Journal of Clinical Microbiology最新文献

Invasive pulmonary infections are a significant cause of morbidity and mortality in patients with hematological malignancies and hematopoietic stem cell transplantation (HCT) recipients. A delay in identifying a causative agent may result in late initiation of appropriate treatment and adverse clinical outcomes. We examine the diagnostic utility of PCR-based assays in evaluating invasive pulmonary infections from bronchoalveolar lavage (BAL). Patients with hematological malignancies and HCT recipients who underwent bronchoscopy with BAL from January 2020 to January 2024 for unexplained pulmonary infiltrates and had ≥1 PCR targeting Aspergillus, Mucorales, or Nocardia (Eurofins-Viracor, KS) were reviewed. Testing for microbiology and pathology except BAL PCRs to identify the etiology of pulmonary infiltrate was defined as standard-of-care. Invasive fungal diseases were defined as per European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium (EORTC/MSGERC) 2020 guidelines. Pulmonary nocardiosis was defined by a combination of clinical, radiographic, and microbiologic criteria. Of 134 patients, 77 were HCT recipients, and 70% were on antifungal agents. Thirty-two were diagnosed with infection with one of the three target pathogens, including 20 with probable or proven invasive pulmonary aspergillosis (IPA), seven with mucormycosis, and three with nocardiosis. For IPA, 19 were diagnosed by standard-of-care, and one (5%) was solely diagnosed by Aspergillus PCR. Mucorales PCR was positive in three of seven cases of proven mucormycosis, but the cultures were negative in all. All three nocardiosis cases were detected by PCR and culture. In our cohort, PCR targeting Mucorales and Nocardia can improve the early detection of invasive pulmonary infection, whereas Aspergillus PCR has a low added value when done in conjunction with standard-of-care, including BAL galactomannan.IMPORTANCEInvasive pulmonary infections are a significant cause of morbidity and mortality in immunocompromised patients. Timely diagnosis of invasive pulmonary infection reduces the time to targeted treatment initiation and improves clinical outcomes. The recent European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium (EORTC/MSGERC) update included the addition of serum or bronchoalveolar lavage (BAL) PCR as a method to determine probable Aspergillus disease. This reflects an increased utilization of PCR-based assays in the diagnosis of fungal diseases. Although PCR assays for Aspergillus diagnosis have been well characterized in the literature, their additive clinical utility in conjunction with BAL galactomannan index measurements remains unclear. Moreover, only a few reports characterize the analytic and clinical performance of Mucorales and Nocardia PCR.

The Burkholderia cepacia complex (BCC) is a group of Gram-negative bacteria that cause opportunistic infections, most notably in people with cystic fibrosis (CF), and have been associated with outbreaks caused by contaminated medical products. Antimicrobial susceptibility testing (AST) is often used to guide treatment for BCC infections, perhaps most importantly in people with CF who are being considered for lung transplant. However, recent studies have highlighted problems with AST methods. Here, we address limitations from previous studies to further evaluate BCC AST methods. We assessed the performance of reference broth microdilution (BMD), disk diffusion (DD) using Mueller-Hinton agar (MHA) from three manufacturers, agar dilution (AD), and gradient diffusion (ETEST) for ceftazidime (CAZ), levofloxacin (LVX), meropenem (MEM), minocycline (MIN), and trimethoprim-sulfamethoxazole (TMP-SMX) on a set of 205 BCC isolates. The isolate set included 100 isolates from people with CF and 105 isolates from people without CF from a variety of sources, which enabled us to systematically evaluate whether specimen source impacts AST performance. For all BCC isolates, BMD reproducibility was 93%, 98%, 99%, 98%, and 96% for CAZ, LVX, MEM, MIN, and TMP-SMX, respectively. Using BMD as the comparator method, we show that DD, AD, and ETEST perform poorly, with neither MHA manufacturer nor specimen source significantly impacting method performance. Based on our data, we recommend that routine AST should not be performed for BCC isolates. If a provider requests AST, clinical microbiology laboratories should perform Clinical and Laboratory Standards Institute reference methodology for BMD (stored frozen) and report MIC only.IMPORTANCEAntimicrobial susceptibility testing for the Burkholderia cepacia complex (BCC) is often used to determine eligibility for lung transplant in people with cystic fibrosis. However, problems with method performance have been reported. Here, we systematically evaluate the performance of reference broth microdilution, disk diffusion, agar dilution, and gradient diffusion (ETEST) for BCC organisms isolated from people with and without cystic fibrosis. We show that broth microdilution reproducibility is acceptable for levofloxacin, meropenem, minocycline, and trimethoprim-sulfamethoxazole, while ceftazidime was just below the acceptability cut-off. Regardless of specimen source, the results from disk diffusion, agar dilution, and ETEST do not correlate with broth microdilution. Based on these findings, we recommend that antimicrobial susceptibility testing should not be routinely performed for BCC, and if requested by the provider, only broth microdilution following Clinical and Laboratory Standards Institute guidelines should be used. Providers should be aware of the significant limitations of antimicrobial susceptibility testing methods for BCC.

