Stephen Morris-Jones

Differential susceptibility of PCR reactions to inhibitors: an important and unrecognised phenomenon

BackgroundPCR inhibition by nucleic acid extracts is a well known yet poorly described phenomenon. Inhibition assessment generally depends on the assumption that inhibitors affect all PCR reactions to the same extent; i.e. that the reaction of interest and the control reaction are equally susceptible to inhibition. To test this assumption we performed inhibition assessment on DNA extracts from human urine samples, fresh urine and EDTA using different PCR reactions.ResultsWhen copurified inhibitors were assessed using two different PCR reactions one reaction appeared to be inhibited whilst the other was not. Further experiments using various concentrations of unextracted urine to inhibit six different PCR reactions revealed that susceptibility to inhibition was highly variable between reactions. Similar results were obtained using EDTA as the PCR inhibitor. We could find no obvious explanation why one reaction should be more susceptible to inhibition than another, although a possible association with amplicon GC content was noted.ConclusionThese findings have serious implications for all PCR-based gene expression studies, including the relatively new PCR array method, and for both qualitative and quantitative PCR-based molecular diagnostic assays, suggesting that careful consideration should be given to inhibition compatibility when conducting PCR analyses. We have demonstrated unequivocally that it is not safe to assume that different PCR reactions are equally susceptible to inhibition by substances co-purified in nucleic acid extracts.

Development and evaluation of a real-time PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveolar lavage fluid of HIV-infected patients

Background: Pneumocystis pneumonia (PCP) is conventionally diagnosed by identifying Pneumocystis jirovecii in lower respiratory tract samples using cytochemical stains. Molecular diagnosis of PCP is potentially more sensitive. Methods: A study was undertaken to use an extensively optimised real-time polymerase chain reaction (PCR) using primers designed to hybridise with the P jirovecii heat shock protein 70 (HSP70) gene to quantify P jirovecii DNA in bronchoalveolar lavage (BAL) fluid from HIV-infected patients with and without PCP, and to compare this assay with conventional PCR targeting the P jirovecii mitochondrial large subunit rRNA gene sequence (mt LSU rRNA). Results: Sixty-one patients had 62 episodes of PCP (defined by detection of P jirovecii in BAL fluid by cytochemical stains and typical clinical presentation). Quantifiable HSP70 DNA was detected in 61/62 (range ∼13–18 608 copies/reaction; median ∼332) and was detectable but below the limit of quantification (∼5 copies/reaction) in 1/62. Seventy-one other patients had 74 episodes with alternative diagnoses. Quantifiable HSP70 DNA was detectable in 6/74 (8%) episodes (range ∼6–590 copies/reaction; median ∼14) and detectable but below the limit of quantification in 34/74 (46%). Receiver-operator curve analysis (cut-off >10 copies/reaction) showed a clinical sensitivity of 98% (95% 91% to 100%) and specificity of 96% (95% CI 87% to 99%) for diagnosis of PCP. By contrast, clinical sensitivity of mt LSU rRNA PCR was 97% (95% CI 89% to 99%) and specificity was 68% (95% CI 56% to 78%). Conclusion: The HSP70 real-time PCR assay detects P jirovecii DNA in BAL fluid and may have a diagnostic application. Quantification of P jirovecii DNA by real-time PCR may also discriminate between colonisation with P jirovecii and infection.

Imported enteric fever: case series from the hospital for tropical diseases, London, United Kingdom.

Our current knowledge of the clinical characteristics of enteric fever is drawn mainly from population-based studies in disease-endemic countries, and there are limited data published on cases in returning travelers. We report the clinical characteristics of enteric fever in 92 travelers returning to London, United Kingdom. Salmonella typhi and S. paratyphi resulted in an almost indistinguishable clinical picture. Rose spots and relative bradycardia were found only in a few patients. A total of 91% of the patients had a normal leukocyte count, which was associated with a markedly increased level of alanine aminotransferase in 82%. A total of 57% of the S. typhi isolates had decreased susceptibility to ciprofloxacin and resistance to nalidixic acid; these isolates were from southern Asia. Thirty percent were multidrug resistant; all were from southern Asia and Nigeria. None of the paratyphoid isolates were multidrug resistant but rates of decreased susceptibility to fluoroquinolones were higher than in S. typhi (74%).

Thoracic empyema: current opinions in medical and surgical management.

