Recognition of Diagnostic Gaps for Laboratory Diagnosis of Fungal Diseases: Expert Opinion from the Fungal Diagnostics Laboratories Consortium (FDLC).

Fungal infections are a rising threat to our immunocompromised patient population, as well as other nonimmunocompromised patients with various medical conditions. However, little progress has been made in the past decade to improve fungal diagnostics. To jointly address this diagnostic challenge, the Fungal Diagnostics Laboratory Consortium (FDLC) was recently created. The FDLC consists of 26 laboratories from the United States and Canada that routinely provide fungal diagnostic services for patient care. A survey of fungal diagnostic capacity among the 26 members of the FDLC was recently completed, identifying the following diagnostic gaps: lack of molecular detection of mucormycosis; lack of an optimal diagnostic algorithm incorporating fungal biomarkers and molecular tools for early and accurate diagnosis of Pneumocystis pneumonia, aspergillosis, candidemia, and endemic mycoses; lack of a standardized molecular approach to identify fungal pathogens directly in formalin-fixed paraffin-embedded tissues; lack of robust databases to enhance mold identification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; suboptimal diagnostic approaches for mold blood cultures, tissue culture processing for Mucorales, and fungal respiratory cultures for cystic fibrosis patients; inadequate capacity for fungal point-of-care testing to detect and identify new, emerging or underrecognized, rare, or uncommon fungal pathogens; and performance of antifungal susceptibility testing. In this commentary, the FDLC delineates the most pressing unmet diagnostic needs and provides expert opinion on how to fulfill them. Most importantly, the FDLC provides a robust laboratory network to tackle these diagnostic gaps and ultimately to improve and enhance the clinical laboratory's capability to rapidly and accurately diagnose fungal infections.

Detection of toxigenic Clostridium difficile colonization in patients admitted to the hospital for chemotherapy or haematopoietic cell transplantation.

Increasing evidence suggests that asymptomatic carriers are an important source of healthcare-associated Clostridium difficile infection. However, it is not known which test for the detection of C. difficile colonization is most sensitive in patients with haematological malignancies. We performed a prospective cohort study of 101 patients with haematological malignancies who had been admitted to the hospital for scheduled chemotherapy or haematopoietic cell transplantation. Each patient provided a formed stool sample. We compared the performance of five different commercially available assays, using toxigenic culture as the reference method. The prevalence of toxigenic C. difficile colonization as determined by toxigenic culture was 14/101 (14 %). The Cepheid Xpert PCR C. difficile/Epi was the most sensitive test for the detection of toxigenic C. difficile colonization, with 93 % sensitivity and 99 % negative predictive value. Our findings suggest that the Xpert PCR C. difficile/Epi could be used to rule out toxigenic C. difficile colonization in this population.

Frequency and clinical correlates of elevated plasma Epstein-Barr virus DNA at diagnosis in peripheral T-cell lymphomas.

Epstein-Barr virus (EBV)-encoded RNAs (EBER) in tumor tissue and cell-free plasma EBV-DNA (pEBVd) are detected in EBV-associated lymphomas. Studies have suggested that EBER+ peripheral T-cell lymphomas (PTCL) have worse prognosis but the role of EBV in these neoplasms remains unclear. pEBVd is quantitative and more easily amenable to standardization than EBER, but frequency of pEBVd detection, clinical impact and agreement with EBER status in PTCL are unknown. We retrospectively assessed frequency of detectable pre-treatment pEBVd, presence of EBER in tumor tissue, and outcomes in 61 of 135 EBV-assessable PTCL patients. Fifteen of 61 patients (24.5%, 95% CI: 14-37%) were pre-treatment pEBVd+, with no significant differences in baseline characteristics or treatment between pEBVd+ and pEBVd- patients. EBER-ISH was performed on 10 pEBVd+ and 35 pEBVd- tumors. All 10 pEBVd+ patients were EBER+, but 9 pEBVd- patients were also EBER+. With median follow up of 24 months (range 1-96), overall survival (OS) was shorter in pEBVd+ compared to pEBVd- patients (13 vs. 72 months; p = 0.04). In our retrospective study, pre-treatment pEBVd was elevated in 25% of PTCL patients, was highly specific for EBER+ tumors, and was associated with shorter survival. pEBVd should be further explored as a prognostic variable and tumor biomarker in PTCL.

Increased Levels of Plasma Epstein Barr Virus DNA Identify a Poor-Risk Subset of Patients With Advanced Stage Cutaneous T-Cell Lymphoma.

