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Infections in Immunocompromised Patients
Abstract
In humans, both the innate and the specific immune systems are important in defence against microorganisms and they combine to ensure that most healthy adults only experience infectious diseases occasionally. Human immunodeficiency virus (HIV) is the most prevalent of these, while another retrovirus, human T-cell lymphotropic virus type 1 (HTLV-1) is associated with adult T-cell leukaemia/lymphoma. This chapter presents a list of the key components of the human immune system. It provides the physiological and immunological consequences for the host of a deficiency in each one. The chapter discusses some of the key organisms to consider in immunocompromised patients in detail. Cytomegalovirus (CMV) is a virus in the Herpesviridae family. It is considered that CMV spreads widely throughout the body during primary infection. CMV appears to be a ubiquitous human pathogen.
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Variability in the performance of nucleic acid amplification technology (NAT)-based assays presents a significant problem in the diagnosis and management of human cytomegalovirus (HCMV) infections. Here we describe a collaborative study to evaluate the suitability of candidate reference materials to harmonize HCMV viral load measurements in a wide range of NAT assays. Candidate materials comprised lyophilized Merlin virus, liquid Merlin virus, liquid AD169 virus, and purified HCMV Merlin DNA cloned into a bacterial artificial chromosome. Variability in the laboratory mean HCMV concentrations determined for virus samples across the different assays was 2 log10. Variability for the purified DNA sample was higher (>3 log10). The agreement between laboratories was markedly improved when the potencies of the liquid virus samples were expressed relative to the lyophilized virus candidate. In contrast, the agreement between laboratories for the purified DNA sample was not improved. Results indicated the suitability of the lyophilized Merlin virus preparation as the 1st WHO International Standard for HCMV for NAT. It was established in October 2010, with an assigned potency of 5 × 106 International Units (IU) (NIBSC code 09/162). It is intended to be used to calibrate secondary references, used in HCMV NAT assays, in IU.
Objectives:
HIV-associated cryptococcal meningitis is commonly caused by Cryptococcus neoformans, whilst infections with C. gattii sensu lato are historically rare. Despite available studies, little is known about the occurrence of C. gattii sensu lato infections among HIV-infected individuals in Zimbabwe.
Methods:
In a prospective cohort, we investigated the prevalence of C. gattii sensu lato meningitis among HIV-infected patients (n=74) in Harare, Zimbabwe.
Results:
Of the 66/74 isolates confirmed by molecular characterization, 16.7% (11/66) were found to be C. gattii sensu lato and 83.3% (55/66) C. neoformans sensu stricto. From one patient two phenotypically different C. gattii sensu lato colonies were cultured. The majority (n=9/12; 75%) of the C. gattii sensu lato isolates were C. tetragattii (AFLP7/VGIV), which has been an infrequently reported pathogen. In-hospital mortality associated with C. gattii sensu lato was 36.4%.
Conclusions:
Our data suggests that C. tetragattii (AFLP7/VGIV) is a more common cause of disease than C. gattii sensu stricto (genotype AFLP4/VGI) among patients with HIV-associated cryptococcal meningitis in Harare, Zimbabwe and possibly underreported in sub-Saharan Africa.
Blood Science is a relatively new discipline which merges biochemistry, haematology, immunology, transfusion science and genetics. This bringing together of traditional disciplines requires a corresponding change in education and training for healthcare scientists and Blood Science: Principles and Pathology is written in response to this emerging need.
It is not known whether psychological stress suppresses host resistance to infection. To investigate this issue, we prospectively studied the relation between psychological stress and the frequency of documented clinical colds among subjects intentionally exposed to respiratory viruses.
After completing questionnaires assessing degrees of psychological stress, 394 healthy subjects were given nasal drops containing one of five respiratory viruses (rhinovirus type 2, 9, or 14, respiratory syncytial virus, or coronavirus type 229E), and an additional 26 were given saline nasal drops. The subjects were then quarantined and monitored for the development of evidence of infection and symptoms. Clinical colds were defined as clinical symptoms in the presence of an infection verified by the isolation of virus or by an increase in the virus-specific antibody titer.
The rates of both respiratory infection (P less than 0.005) and clinical colds (P less than 0.02) increased in a dose-response manner with increases in the degree of psychological stress. Infection rates ranged from approximately 74 percent to approximately 90 percent, according to levels of psychological stress, and the incidence of clinical colds ranged from approximately 27 percent to 47 percent. These effects were not altered when we controlled for age, sex, education, allergic status, weight, the season, the number of subjects housed together, the infectious status of subjects sharing the same housing, and virus-specific antibody status at base line (before challenge). Moreover, the associations observed were similar for all five challenge viruses. Several potential stress-illness mediators, including smoking, alcohol consumption, exercise, diet, quality of sleep, white-cell counts, and total immunoglobulin levels, did not explain the association between stress and illness. Similarly, controls for personality variables (self-esteem, personal control, and introversion-extraversion) failed to alter our findings.
Psychological stress was associated in a dose-response manner with an increased risk of acute infectious respiratory illness, and this risk was attributable to increased rates of infection rather than to an increased frequency of symptoms after infection.
The link between Plasmodium falciparum malaria and endemic Burkitt’s lymphoma (eBL) has been an enigma for more than 50 years, since it was first observed that the occurrence of the two coincided [1,2]. So convincing was the association that it led to the prediction that eBL was caused by an infectious agent that was spread by malarial mosquitoes. The subsequent search turned up the first human oncogenic virus, Epstein-Barr virus (EBV) [3,4]. EBV is found in nearly all cases of eBL [5] and has been shown to be a potent transforming virus for human B cells [4]. eBL is believed to arise from germinal center (GC) B cells [6] and is characterized by a typical translocation of the c-myc oncogene into one of the immunoglobulin loci [7,8]. The subsequent deregulation of c-myc expression would normally lead to rapid apoptosis of the cell, but the presence of EBV is thought to rescue these cells and allow them to survive [9]. The enigma remained, however, because EBV is not spread by mosquitoes. eBL therefore represents an intriguing and unusual situation in which interactions between a protozoan parasite (P. falciparum) and a mammalian virus (EBV) combine to cause cancer. While the mechanisms linking EBV to lymphoma development are becoming better understood, the link between P. falciparum malaria and eBL has remained completely unexplained until now. Finally, we have a breakthrough in this longstanding issue from two separate studies: one through investigation of human tissues and one in a mouse model.