Purpose of review Empyema is defined as pus in the thoracic cavity due to pleural space infection and has a multifactorial underlying cause, although a majority of them are post-bacterial pneumonia caused by tuberculosis or by infection following penetrating chest injuries or surgical procedures. It is still associated with significant morbidity and mortality in adults and children despite optimal management according to current guidelines. Historically, empyema management has been empirical, but more recent data are leading to more focused management guidelines. Recent findings The number of therapeutic agents licensed for intrapleural use or undergoing clinical trials in the management of empyema continues to expand, although their use is currently controversial and probably best limited to trials and specialist centers. Although their use is limited by availability, ultrasound and guided aspiration is the investigation of choice in suspected empyema. It is safer, more sensitive, provides more information, and, in the case of guided-drainage, is more likely to be effective. Finally, there is a growing body of literature that supports very early involvement of thoracic surgeons in empyema management. An emerging question for the future is whether some or indeed all patients with empyema should now bypass medical thoracostomy and proceed directly to video-assisted thoracoscopic surgery for both acute and chronic empyemas. Summary A summary of the most recent opinions and results in thoracic empyema management is outlined. Treatment of empyema can be summarized as appropriate antibiotic therapy combined with medical or surgical pleural space drainage, management of any underlying factors, with further surgery indicated for chronic disease.

A cross-sectional study of Mycoplasma genitalium infection and correlates in women undergoing population-based screening or clinic-based testing for Chlamydia infection in London

Objective To determine Mycoplasma genitalium infection and correlates among young women undergoing population-based screening or clinic-based testing for Chlamydia infection. Design Cross-sectional study. Setting National Chlamydia Screening Programme (NCSP) and two London sexually transmitted infection (STI) clinics. Participants 2441 women aged 15–64 years who participated in the NCSP and 2172 women who attended two London STI clinics over a 4-month period in 2009. Outcome measures (1) M genitalium prevalence in defined populations (%). (2) Age-adjusted ORs (aORs) for correlates of M genitalium infection. Results The overall frequency of M genitalium and Chlamydia trachomatis was 3% and 5.4%, respectively. Co-infection was relatively uncommon (0.5% of all women); however 9% of women with C trachomatis also had M genitalium infection. M genitalium was more frequently detected in swab than urine samples (3.9 vs 1.3%, p<0.001) with a significantly higher mean bacterial load (p ≤ 0.001). Among NCSP participants, M genitalium was significantly more likely to be diagnosed in women of black/black British ethnicity (aOR 2.3, 95% CI 1.2 to 4.5, p=0.01). M genitalium and C trachomatis and were both significantly associated with multiple sexual partners in the past year (aOR 2.4, 95% CI 1.3 to 4.4, p=0.01 and aOR 2.0, 95% CI 1.4 to 2.8, p<0.01). Among STI clinic attendees, M genitalium was more common in women who were less than 25 years in age. Conclusions M genitalium is a relatively common infection among young women in London. It is significantly more likely to be detected in vulvovaginal swabs than in urine samples. Co-infection with Chlamydia is uncommon. The clinical effectiveness of testing and treatment strategies for M genitalium needs further investigation.

Mycotic aneurysms: a case report, clinical review and novel imaging strategy

A 62-year-old man presented to a district general hospital with a 4-week history of fever, drenching sweats, lethargy and intermittent thoracic back pain. Two weeks prior to onset, a pacemaker had been inserted for recurrent syncope secondary to carotid sinus hypersensitivity. His other past medical history included a permanent tracheostomy following resection of a laryngeal carcinoma 8 years previously, and ischaemic heart disease. The latter culminated in an acute coronary syndrome 7 months before the current presentation, for which primary angioplasty was undertaken with insertion of six drug-eluting coronary artery stents requiring dual antiplatelet therapy for at least 1 year. On admission , he was febrile at 38.5°C. There were no peripheral stigmata of infective endocarditis, auscultation of the pre-cordium identified no murmurs and the pacemaker site was not inflamed. Systemic examination was otherwise unremarkable, as were chest radiography and urinalysis. Transthoracic echocardiography demonstrated good systolic function, with no valvular regurgitation or pacemaker lead vegetations. In the absence of a clear septic focus, he was commenced on piperacillin–tazobactam. Blood tests showed haemoglobin of 11.6 g/dl, white cell count of 5.7 × 109 cells/l and platelet count of 52 × 109 cells/l. C-reactive protein (CRP) was elevated at 272 mg/l. Coagulation screen was normal. Multiple blood cultures were drawn, which grew methicillin-sensitive Staphylococcus aureus . Intravenous flucloxacillin and rifampicin were commenced. He was consequently transferred to the London Heart Hospital for further management. The pacemaker was explanted and there was marked clinical improvement. Nonetheless, despite antibiotic therapy he continued to have high-grade pyrexias, without diurnal variation, and elevated serum CRP concentrations. An extensive septic screen, including urine and blood cultures, chest radiographs and repeated transthoracic and transoesophageal echocardiograms revealed no abnormalities. Computed tomography (CT) scan of the thorax, abdomen and pelvis to identify any occult septic focus revealed a large infrarenal aortic aneurysm, as well …