Tongue swab (TS) sampling combined with quantitative PCR (qPCR) to detect Mycobacterium tuberculosis (MTB) DNA is a promising alternative to sputum testing for tuberculosis (TB) diagnosis. In prior studies, the sensitivity of tongue swabbing has usually been lower than sputum. In this study, we evaluated two strategies to improve sensitivity. In one, centrifugation was used to concentrate tongue dorsum bacteria from 2-mL suspensions eluted from high-capacity foam swab samples. The pellets were resuspended as 500-µL suspensions, and then mechanically lysed prior to dual-target qPCR to detect MTB insertion elements IS6110 and IS1081. Fractionation experiments demonstrated that most of the MTB DNA signal in clinical swab samples (99.22% ± 1.46%) was present in the sedimentable fraction. When applied to archived foam swabs collected from 124 South Africans with presumptive TB, this strategy exhibited 83% sensitivity (71/86) and 100% specificity (38/38) relative to sputum microbiological reference standard (MRS; sputum culture and/or Xpert Ultra). The second strategy used sequence-specific magnetic capture (SSMaC) to concentrate DNA released from MTB cells. This protocol was evaluated on archived Copan FLOQSwabs flocked swab samples collected from 128 South African participants with presumptive TB. Material eluted into 500 µL buffer was mechanically lysed. The suspensions were digested by proteinase K, hybridized to biotinylated dual-target oligonucleotide probes, and then concentrated ~20-fold using magnetic separation. Upon dual-target qPCR testing of concentrates, this strategy exhibited 90% sensitivity (83/92) and 97% specificity (35/36) relative to sputum MRS. These results point the way toward automatable, high-sensitivity methods for detecting MTB DNA in TS.

Importance: Improved testing for tuberculosis (TB) is needed. Using a more accessible sample type than sputum may enable the detection of more cases, but it is critical that alternative samples be tested appropriately. Here, we describe two new, highly accurate methods for testing tongue swabs for TB DNA.

Determination of antimicrobial resistance (AMR) in pneumococcal isolates is important for surveillance purposes and in a clinical context. Antimicrobial susceptibility testing (AST) of pneumococci is complicated by the need for exact minimal inhibitory concentrations (MICs) of beta-lactam antibiotics. Two next-generation sequencing (NGS) analysis tools have implemented the prediction of AMR in their analysis workflow, including the prediction of MICs: Pathogenwatch (https://pathogen.watch/) and AREScloud (OpGen). The performance of these tools in comparison to phenotypic AST following EUCAST guidelines is unknown. A total of 538 Streptococcus pneumoniae isolates were used to compare both tools with phenotypic AST for penicillin, amoxicillin, cefotaxime/ceftriaxone, erythromycin, trimethoprim-sulfamethoxazole, and tetracycline. Disk diffusion was performed for all isolates, and broth microdilution was performed for isolates with reduced beta-lactam susceptibility. Demultiplexed FASTQ files from Illumina sequencing, covering the whole genome of pneumococci, were used as input for the NGS tools. Categorical agreement (CA), major error (ME), and very major error (VME) rates were calculated. For beta-lactam antibiotics, CA was high (>94%) associated with none or only one ME and VME (<1%). For erythromycin and tetracycline, CA was >93% for predictions by AREScloud, while for Pathogenwatch, this ranged around 88%. For trimethoprim-sulfamethoxazole, CA was for both tools <86%. High VME rates were observed for erythromycin and tetracycline, higher for Pathogenwatch (53.6% and 47.0%, respectively) compared to AREScloud (14.3% and 19.1%, respectively). Both tools performed excellently despite the complexity of predicting beta-lactam resistance in pneumococci. Further optimization and validation are needed for non-beta-lactams since high (very) major error rates were observed.

