Achromobacter spp. are increasingly reported among cystic fibrosis patients. Genotyping requires time-consuming methods such as multilocus sequence typing or pulsed-field gel electrophoresis. Therefore, data on the prevalence of multiresistant epidemic clones, especially A. xylosoxidans ST137 (AxST137) and the Danish epidemic strain A. ruhlandii (DES), are lacking. We recently developed and published a database for Achromobacter species identification by matrix-assisted laser desorption-ionization-time of flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics). The aim of this study was to evaluate the ability of the MALDI-TOF MS to distinguish these multiresistant epidemic clones within Achromobacter species. All the spectra of A. xylosoxidans (n = 1,571) and A. ruhlandii (n = 174) used to build the local database were analyzed by ClinProTools, MALDI Biotyper PCA, MALDI Biotyper dendrogram, and flexAnalysis software for biomarker peak detection. Two hundred two isolates (including 48 isolates of AxST137 and 7 of DES) were tested. Specific biomarker peaks were identified: absent peak at m/z 6,651 for AxST137 isolates and present peak at m/z 9,438 for DES isolates. All tested isolates were well typed by our local database and clustered within distinct groups (ST137 or non-ST137 and DES or non-DES) no matter the MALDI-TOF software or only by simple visual inspection of the spectra by any user. The use of MALDI-TOF MS allowed us to identify isolates of A. xylosoxidans belonging to the AxST137 clone that spread in France and Belgium (the Belgian epidemic clone) and of A. ruhlandii belonging to the DES clone. This tool will help the implementation of segregation measures to avoid interpatient transmission of these resistant clones.
Achromobacter spp. are nonfermenting Gram-negative bacilli mainly studied among cystic fibrosis (CF) patients. The identification of the 19 species within the genus is time-consuming (nrdA-sequencing), thus data concerning the distribution of the species are limited to specific studies. Recently, we built a database using MALDI-TOF mass spectrometry (MS) (Bruker) that allows rapid and accurate species identification and detection of the multiresistant epidemic clones: A. xylosoxidans ST137 spreading among CF patients in various French and Belgium centers, and A. ruhlandii DES in Denmark. Here, we first assessed whether species identification could be achieved with our database solely by analysis of MS spectra without availability of isolates. Then, we conducted a multicentric study describing the distribution of Achromobacter species and of the clone ST137 among French CF centers. We collected and analyzed with our local database the spectra of Achromobacter isolates from 193 patients (528 samples) from 12 centers during 2020. In total, our approach enabled to conclude for 502/528 samples (95.1%), corresponding to 181 patients. Eleven species were detected, only five being involved in chronic colonization, A. xylosoxidans (86.4%), A. insuavis (9.1%), A. mucicolens (2.3%), A. marplatensis (1.1%) and A. genogroup 3 (1.1%). This study confirmed the high prevalence of A. xylosoxidans in chronic colonizations and the circulation of the clone A. xylosoxidans ST137 in France: four patients in two centers. The present study is the first to report the distribution of Achromobacter species from CF patients samples using retrospective MALDI-TOF/MS data. This easy approach could enable future large-scale epidemiological studies.
Laboratory diagnosis of histoplasmosis is based on various methods, including microscopy, culture, antigen, and DNA detection of Histoplasma capsulatum var. capsulatum or Histoplasma capsulatum var. duboisii. To improve sensitivity of existing real-time quantitative PCR (qPCR) assays, we developed a new RT-qPCR assay that allows amplification of whole nucleic acids of Histoplasma spp. validated on suspected cases. The limit of detection was 20 copies, and the specificity against 114 fungal isolates/species was restricted to Histoplasma spp. Whole nucleic acids of 1319 prospectively collected consecutive samples from 907 patients suspected of having histoplasmosis were tested routinely between May 2015 and May 2019 in parallel with standard diagnostic procedures performed in parallel. Forty-four had proven histoplasmosis attributable to H. capsulatum var. capsulatum (n = 40) or H. capsulatum var. duboisii (n = 4) infections. The results of RT-qPCR were positive in 43 of 44 patients (97.7% sensitivity) in at least one specimen. Nine of 863 cases (99% specificity) were RT-qPCR positive and therefore classified as possible cases. RT-qPCR was positive in 13 of 30 (43.3%) blood samples tested in proven cases. A positive RT-qPCR result in blood was significantly associated with H. capsulatum var. capsulatum progressively disseminated histoplasmosis with a positive RT-qPCR result in 92.3% of the immunocompromised patients with disseminated disease. This new Histoplasma RT-qPCR assay enabling amplification of H. capsulatum var. capsulatum and H. capsulatum var. duboisii is highly sensitive and allows the diagnosis of histoplasmosis advantageously from blood and bronchoalveolar lavage fluid.