Chronic pulmonary aspergillosis (CPA) is an uncommon and problematic pulmonary disease, complicating many other respiratory disorders, thought to affect ∼240 000 people in Europe. The most common form of CPA is chronic cavitary pulmonary aspergillosis (CCPA), which untreated may progress to chronic fibrosing pulmonary aspergillosis. Less common manifestations include: Aspergillus nodule and single aspergilloma. All these entities are found in non-immunocompromised patients with prior or current lung disease. Subacute invasive pulmonary aspergillosis (formerly called chronic necrotising pulmonary aspergillosis) is a more rapidly progressive infection (<3 months) usually found in moderately immunocompromised patients, which should be managed as invasive aspergillosis. Few clinical guidelines have been previously proposed for either diagnosis or management of CPA. A group of experts convened to develop clinical, radiological and microbiological guidelines. The diagnosis of CPA requires a combination of characteristics: one or more cavities with or without a fungal ball present or nodules on thoracic imaging, direct evidence of Aspergillus infection (microscopy or culture from biopsy) or an immunological response to Aspergillus spp. and exclusion of alternative diagnoses, all present for at least 3 months. Aspergillus antibody (precipitins) is elevated in over 90% of patients. Surgical excision of simple aspergilloma is recommended, if technically possible, and preferably via video-assisted thoracic surgery technique. Long-term oral antifungal therapy is recommended for CCPA to improve overall health status and respiratory symptoms, arrest haemoptysis and prevent progression. Careful monitoring of azole serum concentrations, drug interactions and possible toxicities is recommended. Haemoptysis may be controlled with tranexamic acid and bronchial artery embolisation, rarely surgical resection, and may be a sign of therapeutic failure and/or antifungal resistance. Patients with single Aspergillus nodules only need antifungal therapy if not fully resected, but if multiple they may benefit from antifungal treatment, and require careful follow-up.
The diagnosis of invasive candidiasis (IC) and cryptococcosis is often complicated by slow and insensitive culture-based methods. Such delay results in poor outcomes due to the lack of timely therapeutic interventions. Advances in serological, biochemical, molecular and proteomic approaches have made a favorable impact on this process, improving the timeliness and accuracy of diagnosis with resultant improvements in outcome. This paper will serve as an overview of recent developments in the diagnostic approaches to infections due to these important yeast-fungi.
Background:
The use of a cytomegalovirus (CMV)-seronegative donor for a CMV-seronegative allogeneic hematopoietic stem cell transplant (HSCT) recipient is generally accepted. However, the importance of donor serostatus in CMV-seropositive patients is controversial.
Methods:
A total of 49 542 HSCT patients, 29 349 seropositive and 20 193 seronegative, were identified from the European Group for Blood and Marrow Transplantation database. Cox multivariate models were fitted to estimate the effect of donor CMV serological status on outcome.
Results:
Seronegative patients receiving seropositive unrelated-donor grafts had decreased overall survival (hazard ratio [HR], 1.13; 95% confidence interval [CI], 1.06-1.21; P < .0001) compared with seronegative donors, whereas no difference was seen in patients receiving HLA-matched sibling grafts. Seropositive patients receiving grafts from seropositive unrelated donors had improved overall survival (HR, 0.92; 95% CI, .86-.98; P < .01) compared with seronegative donors, if they had received myeloablative conditioning. This effect was absent when they received reduced-intensity conditioning. No effect was seen in patients grafted from HLA-identical sibling donors. The same association was found if the study was limited to patients receiving transplants from the year 2000 onward.
Conclusions:
We confirm the negative impact on overall survival if a CMV-seropositive unrelated donor is selected for a CMV-seronegative patient. For a CMV-seropositive patient, our data support selecting a CMV-seropositive donor if the patient receives a myeloablative conditioning regimen.
Staphylococcus aureus is a major human pathogen that causes a wide range of clinical infections. It is a leading cause of bacteremia and infective endocarditis as well as osteoarticular, skin and soft tissue, pleuropulmonary, and device-related infections. This review comprehensively covers the epidemiology, pathophysiology, clinical manifestations, and management of each of these clinical entities. The past 2 decades have witnessed two clear shifts in the epidemiology of S. aureus infections: first, a growing number of health care-associated infections, particularly seen in infective endocarditis and prosthetic device infections, and second, an epidemic of community-associated skin and soft tissue infections driven by strains with certain virulence factors and resistance to β-lactam antibiotics. In reviewing the literature to support management strategies for these clinical manifestations, we also highlight the paucity of high-quality evidence for many key clinical questions.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Established preoperative antibiotic prophylaxis in cardiac surgery is ineffective against methicillin-resistant coagulase-negative staphylococci (CoNS). This case-control study aimed to determine factors predicting deep sternal wound infections due to methicillin-resistant CoNS. All cardiac surgery patients undergoing sternotomy between June 2009 and March 2013 prospectively documented in a Swiss tertiary care center were included. Among 1999 patients, 82 (4.1%) developed deep sternal wound infection. CoNS were causal in 36 (44%) patients, 25/36 (69%) being methicillin-resistant. Early re-intervention for noninfectious causes (OR 4.3; 95% CI 1.9-9.5) was associated with methicillin-resistant CoNS deep sternal wound infection. Among CoNS deep sternal wound infection, perioperative antimicrobial therapy (P=.002), early re-intervention for noninfectious causes (OR 7.9; 95% CI 0.9-71.1), and time between surgery and diagnosis of infection over 21 days (OR 10.8; 95% CI 1.2-97.8) were associated with methicillin resistance. These findings may help to better tailor preoperative antimicrobial prophylaxis.
The use of MALDI-TOF MS for staphylococcal identification is now considered routine in laboratories compared with the conventional phenotypical methods previously used. We verified its microbiological relevance for the main species of coagulase negative staphylococci (CoNS) by randomly selecting 50 isolates. From January 1, 2007 to August 31, 2008, 12,479 staphylococci were isolated with phenotypic methods, of which 4,594 were identified as Streptococcus aureus and 7,885 were coagulase negative. Using MALDI-TIF MS from January 1, 2011 to August 31, 2012, 14,913 staphylococci were identified, 5,066 as S. aureus and 9,847 were CoNS. MALDI-TOF MS allowed the identification of approximately 85% of CoNS, whereas only 14% of CoNS were identified to the species level with phenotypic methods because they were often considered contaminants. Furthermore, the use of MALDI-TOF MS revealed the occurrence of recently characterized species, such as S. pettenkoferi, S. condimenti, and S. piscifermentans. Microbiological relevance analysis further revealed that some species displayed a high rate of microbiological significance, i.e., 40% of the S. lugdunensis included in the analysis were associated with an infection risk. This retrospective microbiological study confirms the role of MALDI-TOF MS in clinical settings for staphylococcal identification with clinical consequences. Species distribution reveals the occurrence of the recently identified species S. pettenkoferi as well as putative virulent species, including S. lugdunensis.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Background:
Cryptococcus neoformans is the most common cause of adult meningitis in sub-Saharan Africa. The cryptococcal antigen (CRAG) lateral flow assay (LFA) has simplified diagnosis as a point-of-care test approved for serum or cerebrospinal fluid (CSF). We evaluated the accuracy of the CRAG LFA using fingerstick whole blood compared with serum/plasma and CSF for diagnosing meningitis.
