results for the panel of samples.

Diagnostic accuracy of loop-mediated isothermal amplification as a near-patient test for meningococcal disease in children: an observational cohort study

BACKGROUND Diagnosis of meningococcal disease relies on recognition of clinical signs and symptoms that are notoriously non-specific, variable, and often absent in the early stages of the disease. Loop-mediated isothermal amplification (LAMP) has previously been shown to be fast and effective for the molecular detection of meningococcal DNA in clinical specimens. We aimed to assess the diagnostic accuracy of meningococcal LAMP as a near-patient test in the emergency department. METHODS For this observational cohort study of diagnostic accuracy, children aged 0-13 years presenting to the emergency department of the Royal Belfast Hospital for Sick Children (Belfast, UK) with suspected meningococcal disease were eligible for inclusion. Patients underwent a standard meningococcal pack of investigations testing for meningococcal disease. Respiratory (nasopharyngeal swab) and blood specimens were collected from patients and tested with near-patient meningococcal LAMP and the results were compared with those obtained by reference laboratory tests (culture and PCR of blood and cerebrospinal fluid). FINDINGS Between Nov 1, 2009, and Jan 31, 2012, 161 eligible children presenting at the hospital underwent the meningococcal pack of investigations and were tested for meningococcal disease, of whom 148 consented and were enrolled in the study. Combined testing of respiratory and blood specimens with use of LAMP was accurate (sensitivity 89% [95% CI 72-96], specificity 100% [97-100], positive predictive value 100% [85-100]; negative predictive value 98% [93-99]) and diagnostically useful (positive likelihood ratio 213 [95% CI 13-infinity] and negative likelihood ratio 0·11 [0·04-0·32]). The median time required for near-patient testing from sample to result was 1 h 26 min (IQR 1 h 20 min-1 h 32 min). INTERPRETATION Meningococcal LAMP is straightforward enough for use in any hospital with basic laboratory facilities, and near-patient testing with this method is both feasible and effective. By contrast with existing UK National Institute of Health and Care Excellence guidelines, we showed that molecular testing of non-invasive respiratory specimens from children is diagnostically accurate and clinically useful. FUNDING Health and Social Care Research and Development, Public Health Agency, Northern Ireland.

The common vaginal commensal bacterium Ureaplasma parvum is associated with chorioamnionitis in extreme preterm labor

Abstract Objective: To assess the association of vaginal commensal and low-grade pathogenic bacteria including Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, Group B streptococcus (GBS), and Gardnerella vaginalis, in women who delivered preterm at less than 37-week gestation in the presence or absence of inflammation of the chorioamnionitic membranes. Methods: A case control study involving women who delivered before 37-week gestation with and without inflammation of chorioamnionitic membranes. A total of 57 placental samples were histologically examined for polymorphonuclear leukocyte infiltration of placental tissue for evidence of chorioamnionitis, and by type-specific nucleic acid amplification for evidence of infection with one or more of the target bacteria. Demographic data were collected for each mother. Results: Among the 57 placental samples, 42.1% had chorioamnionitis and 24.6% delivered in the second trimester of pregnancy; U. parvum, U. urealyticum, G. vaginalis, and GBS were all detected in the study with respective prevalence of 19.3%, 3.5%, 17.5%, and 15.8%; M. genitalium and M. hominis were not detected. U. parvum was significantly associated with chorioamnionitis (p = 0.02; OR 5.0; (95% CI 1.2–21.5) and was more common in women who delivered in the second (35.7%) compared to the third trimester of pregnancy (13.9%). None of the other bacteria were associated with chorioamnionitis or earlier delivery, and all G. vaginalis-positive women delivered in the third trimester of pregnancy (p = 0.04). Conclusions: The detection of U. parvum in placental tissue was significantly associated with acute chorioamnionitis in women presenting in extreme preterm labor.

Loop Mediated Isothermal Amplification (LAMP) assay for Rapid detection of Streptococcus agalactiae (Group B Streptococcus - GBS) in vaginal swabs - A Proof of Concept Study.

Purpose. Neonatal sepsis caused by Streptococcus agalactiae [group B streptococcus (GBS)] is a life‐threatening condition, which is preventable if colonized mothers are identified and given antibiotic prophylaxis during labour. Conventional culture is time consuming and unreliable, and many available non‐culture diagnostics are too complex to implement routinely at point of care. Loop‐mediated isothermal amplification (LAMP) is a method that, enables the rapid and specific detection of target nucleic acid sequences in clinical materials without the requirement for extensive sample preparation. Methodology. A prototype LAMP assay targeting GBS sip gene is described. Results. The assay was 100% specific for GBS, with a limit of detection of 14 genome copies per reaction. The clinical utility of the LAMP assay for rapid direct molecular detection of GBS was determined by testing a total of 157 vaginal swabs with minimal sample processing using a rapid lysis solution. Compared to a reference quantitative real‐time PCR assay, the direct LAMP protocol had a sensitivity and specificity of 95.4 and 100%, respectively, with positive and negative predictive values of 100 and 98.3%, respectively. Positive and negative likelihood ratios were infinity and 0.05, respectively. The direct LAMP method required a mean time of 45 min from the receipt of a swab to generation of a confirmed result, compared to 2 h 30 min for the reference quantitative real‐time PCR test. Conclusion. The direct LAMP protocol described is easy to perform, facilitating rapid and accurate detection of GBS in vaginal swabs. This test has a potential for use at point of care.

