Polymerase Chain Reaction on Respiratory Tract Specimens of Immunocompromised Patients to Diagnose Pneumocystis Pneumonia: A Systematic Review and Meta-analysis.

BACKGROUND: This meta-analysis examines the comparative diagnostic performance of polymerase chain reaction (PCR) for the diagnosis of Pneumocystis pneumonia (PCP) on different respiratory tract samples, in both human immunodeficiency virus (HIV) and non-HIV populations. METHODS: A total of 55 articles met inclusion criteria, including 11 434 PCR assays on respiratory specimens from 7835 patients at risk of PCP. QUADAS-2 tool indicated low risk of bias across all studies. Using a bivariate and random-effects meta-regression analysis, the diagnostic performance of PCR against the European Organisation for Research and Treatment of Cancer-Mycoses Study Group definition of proven PCP was examined. RESULTS: Quantitative PCR (qPCR) on bronchoalveolar lavage fluid provided the highest pooled sensitivity of 98.7% (95% confidence interval [CI], 96.8%-99.5%), adequate specificity of 89.3% (95% CI, 84.4%-92.7%), negative likelihood ratio (LR-) of 0.014, and positive likelihood ratio (LR+) of 9.19. qPCR on induced sputum provided similarly high sensitivity of 99.0% (95% CI, 94.4%-99.3%) but a reduced specificity of 81.5% (95% CI, 72.1%-88.3%), LR- of 0.024, and LR+ of 5.30. qPCR on upper respiratory tract samples provided lower sensitivity of 89.2% (95% CI, 71.0%-96.5%), high specificity of 90.5% (95% CI, 80.9%-95.5%), LR- of 0.120, and LR+ of 9.34. There was no significant difference in sensitivity and specificity of PCR according to HIV status of patients. CONCLUSIONS: On deeper respiratory tract specimens, PCR negativity can be used to confidently exclude PCP, but PCR positivity will likely require clinical interpretation to distinguish between colonization and active infection, partially dependent on the strength of the PCR signal (indicative of fungal burden), the specimen type, and patient population tested.

Population genomics of the pathogenic yeast Candida tropicalis identifies hybrid isolates in environmental samples.

Candida tropicalis is a human pathogen that primarily infects the immunocompromised. Whereas the genome of one isolate, C. tropicalis MYA-3404, was originally sequenced in 2009, there have been no large-scale, multi-isolate studies of the genetic and phenotypic diversity of this species. Here, we used whole genome sequencing and phenotyping to characterize 77 isolates of C. tropicalis from clinical and environmental sources from a variety of locations. We show that most C. tropicalis isolates are diploids with approximately 2-6 heterozygous variants per kilobase. The genomes are relatively stable, with few aneuploidies. However, we identified one highly homozygous isolate and six isolates of C. tropicalis with much higher heterozygosity levels ranging from 36-49 heterozygous variants per kilobase. Our analyses show that the heterozygous isolates represent two different hybrid lineages, where the hybrids share one parent (A) with most other C. tropicalis isolates, but the second parent (B or C) differs by at least 4% at the genome level. Four of the sequenced isolates descend from an AB hybridization, and two from an AC hybridization. The hybrids are MTLa/α heterozygotes. Hybridization, or mating, between different parents is therefore common in the evolutionary history of C. tropicalis. The new hybrids were predominantly found in environmental niches, including from soil. Hybridization is therefore unlikely to be associated with virulence. In addition, we used genotype-phenotype correlation and CRISPR-Cas9 editing to identify a genome variant that results in the inability of one isolate to utilize certain branched-chain amino acids as a sole nitrogen source.

The effect of cold storage and cooking on the viability of Clostridioides difficile spores in consumer foods.