Methods:
From August 2013 to August 2014, CRAG LFA (IMMY, Norman, Oklahoma) tests were performed on fingerstick whole blood, plasma/serum, and CSF in 207 HIV-infected adults with suspected meningitis in Kampala, Uganda. Venous blood was also collected and centrifuged to obtain serum and/or plasma. CSF was tested after lumbar puncture.
Results:
Of 207 participants, 149 (72%) had fingerstick CRAG-positive results. There was 100% agreement between fingerstick whole blood and serum/plasma. Of the 149 fingerstick CRAG-positive participants, 138 (93%) had evidence of cryptococcal meningitis with a positive CSF CRAG. Eleven participants (5%) had isolated cryptococcal antigenemia with a negative CSF CRAG and culture, of whom 8 had CSF abnormalities (n = 3 lymphocytic pleocytosis, n = 5 elevated protein, n = 4 increased opening pressure). No persons with cryptococcal meningitis had negative fingersticks.
Conclusions:
The 100% agreement between whole blood, serum, and plasma CRAG LFA results demonstrates that fingerstick CRAG is a reliable bedside diagnostic test. Using point-of-care CRAG testing simplifies screening large numbers of patients and enables physicians to prioritize on whom to measure CSF opening pressure using manometers.
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of bacteraemia. We aimed for a complete picture of severe MRSA infections by characterizing all MRSA isolates from bloodstream infections in the largest German federal state (North Rhine-Westphalia, 18 million inhabitants) using S. aureus protein A (spa) sequence-typing and antimicrobial susceptibility testing. MRSA isolates (n=1,952) were prospectively (2011-2013) collected and spa-typed. Among 181 different spa types, t003 (n=746 isolates; 38.2%) and t032 (n=594; 30.4%) were predominant. Analysis of the geographical occurrence of spa clonal complexes (spa-CCs) and spa types revealed divergent distribution between federal state districts for spa-CCs 003 (p<0.001; including t003, p<0.001 and t264, p<0.001), 008 (p=0.021), 011 (p=0.002), 032 (p<0.001; including t022, p=0.014 and t032, p<0.001) and spa type t2807 (p<0.001).Minimum inhibitory concentrations (MICs) of antimicrobial substances were tested using broth microdilution. Of all isolates, 96% were resistant to fluoroquinolons, 78% to erythromycin, 70% to clindamycin, 4% to gentamicin, 2% to rifampicin, 0.4% to daptomycin, 0.1% to linezolid and 0% to vancomycin, respectively. Vancomycin MICs of 2 mg/L involved 0.5% of the isolates. In conclusion, the detection of regional molecular clusters added valuable information for epidemiological case tracing and allowed for conclusions on the importance of newly emerging MRSA reservoirs, such as livestock (spa-CC011), for MRSA bacteraemia in some parts of the federal state. Susceptibility testing revealed broad resistance to substances used for oral treatment, but demonstrated that those antibiotics that are mostly applied for treatment of MRSA bacteraemia and important combination partners were highly susceptible.
Copyright © 2015. Published by Elsevier Ltd.
Background:
Strict definition of invasive aspergillosis (IA) cases is required to allow precise conclusions about the efficacy of antifungal therapy. The Global Comparative Aspergillus Study (GCAS) compared voriconazole to amphotericin B (AmB) deoxycholate for the primary therapy of IA. Because predefined definitions used for this trial were substantially different from the consensus definitions proposed by the European Organization for Research and Treatment of Cancer/Mycoses Study Group in 2008, we recategorized the 379 episodes of the GCAS according to the later definitions.
Methods:
The objectives were to assess the impact of the current definitions on the classification of the episodes and to provide comparative efficacy for probable/proven and possible IA in patients treated with either voriconazole or AmB. In addition to original data, we integrated the results of baseline galactomannan serum levels obtained from 249 (65.7%) frozen samples. The original response assessment was accepted unchanged.
Results:
Recategorization allowed 59 proven, 178 probable, and 106 possible IA cases to be identified. A higher favorable 12-week response rate was obtained with voriconazole (54.7%) than with AmB (29.9%) (P < .0001). Survival was higher for voriconazole for mycologically documented (probable/proven) IA (70.2%) than with AmB (54.9%) (P = .010). Higher response rates were obtained in possible IA treated with voriconazole vs AmB with the same magnitude of difference (26.2%; 95% confidence interval [CI], 7.2%-45.3%) as in mycologically documented episodes (24.3%; 95% CI, 11.9%-36.7%), suggesting that possible cases are true IA.
Conclusions:
Recategorization resulted in a better identification of the episodes and confirmed the higher efficacy of voriconazole over AmB deoxycholate in mycologically documented IA.
Cryptococcosis has evolved into a major invasive fungal disease over the last century. Its primary epidemiology has been focused on three major outbreaks of disease that reflects both changing environmental exposures and growth of host risk factors. The molecular understandings of yeast pathobiology have been bolstered by identification of the yeast’s dynamic genomic structures and functions. It is during these new insights into epidemiology and pathobiology that we have also improved our diagnosis of this infection with a new point-of-care, simple, cheap test which utilizes a lateral flow assay for antigen detection. With methods for effective identification of Cryptococcus in the host, the principles for management of this deadly infection include both use of old drugs and new insights into treatment strategies to improve outcome. In this review there are a series of recent insights, opinions, and facts which attempt to summarize our present knowledge base for this deadly fungal central nervous system infection with a particular emphasis on its diagnosis and management.
Because infectious diseases are a major source of morbidity and mortality in the majority of patients with primary immunodeficiencies
(PIDs), the application of a prophylactic regimen is often necessary. However, because of the variety of PIDs and pathogens
involved, and because evidence is scarce, practices are heterogeneous. To homogenize practices among centers, the French National
Reference Center for PIDs aimed at elaborating recommendations for anti-infectious prophylaxis for the most common PIDs. We
performed a literature review of infectious complications and prophylactic regimens associated with the most frequent PIDs.