Needles and the damage done: Reasons for admission and financial costs associated with injecting drug use in a Central London Teaching Hospital

OBJECTIVES To establish the clinical reasons for inpatient admissions among injecting drug users. To determine the frequency of behavioural issues during their care and to estimate the financial implications of injecting drug use to the health service. METHODS Retrospective cohort study at University College London Hospital. Clinical, laboratory and financial data were extracted from case notes and electronic records. The cost of each admission was compared to the income received for the period of care. RESULTS 124 injecting drug users required 191 admissions between 2005 and 2009. Skin and soft tissue infections (58%) and pneumonia (18%) were the commonest reasons for admission. Bacteraemia at admission was often not accompanied by an inflammatory response. Exposure to HIV (4%), hepatitis B (49%) and C (84%) was common. Drug misuse (16%) during admission was frequent. The cost to the NHS of treating soft tissue infections in drug users was approximately £77 million per annum. After a median follow-up of 40 months, 10 patients (8%) had died. All deaths were attributable to drug use. CONCLUSIONS Bacterial and viral infections are largely responsible for the significant mortality and morbidity of injecting drug users presenting to secondary care. The financial burden to the NHS is substantial.

Comparison of two interferon-gamma release assays (QuantiFERON-TB Gold In-Tube and T-SPOT.TB) in testing for latent tuberculosis infection among HIV-infected adults.

There is currently no ‘gold standard’ for diagnosis of latent tuberculosis infection (LTBI), and both the tuberculin skin test and interferon-gamma release assays (IGRAs) are used for diagnosis; the latter have a higher sensitivity than tuberculin skin tests for diagnosis of LTBI in HIV-infected individuals with lower CD4 counts. No evidence base exists for selection of IGRA methodology to identify LTBI among human immunodeficiency virus-infected patients in the UK. We prospectively evaluated two commercially available IGRA methods (QuantiFERON-TB Gold In Tube [QFG] and T-SPOT.TB) for testing LTBI among HIV-infected patients potentially nosocomially exposed to an HIV-infected patient with ‘smear-positive’ pulmonary tuberculosis. Among the exposed patients median CD4 count was 550 cells/µL; 105 (90%) of 117 were receiving antiretroviral therapy, of who 104 (99%) had an undetectable plasma HIV load. IGRAs were positive in 12 patients (10.3%); QFG positive in 11 (9.4%) and T-SPOT.TB positive in six (5.1%); both IGRAs were positive in five patients (4.3%). There was one indeterminate QFG and one borderline T-SPOT.TB result. Concordance between the two IGRAs was moderate (κ = 0.56, 95% confidence interval = 0.27–0.85). IGRAs were positive in only 4 (29%) of 14 patients with previous culture-proven tuberculosis. No patient developed tuberculosis during 20 months of follow-up.

A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods

Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These ‘isothermal’ methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

Correspondence to invasive shigellosis in MSM

Dear Editor, We read with interest the case report by Serafino Wani et al. that described invasive shigellosis due to Shigella flexneri in an HIV-infected man who had sex with men. We had observed more severe Shigella infections in our cohort and decided to review our recent experience of HIV-infected patients at Mortimer Market Centre, London with Shigella-positive species. Shigella infections usually affect men and women equally, with links to overseas travel. The UK has seen a rise in Shigella infections in men who have sex with men (MSM) and these are now endemic among MSM in London, with, in 2015, an estimated excess of 275 cases in adult males with no travel history compared with adult females (a three-fold increase since 2011). Recent analysis of Shigella infections from 2003 to 2015 found that 30% of non-travel-related infections were in HIV-positive men, the majority of whom were MSM. Shigella in MSM is associated with high-risk behaviour such as multiple-partner unprotected sex at sex parties and ‘chemsex’ (sex associated with recreational drug use). Shigella sonnei with high levels of antimicrobial resistance was identified in September–October 2015 by Public Health England. With the growing number of cases, this is of concern, particularly regarding treatment options in those with severe infection who may be immune-compromised. As Serafino Wani et al. point out, Shigella infection in HIV can be associated with invasive disease, and severe clinical manifestations including haemolytic uraemic syndrome (HUS) have been described. We reviewed patients with Shigella species isolated from faeces between 2012 and 2015, to describe their clinical characteristics, laboratory abnormalities and outcomes. Forty-eight patients were identified, all MSM. Their demographics, clinical and biochemical history, and treatment are shown in Table 1 No patient had a history of chronic kidney disease. Eight had a previous AIDS-defining illness which did not affect the renal or gastrointestinal systems. Twenty-six patients had S. flexneri infections, 21 had S. sonnei and one had S. dysenteriae. The most