The increased detection of clinical cases of Clostridioides difficile coupled with the persistence of clostridial spores at various stages along the food chain suggest that this pathogen may be foodborne. This study examined C. difficile (ribotypes 078 and 126) spore viability in chicken breast, beef steak, spinach leaves and cottage cheese during refrigerated (4 °C) and frozen (-20 °C) storage with and without a subsequent sous vide mild cooking (60 °C, 1 h). Spore inactivation at 80 °C in phosphate buffer solution, beef and chicken were also investigated to provide D80°C values and determine if PBS was a suitable model system for real food matrices. There was no decrease in spore concentration after chilled or frozen storage and/or sous vide cooking at 60 °C. Non-log-linear thermal inactivation was observed for both C. difficile ribotypes at 80 °C in phosphate buffer solution (PBS), beef and chicken. The predicted PBS D80°C values of 5.72±[2.90, 8.55] min and 7.50±[6.61, 8.39] min for RT078 and RT126, respectively, were in agreement with the food matrices D80°C values of 5.65 min (95% CI range from 4.29 to 8.89 min) for RT078 and 7.35 min (95% CI range from 6.81 to 7.01 min) for RT126. It was concluded that C. difficile spores survive chilled and frozen storage and mild cooking at 60 °C but may be inactivated at 80 °C. Moreover thermal inactivation in PBS was representative of that observed in real food matrices (beef and chicken).

Molecular mechanisms of acquired antifungal drug resistance in principal fungal pathogens and EUCAST guidance for their laboratory detection and clinical implications.

The increasing incidence and changing epidemiology of invasive fungal infections continue to present many challenges to their effective management. The repertoire of antifungal drugs available for treatment is still limited although there are new antifungals on the horizon. Successful treatment of invasive mycoses is dependent on a mix of pathogen-, host- and antifungal drug-related factors. Laboratories need to be adept at detection of fungal pathogens in clinical samples in order to effectively guide treatment by identifying isolates with acquired drug resistance. While there are international guidelines on how to conduct in vitro antifungal susceptibility testing, these are not performed as widely as for bacterial pathogens. Furthermore, fungi generally are recovered in cultures more slowly than bacteria, and often cannot be cultured in the laboratory. Therefore, non-culture-based methods, including molecular tests, to detect fungi in clinical specimens are increasingly important in patient management and are becoming more reliable as technology improves. Molecular methods can also be used for detection of target gene mutations or other mechanisms that predict antifungal drug resistance. This review addresses acquired antifungal drug resistance in the principal human fungal pathogens and describes known resistance mechanisms and what in-house and commercial tools are available for their detection. It is emphasized that this approach should be complementary to culture-based susceptibility testing, given the range of mutations, resistance mechanisms and target genes that may be present in clinical isolates, but may not be included in current molecular assays.

Human and Porcine Transmission of Clostridioides difficile Ribotype 078, Europe.

Genomic analysis of a diverse collection of Clostridioides difficile ribotype 078 isolates from Ireland and 9 countries in Europe provided evidence for complex regional and international patterns of dissemination that are not restricted to humans. These isolates are associated with C. difficile colonization and clinical illness in humans and pigs.

Unconventional T cells - New players in antifungal immunity.

Life-threatening invasive fungal diseases (IFD) are increasing in incidence, especially in immunocompromised patients and successful resolution of IFD requires a variety of different immune cells. With the limited repertoire of available antifungal drugs there is a need for more effective therapeutic strategies. This review interrogates the evidence on the human immune response to the main pathogens driving IFD, with a focus on the role of unconventional lymphocytes e.g. natural killer (NK) cells, gamma/delta (γδ) T cells, mucosal associated invariant T (MAIT) cells, invariant natural killer T (iNKT) cells and innate lymphoid cells (ILC). Recent discoveries and new insights into the roles of these novel lymphocyte groups in antifungal immunity will be discussed, and we will explore how an improved understanding of antifungal action by lymphocytes can inform efforts to improve antifungal treatment options.

Overcoming the Challenges of Pyrazinamide Susceptibility Testing in Clinical Mycobacterium tuberculosis Isolates.