Then, a working group including different specialists systematically debated about chemoprophylaxis, immunotherapy, immunization,
and recommendations for patients. Grading of prophylaxis was done using strength of recommendations (decreasing from A to
D) and evidence level (decreasing from I to III). These might help infectious diseases specialists in the management of PIDs
and improving the outcome of patients with PIDs.
Cryptococcal meningitis causes morbidity and mortality worldwide. The burden of disease is greatest in middle- and low-income countries with a high incidence of human immunodeficiency virus (HIV) infection. Patients taking immunosuppressive drugs and some immunocompetent hosts are also at risk. Treatment of cryptococcal meningitis consists of three phases: induction, consolidation, and maintenance. Effective induction therapy requires potent fungicidal drugs (amphotericin B and flucytosine), which are often unavailable in low-resource, high-endemicity settings. As a consequence, mortality is unacceptably high. Wider access to effective treatment is urgently required to improve outcomes. For human immunodeficiency virus-infected patients, judicious management of asymptomatic cryptococcal antigenemia and appropriately timed introduction of antiretroviral therapy are important.
The complete genome of human cytomegalovirus (HCMV) was elucidated almost 25 years ago using a traditional cloning and Sanger sequencing approach. Analysis of the genetic content of additional laboratory and clinical isolates has lead to a better, albeit still incomplete, definition of the coding potential and diversity of wild-type HCMV strains. The introduction of a new generation of massively parallel sequencing technologies, collectively called next-generation sequencing, has profoundly increased the throughput and resolution of the genomics field. These increased possibilities are already leading to a better understanding of the circulating diversity of HCMV clinical isolates. The higher resolution of next-generation sequencing provides new opportunities in the study of intrahost viral population structures. Furthermore, deep sequencing enables novel diagnostic applications for sensitive drug resistance mutation detection. RNA-seq applications have changed the picture of the HCMV transcriptome, which resulted in proof of a vast amount of splicing events and alternative transcripts. This review discusses the application of next-generation sequencing technologies, which has provided a clearer picture of the intricate nature of the HCMV genome. The continuing development and application of novel sequencing technologies will further augment our understanding of this ubiquitous, but elusive, herpesvirus.
Cryptococcosis is an important systemic mycosis and the third most prevalent disease in human immunodeficiency virus (HIV)-positive individuals. The incidence of cryptococcosis is high among the 25 million people with HIV/acquired immunodeficiency syndrome (AIDS), with recent estimates indicating that there are one million cases of cryptococcal meningitis globally per year in AIDS patients. In Cryptococcus neoformans, resistance to azoles may be associated with alterations in the target enzyme encoded by the gene ERG11, lanosterol 14α-demethylase. These alterations are obtained through mutations, or by overexpressing the gene encoding. In addition, C. gattii and C. neoformans present a heteroresistance phenotype, which may be related to increased virulence. Other species beyond C. neoformans and C. gattii, such as C. laurentii, have been diagnosed mainly in patients with immunosuppression. Infections of C. albidus have been isolated in cats and marine mammals. Recent evidence suggests that the majority of infections produced by this pathogen are associated with biofilm growth, which is also related with increased resistance to antifungal agents. Therefore, there is a great need to search for alternative antifungal agents for these fungi. The search for new molecules is currently occurring from nanoparticle drugs of plant peptide origin. This article presents a brief review of the literature regarding the epidemiology of cryptococcosis, as well as fungal resistance and new alternatives for treatment.
Cryptococcal meningitis (CM)-related mortality may be prevented by screening patients for sub-clinical cryptococcal antigenaemia (CRAG) at antiretroviral-therapy (ART) initiation and pre-emptively treating those testing positive. Prior to programmatic implementation in South Africa we performed a cost-effectiveness analysis of alternative preventive strategies for CM.
Cost-effectiveness analysis.
Using South African data we modelled the cost-effectiveness of four strategies for patients with CD4 cell-counts <100 cells/µl starting ART 1) no screening or prophylaxis (standard of care), 2) universal primary fluconazole prophylaxis, 3) CRAG screening with fluconazole treatment if antigen-positive, 4) CRAG screening with lumbar puncture if antigen-positive and either amphotericin-B for those with CNS disease or fluconazole for those without. Analysis was limited to the first year of ART.
The least costly strategy was CRAG screening followed by high-dose fluconazole treatment of all CRAG-positive individuals. This strategy dominated the standard of care at CRAG prevalence ≥0.6%. Although CRAG screening followed by lumbar puncture in all antigen-positive individuals was the most effective strategy clinically, the incremental benefit of LPs and amphotericin therapy for those with CNS disease was small and additional costs were large (US$158 versus US$51per person year; incremental cost effectiveness ratio(ICER) US$889,267 per life year gained). Both CRAG screening strategies are less costly and more clinically effective than current practice. Primary prophylaxis is more effective than current practice, but relatively cost-ineffective (ICER US$20,495).
CRAG screening would be a cost-effective strategy to prevent CM-related mortality among patients initiating ART in South Africa. These findings provide further justification for programmatic implementation of CRAG screening.
Aims:
In an era when clinical microbiology laboratories are under increasing financial pressure, there is a need for inexpensive, yet effective, rapid microbiology tests. The aim of this study was to evaluate a novel modification of standard methodology for the identification and antimicrobial susceptibility testing (AST) of pathogens in positive blood cultures, reducing the turnaround time of laboratory results by 24 h.
Methods:
277 positive blood cultures had a Gram stain performed and were subcultured and incubated at 37°C in a CO2 atmosphere for 4-6 h. Identification of the visible growth was performed using matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS). Taking a modified approach to the Clinical and Laboratory Standards Institute-standardised AST methodology, an inoculum density of 0.5 McFarland was prepared from the early growth for disc diffusion testing. The standard AST method was also performed on the 18-24 h culture.
Results:
96% (n=73/76) of gram-negative organisms were correctly identified by MALDI-TOF MS. Comparative analysis of the rapid and standard AST results showed an overall interpretive category error rate of 7.7% (6.7% minor errors, 0.6% major errors and 0.4% very major errors). 100% of Staphylococcus aureus (n=41) and enterococcus isolates (n=9) were correctly identified after 4-6 h incubation. The overall AST categorical agreement was also 100% for these isolates.
Conclusions:
An incubation of 4-6 h directly from positive blood cultures allowed for both a rapid species identification and an antimicrobial susceptibility result approximately 24 h earlier than is possible using standard methodology.
Using 30 clinical isolates of S. aureus representative of the most prevalent clones circulating in France, the performance of the AlereTM PBP2a Culture Colony Test (CCT) and the Slidex® MRSA detection kit (SMD) were compared in 5 different labs. CCT demonstrated better performance and was easier to conduct in routine.