Aspergillus fumigatus is a common cause of pulmonary and invasive mold infections among immunocompromised hosts. Mortality in immunocompromised hosts with invasive Aspergillus infections (IAI) has been reported to be as high as 80%. Therefore, appropriate therapy is essential in treating IAI. Both isavuconazole and voriconazole are first-line agents in treatment guidelines for IAI, but isavuconazole has favorable properties, often leading it to be preferred over voriconazole, given the lengthy duration of treatment. It is difficult to perform mold antifungal susceptibility testing, which often requires a reference lab and several weeks to determine results. Therefore, use of surrogate markers can be helpful to infer susceptibility when testing is not possible or delayed. We performed isavuconazole and voriconazole broth microdilution susceptibility testing by the Clinical and Laboratory Standards Institute (CLSI) method on a collection of 976 non-duplicate A. fumigatus isolates from a global surveillance program between 2017 and 2022. We found that voriconazole and isavuconazole have a very high essential agreement within two doubling dilutions at 99.9% and a categorical agreement of 92.7% with no very major errors, one major error (0.11%), and <10% minor errors. Many of the minor errors were in the setting of voriconazole testing at a MIC of 0.5 mg/L (susceptible) but isavuconazole at 2 mg/L (intermediate). Genetic analysis of cyp51 genes confirmed that isavuconazole and voriconazole susceptibility testing identified isolates with cyp51A and cyp51B mutations. Voriconazole can be used to predict the isavuconazole susceptibility testing result when A. fumigatus is tested by CLSI broth microdilution methodology.

Rapid and accurate diagnosis of sepsis is of paramount importance to reduce associated morbidity and mortality. The Qvella FAST System is a new instrument that concentrates and purifies bacteria from positive-flagged blood culture bottles (PFBCBs) to produce a "liquid" colony comparable to a subcultured colony in less than 40 min for rapid ID and calibrated antibiotic susceptibility testing (AST). In this study, we evaluated performances of the FAST System workflow and our rapid routine manual workflow (bacterial pellet obtained after lysis, cleaning, washing, and centrifugation for ID; AST by disc diffusion by direct inoculation after dilution) by comparison to the reference method based on 24-h bacterial subcultures. Two panels of PFBCBs were studied: panel A (including 107 prospective BCs from septic patients, October-November 2022) and panel B (including 102 BCs spiked with difficult-to-identify bacteria [mostly streptococci] and multidrug-resistant isolates), resulting in a total of 209 evaluable samples. The FAST System provided a correct ID to the species level in 178/209 (85.2%) of cases. For AST, the categorical agreement (CA) of the FAST System was 99.4%, with rates of very major (VME), major (ME), and minor (mE) errors of 0.59%, 0.20%, and 0.26%, respectively. Our rapid routine workflow based on manual methods show similar results for ID (86.2%) and AST (CA, 99.6%; VME, 0.50%; ME, 0.16%; mE, 0.13%). In conclusion, the Qvella FAST system, a promising tool that can reduce diagnostic time by approximately 1 day, shows excellent performances for rapid ID and AST.

Urinary tract infections (UTIs) impose a substantial burden on patient quality of life and urine testing accounts for the majority of workload in many clinical microbiology laboratories. Traditional UTI diagnosis relies on symptoms, urinalysis, and culture which are interpreted based on historical guidelines. This approach, while foundational, presents limitations, particularly in complex cases. Low-level bacteriuria and the presence of fastidious organisms are often overlooked or entirely missed in standard urine culture, stressing the need for novel diagnostic methods and technologies. This mini-review summarizes the existing state of UTI diagnostics in 2024 and covers current and upcoming technologies including rapid molecular-based pathogen identification, next-generation sequencing, and advanced antimicrobial susceptibility testing. However, these methods represent unique challenges, and as they are implemented, they will require the field to adapt to new concepts to avoid misdiagnosis and overtreatment.

Enterobacterales (mostly Klebsiella pneumoniae, Escherichia coli) with OXA-48-like carbapenemases (e.g., OXA-48, -181, -232, -244) are undermining the global efficiency of carbapenem therapy. In the Middle East, North Africa, and some European countries, OXA-48-like carbapenemases are the most common types of carbapenemases among Enterobacterales. Currently, OXA-48 is endemic in the Middle East, North Africa, Spain, France, and Belgium; OXA-181 is endemic in Sub-Saharan Africa and the Indian Subcontinent, while OXA-232 has been increasing in the Indian Subcontinent. European countries (e.g., Germany, Denmark, Switzerland, France) are experiencing community outbreaks with E. coli ST38 that produce OXA-244, and these strains have been introduced into Norwegian, Polish, and Czech hospitals. The global ascendancy of OXA-48-like genes is due to the combination of carbapenemases with horizontal spread through promiscuous plasmids (e.g., IncL, IncX3, ColE2) and vertical spread with certain high-risk multidrug-resistant clones (e.g., K. pneumoniae ST14, ST15, ST147, ST307; E. coli ST38, ST410). This is a powerful "gene survival strategy" that has assisted with the survival of OXA-48-like genes in different environments including the community setting. The laboratory diagnosis is complex; therefore, bacteria with "difficult to detect" variants (e.g., OXA-244, OXA-484) are likely underreported and are spreading silently "beneath the radar" in hospital and community settings. K. pneumoniae and E. coli with OXA-48-like carbapenemases are forces to be reckoned with.