Pyrazinamide (PZA) is one of the first-line agents used for the treatment of tuberculosis. However, current phenotypic PZA susceptibility testing in the Bactec MGIT 960 system is unreliable, and false resistance is well documented. Rapid identification of resistance-associated mutations can confirm the phenotypic result. This study aimed to investigate the use of genotypic methods in combination with phenotypic susceptibility testing for confirmation of PZA-resistant Mycobacterium tuberculosis isolates. Sanger sequencing and/or whole-genome sequencing were performed to detect mutations in pncA, rpsA, panD, and clpC1. Isolates were screened for heteroresistance, and PZA susceptibility testing was performed using the Bactec MGIT 960 system using a reduced inoculum to investigate false resistance. Overall, 40 phenotypically PZA-resistant isolates were identified. Of these, PZA resistance was confirmed in 22/40 (55%) isolates by detecting mutations in the pncA, rpsA, and panD genes. Of the 40 isolates, 16 (40%) were found to be susceptible using the reduced inoculum method (i.e., false resistance). No mutations were detected in two PZA-resistant isolates. False resistance was observed in isolates with MICs close to the critical concentration. In particular, East African Indian strains (lineage 1) appeared to have an elevated MIC that is close to the critical concentration. While this study illustrates the complexity and challenges associated with PZA susceptibility testing of M. tuberculosis, we conclude that a combination of genotypic and phenotypic drug susceptibility testing methods is required for accurate detection of PZA resistance.

Genomic Analysis of Mycobacterium abscessus Complex Isolates Collected in Ireland between 2006 and 2017.

Members of the Mycobacterium abscessus complex (MABC) are multidrug-resistant nontuberculous mycobacteria and cause opportunistic pulmonary infections in individuals with cystic fibrosis (CF). In this study, genomic analysis of MABC isolates was performed to gain greater insights into the epidemiology of circulating strains in Ireland. Whole-genome sequencing (WGS) was performed on 70 MABC isolates that had been referred to the Irish Mycobacteria Reference Laboratory between 2006 and 2017 across nine Irish health care centers. The MABC isolates studied comprised 52 isolates from 27 CF patients and 18 isolates from 10 non-CF patients. WGS identified 57 (81.4%) as M. abscessus subsp. abscessus, 10 (14.3%) as M. abscessus subsp. massiliense, and 3 (4.3%) as M. abscessus subsp. bolletii Forty-nine (94%) isolates from 25 CF patients were identified as M. abscessus subsp. abscessus, whereas 3 (6%) isolates from 2 CF patients were identified as M. abscessus subsp. massiliense Among the isolates from non-CF patients, 44% (8/18) were identified as M. abscessus subsp. abscessus, 39% (7/18) were identified as M. abscessus subsp. massiliense, and 17% (3/18) were identified as M. abscessus subsp. bolletii WGS detected two clusters of closely related M. abscessus subsp. abscessus isolates that included isolates from different CF centers. There was a greater genomic diversity of MABC isolates among the isolates from non-CF patients than among the isolates from CF patients. Although WGS failed to show direct evidence of patient-to-patient transmission among CF patients, there was a predominance of two different strains of M. abscessus subsp. abscessus Furthermore, some MABC isolates were closely related to global strains, suggesting their international spread. Future prospective real-time epidemiological and clinical data along with contemporary MABC sequence analysis may elucidate the sources and routes of transmission among patients infected with MABC.

Prospective observational study of respiratory Aspergillus colonization or disease in patients with various stages of chronic obstructive pulmonary disease utilizing culture versus nonculture techniques.

Chronic obstructive pulmonary disease (COPD) patients have been recognized to be at increased risk of Aspergillus spp. colonization, which may progress to invasive pulmonary aspergillosis (IPA). The objective of this study was to determine the frequency of Aspergillus colonization, or disease, in a cohort of COPD patients. A prospective observational study was undertaken to determine Aspergillus colonization, or disease, in consecutive COPD patients undergoing bronchoscopy. Fungal culture as well as galactomannan antigen (GM) and Aspergillus nucleic acid detection (PCR) were performed on bronchoalveolar lavage fluid (BAL) samples. One hundred and fifty patients were recruited. One hundred and twelve (74.7%) were outpatients, 38 (25.33%) were inpatients, of whom 6 (4%) were in the intensive care unit. Most patients (N = 122, 81.3%) were either COPD GOLD (Global Initiative for Chronic Obstructive Lung Disease) stages 1 or 2. Nine (6%) patients were on systemic steroids, 64 (42.7%) on inhaled steroids, and 9 (6%) on both. Seventeen patients (11.3%) had at least one positive test for Aspergillus detection (culture ± galactomannan ± polymerase chain reaction [PCR]), 13 (76.4%) of whom were COPD GOLD stages 1 or 2.  Five patients had probable or putative IPA. Aspergillus sp. was detected in five patients (3.3%) by culture, but detection increased to 17 (11.3%) by the additional testing for GM or Aspergillus DNA. The frequency of Aspergillus detection in this cohort of COPD patients may reflect the predominance of early GOLD stages among the study population but deserves further investigation to determine its relevance as a predictive risk factor for IPA. LAY SUMMARY: COPD is a risk factor for Aspergillus spp. colonization. Bronchoalveolar lavage samples of 150 COPD patients were tested for presence of Aspergillus fumigatus, which was detected in five patients (3.3%) by culture, but detection of Aspergillus increased to 17 (11.3%) by additional GM and PCR testing.