INTRODUCTION: Outcomes in advanced stage (AS) cutaneous T-cell lymphomas (CTCL) are poor but with great variability. Epstein-Barr virus (EBV) is associated with a subset of non-Hodgkin lymphomas. Frequency of plasma EBV-DNA (pEBVd) detection, concordance with EBV RNA (EBER) in tumor tissue, codetection of plasma cytomegalovirus DNA (pCMVd), and prognostic effect in AS CTCL are unknown. PATIENTS AND METHODS: Patients (n = 46; 2006-2013) with AS CTCL (≥IIB) were retrospectively studied. pEBVd and pCMVd were longitudinally measured using quantitative real-time polymerase chain reaction. EBER in situ hybridization (ISH) was performed on tumor samples. Survival from time of diagnosis (ToD) and time of progression to AS was assessed. RESULTS: Plasma EBV-DNA and pCMVd were detected in 37% (17 of 46) and 17% (8 of 46) of AS CTCL patients, respectively. pCMVd detection was significantly more frequent in pEBVd-positive (pEBVd(+)) than pEBVd(-) patients (35% vs. 7%; P = .038). Tumor tissue for EBER-ISH was available in 14 of 17 pEBVd(+) and 22 of 29 pEBVd(-) patients; 12 of 14 (85.7%) pEBVd(+) patients were EBER(+) versus 0 of 22 pEBVd(-) patients. Frequency of large cell transformation (LCT) tended to be greater in pEBVd(+) patients, but was not significant (10 of 14 pEBVd(+) vs. 10 of 23 pEBVd(-); P = .17). No notable differences in rates of increased levels of serum lactate dehydrogenase (LDH) were observed (17 of 17 pEBVd(+) vs. 27 of 29 pEBVd(-)). pEBVd detection was associated with significantly worse survival from ToD (P = .021) and time of progression to AS (P = .0098). CONCLUSION: Detection of cell-free plasma EBV-DNA was highly concordant with the presence of EBERs in tumor tissue, predicted survival independent of LDH and LCT, and should be further studied as a biomarker in AS CTCL.

Clinical and microbiological outcomes in patients with Streptococcus anginosus group bacteraemia identified through use of a rapid microarray assay.

Limited data exist evaluating outcomes in patients with serious Streptococcus anginosus group infections, particularly bacteraemia. A retrospective, single-centre cohort study was conducted to characterize potential risk factors along with clinical and microbiological outcomes in patients with S. anginosus group bacteraemia (SAGB). Adult inpatients with SAGB identified using the Verigene Gram-positive blood culture assay between March 2013 and April 2014 were included. Patients aged ≤ 18 or >89 years, those with SAGB identified at an outside facility and those who were incarcerated were excluded. Differences between groups were explored using a Wilcoxon rank-sum test, χ2 test, Student's t-test or Fisher's exact test as appropriate and a two-tailed P value of ≤ 0.05 was considered statistically significant. The 34 patients who met the inclusion criteria were 57 ± 14 (mean ± SD) years old and had a median Charlson co-morbidity index of 4 [interquartile range (IQR) 1-6] and 10 (29%) were immunosuppressed at baseline. Almost half (47%) had received antibiotics in the previous 90 days. Twelve (35%) patients had gastrointestinal malignancies and the commonest source of bacteraemia was the gastrointestinal tract (53%). The primary species responsible for SAGB was S. anginosus (68%), and overall susceptibility to penicillin was 91%. Patients were most often treated with a ß-lactam/ß-lactamase inhibitor combination (36%) for a duration of 8 (IQR 4-13) days. Length of stay (LOS) and infection-related LOS were 10 (IQR 5-17) and 9 (IQR 4-12) days, respectively. Twenty [59%] patients achieved a clinical cure, while 29 (85%) achieved a microbiological cure. Four (12%) patients died and one patient was readmitted within 30  days. In the largest cohort of patients with SAGB to date, gastrointestinal malignancies may have been an important risk factor for SAGB, while rapid identification via a microarray assay likely contributed to improved disease recognition and timely pharmacological and non-pharmacological therapy.

The Streptococcus pyogenes orphan protein tyrosine phosphatase, SP-PTP, possesses dual specificity and essential virulence regulatory functions.