Principles and Practice of Clinical Virology is the bible for all working in the field of clinical virology - from the trainee to the expert because there's always something new to learn! As before, the book provides a detailed account of the diagnosis and treatment of virus infections, with a stronger emphasis on clinical expertise and management. Each chapter deals with a single virus or group or viruses and is written by leading international experts in the field. What's new in this edition Showcases the wealth of new knowledge acquired on virus infections and reflects the discovery of newly recognized emerging infections, the improvement or development of new vaccines, and an increasing repertoire of antiviral agents for treatment. All chapters have been thoroughly revised and there are a number of new contributors, joining the cadre of internationally-recognized experts. Includes a new chapter on vaccinology covering the principles relating to the development and use of vaccines generally, which complements the specific vaccines described in the other chapters. The two chapters on nosocomial infections have been enlarged and will be particularly useful for those having to advise on the management of hospital-acquired infections. Emphasizes the rapid accumulation of new information in such fields as retroviruses, particularly HIV, SARS, hepatitis C and influenza, including avian influenza.
Background:
The diagnosis of CMV infection is challenging and the quality of serological laboratory testing is critical, especially in pregnancy and in the determination of transplant recipients and donors serostatus.
Objectives:
Evaluate the performances of the new LIAISON(®) CMV II line: LIAISON(®) CMV IgG II, LIAISON(®) CMV IgM II and LIAISON(®) CMV IgG Avidity II in comparison with the routine methods used in our laboratory.
Study design:
The evaluation of LIAISON(®) CMV IgG II and LIAISON(®) CMV IgM II was performed on both prospective routine samples and retrospective selected samples for a total of 383 sera. CMV IgG avidity was assessed with 88 samples.
Results:
The overall agreement was 98.8% for the IgG and 95% for the IgM on the routine population. On selected retrospective samples, excellent agreement was found in the seronegative and past infection groups. In the recent infection group, discordances were observed in 7.1% of IgG and 13.1% of IgM. No recent infection was missed with LIAISON(®). Avidity agreement with VIDAS(®) was 81%. On 51 sera with a known time of infection, no high avidity was found in the group infected for less than 3 months and 82% of the samples showed a high avidity in the group infected for more than 3 months.
Conclusion:
The performances of the fully automated LIAISON(®) CMV II line assays are comparable to those of the reference methods used in our lab for both prospective and selected populations. This new CMV line is a useful tool for the diagnosis of CMV infections and CMV immune status in clinical settings.
Introduction and ClassificationEpidemiologyReplicationHost Genetic Determinants for HIV/AIDSViral Dynamics and PathogenesisImmune ResponsesThe Laboratory Diagnosis of HIV InfectionThe Natural History of HIV Infection and Its Clinical ManifestationsAntiretroviral Therapy—A Historical PerspectiveMonitoring of Antiretroviral Therapy and ResistanceAntiretroviral Drug ClassesTransmission of Drug ResistancePreventionVaccinesReferencesFurther Reading
Parvovirus B19 is a single-stranded DNA virus which preferentially targets the erythroblast resulting in red cell aplasia, which is temporary in immunocompetent persons. Since the discovery of B19 virus in 1975, a wide variety of blood diseases and cytopenias affecting several blood cell lineages have been documented during or following B19 infection. These include cytopenias affecting the erythroid, megakaryoblastoid, myeloid and lymphoid lineages, as well as a variety of bicytopenias, pancytopenia, bone marrow necrosis / fat embolism syndrome, myelodysplastic syndrome, leucoerythroblastopenia, and hemophagocytic lymphohistiocytosis. B19 infection may also complicate and precede the course of acute leukemia, the significance of which remains to be determined. This review describes the current state of knowledge of the abnormalities of individual blood cell lineages encountered during parvovirus B19 infection, over the almost 40 years since its discovery, and reveals some very interesting themes, which improve our understanding of the pathogenesis of B19 infection with particular reference to the bone marrow. Copyright © 2015 John Wiley & Sons, Ltd.
Copyright © 2015 John Wiley & Sons, Ltd.
ABSTRACT Cryptococcosis is caused by the fungal genus Cryptococcus. Cryptococcosis, predominantly meningoencephalitis, emerged with the HIV pandemic, primarily afflicting HIV-infected patients with profound T-cell deficiency. Where in use, combination antiretroviral therapy has markedly reduced the incidence of and risk for disease, but cryptococcosis continues to afflict those without access to therapy, particularly in sub-Saharan Africa and Asia. However, cryptococcosis also occurs in solid organ transplant recipients and patients with other immunodeficiencies as well as those with no known immunodeficiency. This article reviews innate and adaptive immune responses to C. neoformans, with an emphasis on recent studies on the role of B cells, natural IgM and Fc gamma receptor polymorphisms in resistance to cryptococcosis.
Secreted proteins are the frontline between the host and pathogen. In mammalian hosts, secreted proteins enable invasive infection and can modulate the host immune response. Cryptococcosis, caused by pathogenic Cryptococcus species, begins when inhaled infectious propagules establish to produce pulmonary infection, which if not resolved, can disseminate to the central nervous system to cause meningoencephalitis. Strains of Cryptococcus differ in their capacity to cause disease and the mechanisms underlying this are not well understood. To investigate the role of secreted proteins in disease we determined the secretome for three genome strains of Cryptococcus, including a hypovirulent and a hypervirulent strain of C. gattii and a virulent strain of C. neoformans. Sixty-seven unique proteins were identified, with different numbers and types of proteins secreted by each strain. The secretomes of the virulent strains were largely limited to proteolytic and hydrolytic enzymes, while the hypovirulent strain had a diverse secretome, including non-conventionally secreted canonical cytosolic and immunogenic proteins that have been implicated in virulence. The hypovirulent strain cannot establish pulmonary infection in a mouse model, but strains of this genotype have caused human meningitis. To directly test brain infection we used intracranial inoculation and found the hypovirulent strain was substantially more invasive than its hypervirulent counterpart. We suggest that immunogenic proteins secreted by this strain invoke a host response that limits pulmonary infection, but if infection reaches the brain there can be invasive growth and damage. Given their known role in virulence, it is possible that non-conventionally secreted proteins mediate this process.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
With increased use of expanded-spectrum triazoles for antifungal prophylaxis, the epidemiology of invasive fungal infections (IFIs) after allogeneic haematopoietic stem cell transplantation (HSCT) continues to evolve. To define the contemporary epidemiology of IFIs in this population, we reviewed all European Organization for Research and Treatment of Cancer-Mycoses Study Group proven and probable IFIs in adults transplanted from 2002 to 2011 and determined the incidence and risk factors for IFI and post-IFI mortality. All patients received antifungal prophylaxis. Fifty-three (14%) of 378 allogeneic HSCT recipients developed an IFI. There were 62 IFI episodes, of which aspergillosis (n = 31; 50%) and candidaemia (n = 15; 24%) were most common. Sixteen episodes (26%) were caused by other fungi, including Mucorales (n = 6; 10%) and the following uncommon pathogens: Trichosporon asahii, Arthrographis sp., Cladosporium sp., Geosmithia argillacea and Hormographiella aspergillata. Independent IFI risk factors were hospitalisation in an intensive care unit [ICU; odds ratio (OR) = 6.0], graft-versus-host disease (OR = 5.3), central venous catheter use (OR = 5.2) and hypoalbuminaemia (OR = 0.3 g(-1) dl(-1) increase in albumin). The 90-day mortality rate after IFI was 57%. Non-cytomegalovirus systemic viral co-infection (OR = 3.5) and stay in an ICU (OR = 2.9) were independent risk factors for death. Despite antifungal prophylaxis, IFIs remain common after allogeneic HSCT and previously uncommon pathogens are emerging.