Whole-Genome Sequencing of Aspergillus terreus Species Complex.

Aspergillus terreus species complex is an opportunistic fungal pathogen increasingly implicated in invasive infection, as well as chronic respiratory disease. Currently, an understanding of A. terreus pathogenicity is impeded by a limited number of whole-genome sequences of this fungal pathogen. We here describe a high-quality whole-genome assembly of European A. terreus clinical isolate M6925, derived by single-molecule real-time sequencing with short-read polishing.

Antimicrobial Susceptibility of Clinical and Environmental Mycobacterium chimaera Isolates.

Mycobacterium chimaera is a slow-growing nontuberculous Mycobacterium species belonging to the Mycobacterium avium complex (MAC). It has been identified globally as the cause of a large outbreak of cardiovascular infections following open heart surgery, but it can also cause respiratory infections in individuals with underlying structural pulmonary disease. Invasive M. chimaera infections are associated with poor clinical responses, and the optimal antibiotic treatment regimen for these infections is not known. In this study, the drug susceptibility profiles of clinical and environmental M. chimaera isolates for antimicrobial agents that are commonly considered for treatment of MAC infections were determined. All M. chimaera isolates were susceptible to clarithromycin, with a median MIC of 2 µg/ml, while 98% (85/87 isolates) were susceptible to amikacin. Twenty-five percent of isolates (22/87 isolates) had intermediate susceptibility and 52% (46/87 isolates) were resistant to moxifloxacin. Similarly, 39% of isolates (34/87 isolates) had intermediate susceptibility and 39% (34/87 isolates) were resistant to linezolid. MIC breakpoints derived from the literature were used to determine resistance to rifampin (16/87 isolates [18%]), ethambutol (10/87 isolates [11%]), rifabutin (2/87 isolates [2%]), and streptomycin (1/87 isolates [1%]). In conclusion, our results showed that clarithromycin, amikacin, rifabutin, and streptomycin had the best activity against M. chimaera isolates, while susceptibility rates were lower for rifampin and ethambutol. In contrast, there was a high prevalence of isolates that were not susceptible to moxifloxacin or linezolid. While factors in addition to antibiotic susceptibility may determine the outcomes of treatment of M. chimaera infections, our results should inform the selection of antimicrobials as part of the overall therapeutic strategy.

Diagnosis of aspergillosis by PCR: Clinical considerations and technical tips.

Standardization of Aspergillus polymerase chain reaction (PCR) protocols has progressed, and analytical validity of blood-based assays has been formally established. It remains necessary to consider how the tests can be used in practice to maximize clinical utility. To determine the optimal diagnostic strategies and influence on patient management, several factors require consideration, including the patient population, incidence of invasive aspergillosis (and other fungal disease), and the local antifungal prescribing policy. Technical issues such as specimen type, ease of sampling, frequency of testing, access to testing centers, and time to reporting will also influence the use of PCR in clinical practice. Interpretation of all diagnostic tests is dependent on the clinical context and molecular assays are no exception, but with the proposal to incorporate Aspergillus PCR into the second revision of the consensus guidelines for defining invasive fungal disease the acceptance and understanding of molecular tests should improve.

Post-influenzal triazole-resistant aspergillosis following allogeneic stem cell transplantation.