Group A Streptococcus (GAS) is a human pathogen that causes high morbidity and mortality. GAS lacks a gene encoding tyrosine kinase but contains one encoding tyrosine phosphatase (SP-PTP). Thus, GAS is thought to lack tyrosine phosphorylation, and the physiological significance of SP-PTP is, therefore, questionable. Here, we demonstrate that SP-PTP possesses dual phosphatase specificity for Tyr- and Ser/Thr-phosphorylated GAS proteins, such as Ser/Thr kinase (SP-STK) and the SP-STK-phosphorylated CovR and WalR proteins. Phenotypic analysis of GAS mutants lacking SP-PTP revealed that the phosphatase activity per se positively regulates growth, cell division and the ability to adhere to and invade host cells. Furthermore, A549 human lung cells infected with GAS mutants lacking SP-PTP displayed increased Ser-/Thr-/Tyr-phosphorylation. SP-PTP also differentially regulates the expression of ∼50% of the total GAS genes, including several virulence genes potentially through the two-component regulators, CovR, WalR and PTS/HPr regulation of Mga. Although these mutants exhibit attenuated virulence, a GAS mutant overexpressing SP-PTP is hypervirulent. Our study provides the first definitive evidence for the presence and importance of Tyr-phosphorylation in GAS and the relevance of SP-PTP as an important therapeutic target.

Detection of Group A Streptococcus in Pharyngeal Swab Specimens by Use of the AmpliVue GAS Isothermal Helicase-Dependent Amplification Assay.

We evaluated the clinical performance (sensitivity and specificity) of the AmpliVue group A Streptococcus (GAS) isothermal helicase-dependent amplification assay using 1,192 pharyngeal swab specimens. AmpliVue GAS assay results were compared to the results of routine throat cultures on selective streptococcal blood agar plates. The sensitivity and specificity of the AmpliVue GAS assay were 98.3% (95% confidence interval [CI], 95 to 100%) and 93.2% (95% CI, 91 to 95%), respectively.

Molecular and clinical characteristics of hospital and community onset methicillin-resistant Staphylococcus aureus strains associated with bloodstream infections.

Methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infections (BSI) are classified epidemiologically as health care-associated hospital onset (HAHO)-, health care-associated community onset (HACO)-, or community-associated (CA)-MRSA. Clinical and molecular differences between HAHO- and HACO-MRSA BSI are not well known. Thus, we evaluated clinical and molecular characteristics of MRSA BSI to determine if distinct features are associated with HAHO- or HACO-MRSA strains. Molecular genotyping and medical record reviews were conducted on 282 MRSA BSI isolates from January 2007 to December 2009. MRSA classifications were 38% HAHO-, 54% HACO-, and 8% CA-MRSA. Comparing patients with HAHO-MRSA to those with HACO-MRSA, HAHO-MRSA patients had significantly higher rates of malignancy, surgery, recent invasive devices, and mortality and longer hospital stays. Patients with HACO-MRSA were more likely to have a history of renal failure, hemodialysis, residence in a long-term-care facility, long-term invasive devices, and higher rate of MRSA relapse. Distinct MRSA molecular strain differences also were seen between HAHO-MRSA (60% staphylococcal cassette chromosome mec type II [SCCmec II], 30% SCCmec III, and 9% SCCmec IV) and HACO-MRSA (47% SCCmec II, 35% SCCmec III, and 16% SCCmec IV) (P < 0.001). In summary, our study reveals significant clinical and molecular differences between patients with HAHO- and HACO-MRSA BSI. In order to decrease rates of MRSA infection, preventive efforts need to be directed toward patients in the community with health care-associated risk factors in addition to inpatient infection control.

Design and synthesis of novel anti-tuberculosis agents from the celecoxib pharmacophore.

The identification of compounds with anti-mycobacterial activity within classes of molecules that have been developed for other purposes is a fruitful approach for the development of anti-tuberculosis (TB) agents. In this study we used the scaffold of celecoxib which exhibits several activities against different pathogens, for the design and focused synthesis of a library of 64 compounds. For the primary screen, we used a bioluminescence-based method by constructing a luciferase-expressing reporter M.tb strain which contains the entire bacterial Lux operon cloned in a mycobacterial integrative expression vector. Through the screening of this library, we identified 6 hit compounds with high in vitro anti-mycobacterial activity (IC50 ∼0.18-0.48 µM). In particular, compounds 41, 51 and 53 were capable of inhibiting M.tb as effectively as the anti-TB drug isoniazid (INH) at 5 µM over a 72-h period, as analyzed by both bioluminescence- and colony forming unit (CFU)-based assays. All hit compounds also showed anti-M.tb activities against several multi-drug-resistant (MDR) strains. Most of the hit compounds showed no cytotoxicity for human macrophages at concentrations as high as 40 µM, setting the stage for further optimization and development of these anti-TB hit compounds both ex vivo and in vivo.

Mycobacterial skin and soft tissue infection.