© 2015 Blackwell Verlag GmbH.
Background: Cryptococcal meningitis (CM) is one of the most important HIV-related opportunistic infections, especially in the developing world, where overall mortality is very high. In order to help develop global strategies for prevention and treatment, it is important to estimate the burden of CM. Methods: We searched the English literature for studies reporting an estimate of CM among HIV populations. The median CM incidence of each UNAIDS geographic region was multiplied by the HIV population to estimate the region-specific CM cases. The range of cases in each region was calculated as ± one standard deviation from the regional estimate, using a median coefficient of variation across the regions. To estimate deaths, we assumed the 3-month case-fatality rate in high-income regions to be 9%, 55% in low- and middle-income regions, and 70% in Sub-Saharan Africa, based on available literature and expert opinion. Results: Of over 9,000 abstracts reviewed, 19 studies met the search criteria; yearly incidence ranged from 0.04%-12% among persons with HIV. Sub-Saharan Africa had the highest estimate (median incidence 3.2%, 720,000 cases, range, 144,000 - 1.3 million), followed by South/ Southeast Asia (median incidence 3.0%, 120,000 cases, range, 24,000 - 216,000). Median incidence was lowest in Western and Central Europe and Oceania (<0.1% each). Globally, approximately 957,900 cases (range, 371,700 - 1,544,000) of CM occur each year, resulting in 624,700 deaths (range, 125,000 - 1,124,900) by 3 months after infection. Conclusions: This study, the first attempt to estimate the global burden of CM, confirms the high burden of CM, particularly in Sub-Saharan Africa and South/ Southeast Asia. Further work is needed to better define and track the epidemiology of this infection, in order to prioritize prevention, diagnosis, and treatment strategies.
Purpose of review:
To consider new treatment options for cytomegalovirus (CMV) infection, review recent trials, and anticipate their use in clinical practice, focussing on bone marrow transplantation, congenital infection, and intervention during pregnancy.
Recent findings:
Three double-blind randomized placebo-controlled phase 2 proof-of-concept studies have each identified a novel antiviral drug with activity against CMV infection in bone marrow transplant patients. One of these (brincidofovir) inhibits the DNA polymerase that is the target of the currently licensed drug ganciclovir. Another new drug (maribavir) inhibits a protein kinase which, coincidentally, is the enzyme responsible for activating ganciclovir through phosphorylation. The third drug (letermovir) inhibits the terminase enzyme complex responsible for packaging unit length DNA into assembling virions.In addition, in a double-blind randomized placebo-controlled trial in neonates with symptomatic congenital CMV infection, a 6-month course of valganciclovir was superior to the standard 6-week course of the same drug. In pregnant women with primary CMV infection, administration of hyperimmune immunoglobulin did not significantly reduce transmission of CMV across the placenta.
Summary:
The ability to diagnose CMV infections reliably in different clinical settings through application of molecular laboratory methods has ushered in new ways of evaluating potential new treatments for CMV. Several of these may help control the diseases caused by this important human pathogen.
The definition of the heterogeneous group of coagulase-negative staphylococci (CoNS) is still based on diagnostic procedures that fulfill the clinical need to differentiate between Staphylococcus aureus and those staphylococci classified historically as being less or nonpathogenic. Due to patient- and procedure-related changes, CoNS now represent one of the major nosocomial pathogens, with S. epidermidis and S. haemolyticus being the most significant species. They account substantially for foreign body-related infections and infections in preterm newborns. While S. saprophyticus has been associated with acute urethritis, S. lugdunensis has a unique status, in some aspects resembling S. aureus in causing infectious endocarditis. In addition to CoNS found as food-associated saprophytes, many other CoNS species colonize the skin and mucous membranes of humans and animals and are less frequently involved in clinically manifested infections. This blurred gradation in terms of pathogenicity is reflected by species- and strain-specific virulence factors and the development of different host-defending strategies. Clearly, CoNS possess fewer virulence properties than S. aureus, with a respectively different disease spectrum. In this regard, host susceptibility is much more important. Therapeutically, CoNS are challenging due to the large proportion of methicillin-resistant strains and increasing numbers of isolates with less susceptibility to glycopeptides.
The human fungal pathogen Cryptococcus neoformans is able to rapidly and effectively adapt to varying conditions, favoring its survival in the environment and in the infected host. Many microbial phenotypes have been specifically correlated with virulence in this opportunistic pathogen, such as capsule production, melanin formation, and the secretion of various proteins. Additionally, cellular features such as the cell wall and morphogenesis play important roles in the interaction of this fungus with host immune recognition and response pathways. Survival in the face of host stress also requires maintaining RNA/DNA integrity. Additionally, aging and senescence of the fungal cells determines resistance to host-derived stresses. New mechanisms regulating the expression of these virulence-associated phenotypes have been recently explored. Importantly, human clinical studies are now confirming the roles of specific microbial factors in human infections.
Human cytomegalovirus (HCMV) is a recognised cause of disease in the fetus, the allograft recipient and AIDS patients. More recently, it has been recognised as a pathogen for those admitted to intensive care units, for the elderly and for the general population. The epidemiology and molecular and cellular pathology of this virus are summarised to provide an overarching model of pathogenesis able to account for these varying clinical presentations. In brief, HCMV has the potential to spread in the bloodstream to all organs, but only produces overt disease if the viral load increases to high levels. This is normally prevented by a robust immune response, so that the infected individual usually remains asymptomatic. However, this benefit comes at the cost of committing more and more immunological resources to controlling HCMV with time, so that the overall function of the immune system is impaired. Fortunately, recent progress in developing novel antiviral drugs and vaccines suggests the possibility that the diverse effects of HCMV may soon become controllable at the individual and population level respectively.