Influenza virus infection is now recognised as a risk factor for invasive pulmonary aspergillosis (IPA). Delays in diagnosis contribute to delayed commencement of antifungal therapy. In addition, the emergence of resistance to first-line triazole antifungal agents puts emphasis on early detection to prevent adverse outcomes. We present 2 allogeneic stem cell transplant patients who developed IPA due to triazole-resistant Aspergillus fumigatus following influenza infection. We underline the challenges faced in the management of these cases, the importance of early diagnosis and need for surveillance given the emergence of triazole resistance.

Surface layer proteins from virulent Clostridium difficile ribotypes exhibit signatures of positive selection with consequences for innate immune response.

BACKGROUND: Clostridium difficile is a nosocomial pathogen prevalent in hospitals worldwide and increasingly common in the community. Sequence differences have been shown to be present in the Surface Layer Proteins (SLPs) from different C. difficile ribotypes (RT) however whether these differences influence severity of infection is still not clear. RESULTS: We used a molecular evolutionary approach to analyse SLPs from twenty-six C. difficile RTs representing different slpA sequences. We demonstrate that SLPs from RT 027 and 078 exhibit evidence of positive selection (PS). We compared the effect of these SLPs to those purified from RT 001 and 014, which did not exhibit PS, and demonstrate that the presence of sites under positive selection correlates with ability to activate macrophages. SLPs from RTs 027 and 078 induced a more potent response in macrophages, with increased levels of IL-6, IL-12p40, IL-10, MIP-1α, MIP-2 production relative to RT 001 and 014. Furthermore, RTs 027 and 078 induced higher expression of CD40, CD80 and MHC II on macrophages with decreased ability to phagocytose relative to LPS. CONCLUSIONS: These results tightly link sequence differences in C. difficile SLPs to disease susceptibility and severity, and suggest that positively selected sites in the SLPs may play a role in driving the emergence of hyper-virulent strains.

Intercountry Transfer of Triazole-Resistant Aspergillus fumigatus on Plant Bulbs.

We investigated whether plants imported to Ireland from the Netherlands might harbor triazole-resistant Aspergillus fumigatus. Samples of plant bulbs were positive for triazole-resistant A. fumigatus with CYP51A mutations. We hypothesize that this represents a route for intercountry transfer of an emerging resistance mechanism in a major opportunistic mold pathogen.

Evaluation of GenoType NTM-DR Assay for Identification of Mycobacterium chimaera.

Identification of species within the Mycobacterium avium complex (MAC) is difficult, and most current diagnostic laboratory tests cannot distinguish between species included in the complex. Differentiation of species within the MAC is important, as Mycobacterium chimaera has recently emerged as a major cause of invasive cardiovascular infections following open heart surgery. A new commercial diagnostic assay, GenoType NTM-DR ver. 1.0, is intended to differentiate between three species within the MAC, namely, Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium chimaera In this study, we investigated an archival collection of 173 MAC isolates using 16S rRNA and 16S-23S internal transcribed spacer (ITS) gene sequencing, and GenoType NTM-DR was evaluated for identifying M. chimaera and other species belonging to the MAC. Species identification of 157/173 (91%) isolates with the GenoType NTM-DR assay was in agreement with 16S rRNA and 16S-23S ITS gene sequencing results. Misidentification occurred with 16 isolates which belonged to four species included in the MAC that are rarely encountered in clinical specimens. Despite some limitations of this assay, GenoType NTM-DR had 100% specificity for identifying M. chimaera This novel assay will enable diagnostic laboratories to differentiate species belonging to the Mycobacterium avium complex and to accurately identify M. chimaera It can produce rapid results and is also more cost efficient than gene sequencing methods.

Memory Th1 Cells Are Protective in Invasive Staphylococcus aureus Infection.