A comparison of different pre-lysis methods and extraction kits for recovery of Streptococcus 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 achromopeptidase pre-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 due to 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.

16S pan-bacterial PCR can accurately identify patients with ventilator-associated pneumonia

Ventilator-associated pneumonia (VAP) remains a challenge to intensive care units, with secure diagnosis relying on microbiological cultures that take up to 72 hours to provide a result. We sought to derive and validate a novel, real-time 16S rRNA gene PCR for rapid exclusion of VAP. Bronchoalveolar lavage (BAL) was obtained from two independent cohorts of patients with suspected VAP. Patients were recruited in a 2-centre derivation cohort and a 12-centre confirmation cohort. Confirmed VAP was defined as growth of >104 colony forming units/ml on semiquantitative culture and compared with a 16S PCR assay. Samples were tested from 67 patients in the derivation cohort, 10 (15%) of whom had confirmed VAP. Using cycles to cross threshold (Ct) values as the result of the 16S PCR test, the area under the receiver operating characteristic (ROC) curve (AUROC) was 0.94 (95% CI 0.86 to 1.0, p<0.0001). Samples from 92 patients were available from the confirmation cohort, 26 (28%) of whom had confirmed VAP. The AUROC for Ct in this cohort was 0.89 (95% CI 0.83 to 0.95, p<0.0001). This study has derived and assessed the diagnostic accuracy of a novel application for 16S PCR. This suggests that 16S PCR in BAL could be used as a rapid test in suspected VAP and may allow better stewardship of antibiotics. Trial registration number VAPRAPID trial ref NCT01972425.

Ureaplasma parvum and Mycoplasma genitalium are found to be significantly associated with microscopy-confirmed urethritis in a routine genitourinary medicine setting

Summary Inflammation of the urethra defined by an excess of polymorphonuclear leukocytes in the absence of sexually transmitted Chlamydia trachomatis and Neisseria gonorrhoeae is called non-chlamydial non-gonococcal urethritis (NCNGU). Although Mycoplasma genitalium is now recognised as causing a sexually transmitted infection, the clinical significance of the other Mollicute species is less clear. This study used specific real-time quantitative polymerase chain reaction assays to detect and quantify four Mollicute species, M. genitalium, M. hominis, Ureaplasma urealyticum and U. parvum, in urine specimens from men with and without NCNGU. A total of 165 urine specimens from male patients attending a genitourinary medicine clinic were eligible for the study, with microscopy-confirmed (≥5 polymorphonuclear leukocytes in urethral swab) NCNGU in 75 (45.5%) and non-confirmed NCNGU in 90 (54.5%). Chi-squared statistical analysis indicated a significantly higher prevalence of U. parvum (17.3% vs. 5.6%; p = 0.03) and M. genitalium (12% vs. 0%; p < 0.001) in NCNGU. In a subset analysis, M. genitalium was also significantly (p = 0.03) higher in men who have sex with men (MSM; 13.5%) compared to non-MSM (3.1%). No significant associations were reported for U. urealyticum and M. hominis. In conclusion, this study supports a clinically significant role in NGNCU for both U. parvum and M. genitalium.

Lack of association between serological evidence of past Coxiella burnetii infection and incident ischaemic heart disease: nested case-control study.

BackgroundCoxiella burnetii causes the common worldwide zoonotic infection, Q fever. It has been previously suggested that patients who had recovered from acute Q fever (whether symptomatic or otherwise) may be at increased risk of ischaemic heart disease. We undertook this study to determine if past infection with Coxiella burnetii, the aetiological agent of Q fever, is a risk factor for the subsequent development of ischaemic heart disease.MethodsA nested case-control study within the Prospective Epidemiological Study of Myocardial Infarction (PRIME). The PRIME study is a cohort study of 10,593 middle-aged men undertaken in France and Northern Ireland in the 1990s. A total of 335 incident cases of ischaemic heart disease (IHD) were identified and each case was matched to 2 IHD free controls. Q fever seropositivity was determined using a commercial IgG ELISA method.ResultsSeroprevalence of Q fever in the controls from Northern Ireland and France were 7.8% and 9.0% respectively. No association was seen between seropositivity and age, smoking, lipid levels, or inflammatory markers. The unadjusted odds ratio (95% CI) for Q fever seropositivity in cases compared to controls was 0.95 (0.59, 1.57). The relationship was substantially unaltered following adjustment for cardiovascular risk factors and potential confounders.ConclusionSerological evidence of past infection with C. burnetii was not found to be associated with an increased risk of IHD.

Clostridium difficile Ribotype 023 Lacks the Ability To Hydrolyze Esculin, Leading to False-Negative Results on Chromogenic Agar

Clostridium difficile strains are toxigenic, spore forming, Gram-positive, anaerobic bacteria responsible for the majority of cases of colitis and antibiotic-associated diarrhea in the developed world ([1][1]). The use of chromogenic media for the rapid and specific detection of clinically important