SUMMARY We report on the follow-up and epidemiological study triggered by the isolation of the first vancomycin-resistant Staphylococcus aureus (VRSA) detected in Europe. The patient and 53 close contacts were screened for S. aureus colonization and all isolates recovered were characterized by multiple molecular typing methods. The VRSA remained confined to the infected foot of the patient and was not detected in any of the close contacts. Nasal colonization with S. aureus was detected in 20 subjects, of whom 15 carried methicilin-susceptible isolates with the remaining five harbouring methicilin-resistant S. aureus (MRSA). The majority of the isolates belonged to clones that have been previously shown to be prevalent in Portugal, both in the hospital setting and in the community. Only one isolate, an MRSA, was closely related to the VRSA. Like most of the characterized VRSA isolates from other countries, the VRSA isolated in Portugal belonged to clonal complex (CC) 5. Despite the absence of VRSA dissemination, the recent increase in the incidence of lineages belonging to CC5 in some European countries, including Portugal, may result in more frequent opportunities for the emergence of VRSA.
A meticillin-resistant Staphylococcus aureus (MRSA) polymerase chain reaction assay (Xpert(®) MRSA, Cepheid) was assessed for point-of-care testing (POCT) used by healthcare assistants in an orthopaedic pre-admission clinic and on a vascular ward to reduce turnaround time. POCT results were compared with the routine swabs taken for culture. The POCT assay was easy to use, the turnaround time for negative results was greatly reduced, and sensitivity was 75.0% in the pre-admission clinic and 80.0% in the vascular ward. There were 27 POCT-positive/culture-negative results, but there was no evidence of MRSA infection or colonization in these patients for at least a year post procedure. POCT for MRSA colonization performed beyond the laboratory has important advantages over laboratory-based methods and should be explored further.
Nowadays transplantation is a consolidated therapeutic option for many kidney, heart, liver, and lung diseases in terminal stadium. During the last two decades, the survival of solid organ transplant (SOT) recipients has increased. Advances in the optimal tissue typing of organ donors, in the pre-transplant risk assessment of donor and recipient, in surgical skills, and in immunosuppressive treatment, have all contributed to this improvement. This article is protected by copyright. All rights reserved.
SUMMARY The negative impact of cytomegalovirus (CMV) infection on transplant outcomes warrants efforts toward improving its prevention, diagnosis, and treatment. During the last 2 decades, significant breakthroughs in diagnostic virology have facilitated remarkable improvements in CMV disease management. During this period, CMV nucleic acid amplification testing (NAT) evolved to become one of the most commonly performed tests in clinical virology laboratories. NAT provides a means for rapid and sensitive diagnosis of CMV infection in transplant recipients. Viral quantification also introduced several principles of CMV disease management. Specifically, viral load has been utilized (i) for prognostication of CMV disease, (ii) to guide preemptive therapy, (iii) to assess the efficacy of antiviral treatment, (iv) to guide the duration of treatment, and (v) to indicate the risk of clinical relapse or antiviral drug resistance. However, there remain important limitations that require further optimization, including the interassay variability in viral load reporting, which has limited the generation of standardized viral load thresholds for various clinical indications. The recent introduction of an international reference standard should advance the major goal of uniform viral load reporting and interpretation. However, it has also become apparent that other aspects of NAT should be standardized, including sample selection, nucleic acid extraction, amplification, detection, and calibration, among others. This review article synthesizes the vast amount of information on CMV NAT and provides a timely review of the clinical utility of viral load testing in the management of CMV in solid organ transplant recipients. Current limitations are highlighted, and avenues for further research are suggested to optimize the clinical application of NAT in the management of CMV after transplantation.
BACKGROUND:Information about the prevalence of Staphylococcus aureus resistance to antimicrobial drugs has mainly been obtained from invasive strains, although the commensal microbiota is thought to be an important reservoir of resistance. We aimed to compare the prevalence of nasal S aureus carriage and antibiotic resistance, including meticillin-resistant S aureus (MRSA), in healthy patients across nine European countries.
METHODS:
In this cross-sectional study, nasal swabs were obtained from 32,206 patients recruited by family doctors participating in existing nationwide family doctor networks in Austria, Belgium, Croatia, France, Hungary, Spain, Sweden, the Netherlands, and the UK. Eligible patients were aged 4 years or older (≥ 18 years in the UK) and presented with a non-infectious disorder. Swabs were sent to national microbiological laboratories for identification and isolation of S aureus. Antibiotic resistance testing was done at one central microbiological laboratory. We established the genotypic structure of the isolated MRSA strains with the spa typing method.
FINDINGS:
S aureus was isolated from 6956 (21 · 6%) of 32,206 patients swabbed. The adjusted S aureus prevalence for patients older than 18 years ranged from 12 · 1% (Hungary) to 29 · 4% (Sweden). Except for penicillin, the highest recorded resistance rate was to azithromycin (from 1 · 6% in Sweden to 16 · 9% in France). In total, 91 MRSA strains were isolated, and the highest MRSA prevalence was reported in Belgium (2 · 1%). 53 different spa types were detected-the most prevalent were t002 (n = 9) and t008 (n = 8).
INTERPRETATION:
The prevalence of S aureus nasal carriage differed across the nine European countries assessed, even after correction for age, sex, and family doctor. Generally, the prevalence of resistance, including that of MRSA, was low. The MRSA strains recorded showed genotypic heterogeneity, both within and between countries.
Since sternal surgical site infections (SSIs) can be life-threatening, every effort should be made to reduce their rate of occurrence.
To measure the rate of sternal SSIs after open heart surgery and to define the efficacy of infection control interventions in reducing this rate.
Surveillance of sternal SSIs was carried out prospectively for adult patients who underwent sternotomy between 2005 and 2012. Infection control interventions that were undertaken during the study period at different time intervals were prophylaxis with cefazolin or vancomycin, surveillance of sternal SSIs and feedback, preoperative nasal Staphylococcus aureus screening and decolonization with mupirocin, isolation of patients infected with or colonized by meticillin-resistant S. aureus, appropriate management of perioperative blood glucose level and chlorhexidine/alcohol usage for skin antisepsis.