Mechanisms of protective immunity to Staphylococcus aureus infection in humans remain elusive. While the importance of cellular immunity has been shown in mice, T cell responses in humans have not been characterised. Using a murine model of recurrent S. aureus peritonitis, we demonstrated that prior exposure to S. aureus enhanced IFNγ responses upon subsequent infection, while adoptive transfer of S. aureus antigen-specific Th1 cells was protective in naïve mice. Translating these findings, we found that S. aureus antigen-specific Th1 cells were also significantly expanded during human S. aureus bloodstream infection (BSI). These Th1 cells were CD45RO+, indicative of a memory phenotype. Thus, exposure to S. aureus induces memory Th1 cells in mice and humans, identifying Th1 cells as potential S. aureus vaccine targets. Consequently, we developed a model vaccine comprising staphylococcal clumping factor A, which we demonstrate to be an effective human T cell antigen, combined with the Th1-driving adjuvant CpG. This novel Th1-inducing vaccine conferred significant protection during S. aureus infection in mice. This study notably advances our understanding of S. aureus cellular immunity, and demonstrates for the first time that a correlate of S. aureus protective immunity identified in mice may be relevant in humans.

Candida albicans stimulates IL-23 release by human dendritic cells and downstream IL-17 secretion by Vδ1 T cells.

γδ T cells expressing the Vδ1 TCR are expanded in patients with HIV infection. We show in this article that circulating Vδ1 T cell numbers are particularly high in patients with HIV and candidiasis, and that these cells expand and produce IL-17 in response to Candida albicans in vitro. Although C. albicans could directly stimulate IL-17 production by a subset of Vδ1 T cells, fungus-treated dendritic cells (DCs) were required to expand C. albicans-responsive Vδ1 T cells to generate sufficient numbers of cells to release IL-17 at levels detectable by ELISA. C. albicans induced the release of IL-1ß, IL-6, and IL-23 by DCs, but addition of these cytokines or supernatants of C. albicans-treated DCs to Vδ1 T cells was not sufficient to induce proliferation. We found that direct contact with DCs was required for Vδ1 T cell proliferation, whereas IL-23R-blocking studies showed that IL-23 was required for optimal C. albicans-induced IL-17 production. Because IL-17 affords protection against both HIV and C. albicans, and because Vδ1 T cells are not depleted by HIV, these cells are likely to be an important source of IL-17 in HIV-infected patients with candidiasis, in whom CD4(+) Th17 responses are impaired. These data show that C. albicans stimulates proliferation and IL-17 production by Vδ1 T cells by a mechanism that involves IL-23 release by DCs.

Proteomic Profiling of Serological Responses to Aspergillus fumigatus Antigens in Patients with Invasive Aspergillosis.

Aspergillus fumigatus is the species that most commonly causes the opportunistic infection invasive aspergillosis (IA) in patients being treated for hematological malignancies. Little is known about the A. fumigatus proteins that trigger the production of Aspergillus-specific IgG antibodies during the course of IA. To characterize the serological response to A. fumigatus protein antigens, mycelial proteins were separated by 2-D gel electrophoresis. The gels were immunoblotted with sera from patients with probable and proven IA and control patients without IA. We identified 49 different fungal proteins, which gave a positive IgG antibody signal. Most of these antigens play a role in primary metabolism and stress responses. Overall, our analysis identified 18 novel protein antigens from A. fumigatus. To determine whether these antigens can be used as diagnostic or prognostic markers or exhibit a protective activity, we employed supervised machine learning with decision trees. We identified two candidates for further analysis, the protein antigens CpcB and Shm2. Heterologously produced Shm2 induced a strongly proinflammatory response in human peripheral blood mononuclear cells after in vitro stimulation. In contrast, CpcB did not activate the immune response of PBMCs. These findings could serve as the basis for the development of an immunotherapy of IA.

Enhanced Tracking of Nosocomial Transmission of Endemic Sequence Type 22 Methicillin-Resistant Staphylococcus aureus Type IV Isolates among Patients and Environmental Sites by Use of Whole-Genome Sequencing.

Whole-genome sequencing (WGS) of 41 patient and environmental sequence type 22 methicillin-resistant Staphylococcus aureus staphylococcal cassette chromosome mec type IV (ST22-MRSA-IV) isolates recovered over 6 weeks in one acute hospital ward in Dublin, Ireland, where ST22-MRSA IV is endemic, revealed 228 pairwise combinations differing by <40 single nucleotide variants corresponding to potential cross-transmission events (CTEs). In contrast, 15 pairwise combinations of isolates representing five CTEs were previously identified by conventional molecular epidemiological typing. WGS enhanced ST22-MRSA-IV tracking and highlighted potential transmission of MRSA via the hospital environment.