New assessment of Anopheles vector species identification using MALDI-TOF MS.

BACKGROUND: Anopheles species identification is essential for an effective malaria vector control programme. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has been developed to identify adult Anopheles species, using the legs or the cephalothorax. The protein repertoire from arthropods can vary according to compartment, but there is no general consensus regarding the anatomic part to be used. METHODS: To determine the body part of the Anopheles mosquitoes best suited for the identification of field specimens, a mass spectral library was generated with head, thorax with wings and legs of Anopheles gambiae, Anopheles arabiensis and Anopheles funestus obtained from reference centres. The MSL was evaluated using two independent panels of 52 and 40 An. gambiae field-collected in Mali and Guinea, respectively. Geographic variability was also tested using the panel from Mali and several databases containing added specimens from Mali and Senegal. RESULTS: Using the head and a database without specimens from the same field collection, the proportion of interpretable and correct identifications was significantly higher than using the other body parts at a threshold value of 1.7 (p < 0.0001). The thorax of engorged specimens was negatively impacted by the blood meal after frozen storage. The addition of specimens from Mali into the database significantly improved the results of Mali panel (p < 0.0001), which became comparable between head and legs. With higher identification scores, the using of the head will allow to decrease the number of technical replicates of protein extract per specimen, which represents a significant improvement for routine use of MALDI-TOF MS. CONCLUSIONS: The using of the head of Anopheles may improve the performance of MALDI-TOF MS. Region-specific mass spectrum databases will have to be produced. Further research is needed to improve the standardization in order to share online spectral databases.

Decision criteria for MALDI-TOF MS-based identification of filamentous fungi using commercial and in-house reference databases.

BACKGROUND: Several Matrix-Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry protocols, which differ in identification criteria, have been developed for mold and dermatophyte identification. Currently, the most widely used approach is Bruker technology, although no consensus concerning the log(score) threshold has been established. Furthermore, it remains unknown how far increasing the number of spots to compare results might improve identification performance. In this study, we used in-house and Bruker reference databases as well as a panel of 422 isolates belonging to 126 species to test various thresholds. Ten distinct identification algorithms requiring one to four spots were tested. RESULTS: Our findings indicate that optimal results were obtained by applying a decisional algorithm in which only the highest score of four spots was taken into account with a 1.7 log(score) threshold. Testing the entire panel enabled identification of 87.41% (in-house database) and 35.15% (Bruker database) of isolates, with a positive predictive value (PPV) of 1 at the genus level for both databases as well as 0.89 PPV (in-house database) and 0.72 PPV (Bruker database) at the species level. Applying the same rules to the isolates for which the species were represented by at least three strains in the database enabled identification of 92.1% (in-house database) and 46.6% (Bruker database) of isolates, with 1 PPV at the genus level for both databases as well as 0.95 PPV (in-house database) and 0.93 PPV (Bruker database) at the species level. CONCLUSIONS: Depositing four spots per extract and lowering the threshold to 1.7, a threshold which is notably lower than that recommended for bacterial identification, decreased the number of unidentified specimens without altering the reliability of the accepted results. Nevertheless, regardless of the criteria used for mold and dermatophyte identification, commercial databases require optimization.

Aspergillus tubingensis: a major filamentous fungus found in the airways of patients with lung disease.

The black Aspergillus group comprises A. niger and 18 other species, which are morphologically indistinguishable. Among this species subset, A. tubingensis, described in less than 30 human cases before 2014, is primarily isolated from ear, nose, and throat samples. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has emerged as a powerful technique to identify microbes in diagnostic settings. We applied this method to identify 1,720 filamentous fungi routinely isolated from clinical samples our laboratory over a two-year study period. Accordingly, we found 85 isolates of A. niger, 58 of A. tubingensis, and six other black Aspergillus (4 A. carbonarius and 2 A. japonicus). A. tubingensis was the fifth most frequent mold isolated in our mycology laboratory, primarily isolated from respiratory samples (40/58 isolates). In this study, we mainly aimed to describe the clinical pattern of Aspergillus tubingensisWe analyzed the clinical features of the patients in whom A. tubingensis had been isolated from 40 respiratory samples. Thirty patients suffered from cystic fibrosis, chronic obstructive pulmonary disease or other types of chronic respiratory failure. Strikingly, 20 patients were experiencing respiratory acute exacerbation at the time the sample was collected. Antifungal susceptibility testing of 36 A. tubingensis isolates showed lower amphotericin B MICs (P < 10(-4)) and higher itraconazole and voriconazole MICs (P < 10(-4) and P = .0331, respectively) compared with 36 A. niger isolates. Further studies are required to better establish the role that this fungus plays in human diseases, especially in the context of cystic fibrosis and chronic pulmonary diseases.

Routine identification and mixed species detection in 6,192 clinical yeast isolates.

The clinical laboratory methods used to diagnose yeast infections should be rapid, reliable, and capable of detecting mixed infections with species exhibiting a distinct antifungal susceptibility profile. In this study, we report the performance of a procedure combining the detection of mixed yeast cultures with a chromogenic medium and MALDI-TOF identification of the colonies. We then evaluated the impact that (i) the isolation medium and (ii) lowering the identification log score (LS) threshold value have on yeast identification performance in the routine laboratory.Among 15,661 clinical samples analyzed, 5,671 tested positive and 6,192 yeasts of 42 distinct species were identified. Overall, 6,117 isolates (98.79%) were identified on the first or second MALDI-TOF Mass Spectrometry (MS) attempt, yielding an average yeast species identification turnaround time of 0.346 days (95% CI [0.326 to 0.364]). The 75 remaining isolates were identified via nucleotide sequencing. Mixed infections accounted for 498 (8.78%) of the positive samples. The MALDI-TOF MS identification procedure performed well, regardless of the culture media tested. Lowering the recommended 2.0 LS threshold value to 1.8 would reduce the number of required (i) second MALDI-TOF MS identification attempts (178 vs. 490) and (ii) ITS2 and D1-D2 sequence-based identifications (17 vs. 75), while achieving an adequate identification rate (6,183/6,192, 99.85%).In conclusion, we propose applying a 1.8 LS threshold combined with chromogenic medium subculture to optimize the yeast identification workflow and detect mixed infection in the clinical laboratory.

Cochliobolus hawaiiensis Sinusitis, a Tropical Disease? A Case Report and Review of the Literature.

A sinusitis caused by Cochliobolus hawaiiensis (anamorph: Bipolaris hawaiiensis) was diagnosed in metropolitan France in a patient originating from New Caledonia. The patient completely recovered after surgical treatment consisting in marsupialization of the mucoceles and removal of the fungus balls located in the left nasal cavity and the left maxilla and ethmoid sinuses. One year after, both endoscopic examination and CT scan of the sinuses were normal. Various clinical presentations of diseases associated with C. hawaiiensis have been reported. A review of the literature indicates that although C. hawaiiensis is very rarely reported in Europe, it is one of the major rhinosinusitis agents in areas with a relatively warmer climate, such as India or Southwestern USA. This is the first report of a sinusitis caused by C. hawaiiensis diagnosed in France, with a total recovery outcome.