There were 479 sternal SSIs in 18,460 patients during the study period (2.59%). The most frequent causes of sternal SSIs were coagulase-negative staphylococci (CoNS) (36%) and S. aureus (31%). Infection control interventions reduced the rate of sternal SSIs from 3.63% in 2005 to 1.65% in 2012 (P < 0.0001).
Our study shows that the rate of sternal SSIs can be decreased with proper infection control interventions. However, the interventions that were undertaken were effective only in reducing the rate of sternal SSIs caused by S. aureus. It is time to find interventions to control sternal SSIs caused by CoNS, the pathogen responsible for most sternal SSIs in hospitals where S. aureus SSIs are successfully controlled.
In this Journal we recently reported the effective and rapid identification and susceptibility testing of microor-ganisms by direct inoculation from positive blood culture bottles by combining MALDI-TOF and Vitek-2 Compact. 1 We have now investigated the use of MALDI-TOF and Xpert Ò MRSA/SA BC assay, for the rapid detection of MRSA and MSSA obtained directly from positive blood culture bottles. Staphylococcus aureus bacteraemia has been associated with high mortality rates, prolonged hospitalization, and increased economic costs. Delay in the initiation of appro-priate antimicrobial therapy has been identified as an impor-tant determinant in clinical outcomes. As a result, rapid identification of staphylococcal species and susceptibilities in bacteraemia patients might assist in the early optimization of therapy that would have a positive clinical impact. 2,3 Confirmatory identification and susceptibility testing of staphylococcal isolates recovered from positive blood cultures require a minimum of 2 days (1 day for culture plus 1 day for identification and susceptibility testing). This period of time delays the administration of pathogen-targeted therapy, promoting the unnecessary use of broad-spectrum antibiotics and therefore increasing the antibiotic selection pressure in the hospital environment. There are a number of methods currently used for identification of staphylococci in clinical samples that attempt to provide same-day definitive identification and differentiation between MSSA and MRSA. These include peptide nucleic acid fluorescence in situ hybridization (FISH), 4 mass spectrometry, 5 rapid immunochromato-graphic assays 6 and nucleic acid amplification methods. 7 In order to reduce the time needed to obtain reliable results, some authors have done direct identification from blood culture broths with matrix assisted laser desorption-ionization/time-of-flight mass spectrometry (MALDI-TOF MS) that yields identification results in 2e3 h. Wolters M et al., have suggested that MALDI-TOF MS could become a valuable tool for rapid typing of MRSA. 5 Nevertheless, further studies will be needed to show the usefulness of this tool for rapid detection of MRSA in the clinical setting. The aim of this study was to evaluate a combined use of MALDI-TOF MS and Xpert Ò MRSA/SA BC assay, for the rapid detection of MRSA and MSSA obtained directly from positive blood culture bottles and comparing it with conventional methods for MRSA detection. Over a period of 12 months, from November 2011 to November 2012, the Xpert Ò MRSA/SA BC test was applied to randomly selected and nonduplicate positive blood culture bottles, where S. aureus (n Z 91) was previously identified by MALDI-TOF MS. Two automatic systems were used in this study: the BacT/ Alert system (bioM erieux, Durham, NC) blood culture bottles, and the Bactec systems (Becton Dickinson, Sparks, MD). Bactec samples included Plus Aerobic, and Plus Anaerobic bottles and BacT/Alert samples included FA (aerobic), FN (anaerobic), and BacT/Alert PF (paediatric) bottles. When a blood culture was flagged positive by the BACTECä or the BacT/ALERT, and a Gram stain confirmed the presence of gram-positive cocci in clusters, direct identification was done by MALDI-TOF mass spectrometry. 1,6 Mass spectra were obtained using a Microflex LT Mass spec-trometer (Bruker Daltonik GmbH) and Flex Control software. For S. aureus identification, the spectra obtained were compared with a reference library of spectra provided by the manufacturer (Reference Library 3.0.10) using the MALDI-Biotyper 2.0 software (Bruker Daltonik GmbH). All positive blood cultures were routinely subcultivated on 3 agar plates: sheep blood agar, chocolate blood agar, and Brucella blood agar. The sheep blood agar and chocolate blood agar plates were incubated at 35 C in an atmosphere containing 5% CO 2 for 24 h. The Brucella blood agar was incu-bated at 35 C in anaerobic atmosphere for 2 days. Identifi-cation of S. aureus cultures was performed by MALDI-TOF MS. The susceptibility studies were done using the susceptibility cards of VITEK 2 AST-P588 (bioM erieux) and Wider Ò microdi-lution system (Fco. Soria-Melguizo, Spain). These were considered the reference methods for assay evaluation. The Xpert Ò MRSA/SA BC (Cepheid, Sunnyvale, CA) is a real-time PCR-based method that can identify S. aureus and methicillin resistance from blood cultures with Gram-positive cocci cluster within approximately 1 h after the positive signal from an automated blood culture instru-ment. 7,8 These assay detect sequences within the staphylo-coccal protein A (spa) gene, the gene for methicillin resistance (mecA), and the staphylococcal cassette chro-mosome (SCCmec) inserted into the S. aureus chromosomal attB insertion site. Inclusion of the attB insertion site and the mecA gene targets enables the assay to discriminate false positive results due to methicillin-resistant coagu-lase-negative staphylococcus species that may occur in mo-lecular tests that target only the SCCmec cassette. The samples were treated according to the manufac-turer's instructions, and placed into the GeneXpert instru-ment. Invalid kit results and mechanical errors were discarded for further analysis. A total of 91 blood culture bottles containing S. aureus were randomly selected and analysed. Of these 25 were methicillin resistant and 66 methicillin susceptible. The Xpert assay correctly detected all 66 MSSA isolates, and 24 out of the 25 MRSA isolates. In the kit-negative specimen, the mecA gene was detected in the cycle num-ber 22.6, but the SCCmec was not detected and the system reported the assay as negative for MRSA. In this sample a pure culture of S. aureus was confirmed and identification and susceptibility were performed by standard methods. Further PCR analysis of the SCCmec type showed that this isolate presented an SCCmec type III cassette. 9 There was no difference between bottles containing charcoal and those without. Four MRSA and five MSSA samples (9.9%) were invalidated due to inhibition of the internal control. These were not further considered. The sensitivity and specificity of the Xpert Ò MRSA/SA BC assay were of 96.0% and 100%, respectively (95% confidence intervals, 77.7%e99.8% and 93.1%e99.9%, respectively). The accuracy of our testing methods using the MALDI-TOF and Xpert MRSA/SA BC systems was 98.9% with a negative Letters to the Editor 91