Mycobacterial skin and soft tissue infection (SSTI) includes nontuberculous mycobacterial (NTM) infections, tuberculosis (TB), and leprosy. Diagnosis of mycobacterial SSTI can be challenging due to diverse clinical presentation, low yield from cultured specimens, and nonspecific histopathology on tissue biopsy. In addition, immunosuppressed patients may present with atypical or disseminated disease. Despite aggressive medical treatment and often with surgical intervention, results may be suboptimal with poor outcomes. Regimens typically require multiple antibiotics for extended periods of time and are often complicated by medication side effects and drug-drug interactions. Biopsy with culture is the gold standard for diagnosis, but newer molecular diagnostics and proteomics such as matrix-assisted laser desorption ionization-time of flight mass spectrometry have improved diagnosis with increased identification of clinically significant mycobacteria species in clinically relevant time frames. We will review updates in diagnostic tests along with clinical presentation and treatment of mycobacterial SSTI for NTM, TB, and leprosy.

Evaluation of commercial ResPlex II v2.0, MultiCode-PLx, and xTAG respiratory viral panels for the diagnosis of respiratory viral infections in adults.

BACKGROUND: Commercial multiplex PCR panels for respiratory viruses (PRV) have been recently developed. ResPlex II Panel v2.0 (Qiagen), MultiCode-PLx (EraGen Biosciences), and xTAG (Luminex) PRV's were studied. All assays detect influenza A and B, adenovirus, parainfluenza 1-3, respiratory syncytial virus A and B, human metapneumovirus and human rhinovirus. The ResPlex II additionally detects coronavirus (229E, OC43, NL63, HKU1), coxsackie/echo virus, bocavirus and differentiates adenoviruses (B, E). The MultiCode-PLX assay detects 229E, OC43, and NL63, differentiates parainfluenza 4a, 4b and adenoviruses (B, C, E). The xTAG additionally subtypes influenza A as seasonal H1 and H3. STUDY DESIGN: 202 specimens collected from adult patients with signs of respiratory infection from November, 2008 to May, 2009 were used for evaluating the performance of the three commercial PRV assays. Viral culture and xTAG were used as the standards to assess sensitivity and specificity. RESULTS: The PRV assays detected more viruses than culture. When compared to culture, the xTAG PRV showed a sensitivity and specificity of 100% and 91%, compared to MultiCode-PLx with 89% and 87%, and ResPlex II with 89% and 94%, respectively. Co-infection was detected in a small subset of patient specimens. Each panel showed differences in sensitivities for individual viruses. CONCLUSIONS: While the ResPlex II and MultiCode-PLx offer a broader virus detection range and greater ease of use, the xTAG PRV showed increased sensitivity to common viral targets represented in the assays, and also had the ability to differentiate human from non-human influenza A H1.

Rapid screening of urine specimens for bacteriuria by the cellenium system.

This study evaluated the performance of the Cellenium 160 US urine screening system in comparison to that of the semiquantitative culture method. The performance characteristics of the Cellenium system for all clinically significant uropathogens were 89.5% sensitivity, 94.4% specificity, 97.1% negative predictive value, and 81% positive predictive value.

DNA immunization with hepatitis C virus (HCV) polycistronic genes or immunization by HCV DNA priming-recombinant canarypox virus boosting induces immune responses and protection from recombinant HCV-vaccinia virus infection in HLA-A2.1-transgenic mice.

We studied immune responses to hepatitis C virus (HCV) genes delivered as DNA encoding the entire HCV protein coding genome in two polycistronic plasmids encoding HCV capsid-E1-E2-NS2-NS3 and HCV NS3-NS4-NS5 in HLA-A2.1-transgenic mice. Immune responses to HCV DNA prime and recombinant canarypox virus boost were also studied with the above constructs. At 8 weeks after a canarypox virus boost, the DNA prime/canarypox virus boosting regimen induced potent cellular immune responses to HCV structural and nonstructural proteins on target cells expressing the HLA-A2.1 allele. High frequencies of gamma interferon-secreting cells, as detected by enzyme-linked immunospot assay, were obtained in response to several endogenously expressed HCV proteins. We also observed cytotoxic-T-lymphocyte reactivity in response to endogenously expressed HCV proteins in fresh spleen cells without in vitro expansion. Upon challenge with a recombinant vaccinia virus expressing HCV proteins at 2 months postimmunization, the HCV DNA prime/canarypox virus-immunized mice showed a complete reduction in vaccinia virus titers compared to HCV DNA prime/boost- and mock-immunized controls. Immune responses were still detectable 4 months after canarypox virus boost in immunized mice. Interestingly, at 10 months postimmunization (8 months after canarypox virus boost), the protection in HCV DNA prime/boost-immunized mice against recombinant HCV-vaccinia virus challenge was higher than that observed in HCV DNA prime/canarypox virus boost-immunized mice.