Nosocomial transmission of Aspergillus flavus in a neonatal intensive care unit: Long-term persistence in environment and interest of MALDI-ToF mass-spectrometry coupled with convolutional neural network for rapid clone recognition.

Aspergillosis of the newborn remains a rare but severe disease. We report four cases of primary cutaneous Aspergillus flavus infections in premature newborns linked to incubators contamination by putative clonal strains. Our objective was to evaluate the ability of matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) coupled to convolutional neural network (CNN) for clone recognition in a context where only a very small number of strains are available for machine learning. Clinical and environmental A. flavus isolates (n = 64) were studied, 15 were epidemiologically related to the four cases. All strains were typed using microsatellite length polymorphism. We found a common genotype for 9/15 related strains. The isolates of this common genotype were selected to obtain a training dataset (6 clonal isolates/25 non-clonal) and a test dataset (3 clonal isolates/31 non-clonal), and spectra were analysed with a simple CNN model. On the test dataset using CNN model, all 31 non-clonal isolates were correctly classified, 2/3 clonal isolates were unambiguously correctly classified, whereas the third strain was undetermined (i.e., the CNN model was unable to discriminate between GT8 and non-GT8). Clonal strains of A. flavus have persisted in the neonatal intensive care unit for several years. Indeed, two strains of A. flavus isolated from incubators in September 2007 are identical to the strain responsible for the second case that occurred 3 years later. MALDI-TOF is a promising tool for detecting clonal isolates of A. flavus using CNN even with a limited training set for limited cost and handling time.

Cutaneous aspergillosis is a rare but potentially fatal disease of the prematurely born infant. We described here several cases due to Aspergillus flavus and have linked them to environnemental strains using MLP genotyping and MALDI-TOF mass spectrometry coupled with artificial intelligence.

Comparison of serum protein profiles of Borrelia burgdorferi-positive Bernese Mountain Dogs and dogs of other breeds using MALDI-TOF technique.

The aim of the study was to compare the serum protein profile of Bernese Mountain Dogs (BMDs) reacting positive for Bb in snap testing with the serum protein profile of dogs of other breeds (healthy and with clinical borreliosis) using the MALDI time-of-flight (MALDI-TOF) technique. The observations included five groups of dogs. BMDs reacting positively to Bb in snap serological testing and showing symptoms of borreliosis (group 1), BMDs for which no borreliosis symptoms were determined but with seropositivity for Bb determined with snap serological tests (group 2), clinically healthy BMDs with no antibodies for Bb found in the serum (group 3), five dogs of different breeds, reacting positively in serological testing, in which borreliosis symptoms were observed (group 4), clinically healthy dogs of different breeds with negative reaction in tests towards Bb (group 5). A proteomic analysis demonstrated the presence of five identical protein fractions among all five groups. An additional two protein fractions of approximately 7.630 and 15.260 kDa were found in all the serum samples obtained from the dogs positive for borrelia in a snap test, both in those exhibiting symptoms of borreliosis, and seropositive BMDs not presenting symptoms of the disease. These two additional protein fractions may be used to differentiate between seropositive and seronegative B. burgdorferi dogs and may be considered a seropositivity marker, however, it cannot be used to differentiate between animals with the clinical form of the disease and those that are only seropositive.

New variant strain of Streptococcus canis with Lancefield group C isolated from canine otitis externa.

Every basic course in microbiology teaches us, Streptococcus canis always tests positive for Lancefield group G. Surprisingly, we identified a strain of S. canis with Lancefield group C, cultured from a dog with otitis externa after lateral ear canal resection. Whole genome sequencing data and analysis points towards a horizontal gene transfer event between S. canis and S. dysgalactiae. Although these species are closely related, gene transfer in this region of the genome of S. canis has not been described before. The value of technologies as MALDI-TOF MS and sequencing in microbiological diagnostics will grow as more diverse streptococci arise that do not always conform anymore to the classical Lancefield group typing.

Rapid identification of canine uropathogens by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and the clinical factors that correlated bacterial species and antimicrobial resistance.

Using the matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) method for bacterial diagnosis, rapid urine sample preparation can reduce time relapsing of diagnosis and improve discriminatory power in coinfection cases. We aimed to evaluate rapid urine preparation procedures before MALDI-TOF MS application using dog clinical urine samples in comparison with standard microbiological diagnostic methods by agreement analysis. We determined the frequency and distribution of bacteria and bacterial resistance and their correlations to clinical history. Three experimental procedures comprising direct centrifugation, 10% sodium dodecyl sulfate digestion, and ultrasonic preparation were performed for method validation and sensitivity. Sterile urine containing Escherichia coli and/or Staphylococcus aureus were used as simulated samples. By ultrasonic preparation, the microorganisms could be detected 1.46-1.51 × 105 CFU, which was considered the most suitable technique. This preparation was significantly consistent with the routine method based on data from Hospital Information Systems for 50 urine samples from canine cystitis. By standard protocol, Enterobacteriaceae and Staphylococcus pseudintermedius were found in most of the 155 urine samples with cystitis. Extended-spectrum beta-lactamase-producing Enterobacteriaceae was found in 25-30% of the samples. Imipenem resistance was found in 70% of Acinetobacter baumannii cases; almost all were resistant to second-generation fluoroquinolones and tetracyclines. The most efficient antibiotic for treating bacterial urinary tract infection was amoxicillin plus clavulanic acid. A. baumannii and Pseudomonas aeruginosa were susceptible to pradofloxacin. Prolonged urine catheterization was linked to lower urinary tract infections by Enterobacter spp., which also correlated with chronic kidney disease.

The need for fast and accurate detection of dermatomycosis.

Dermatomycosis of the hair, skin, or nails is one of the most common fungal infections worldwide. Beyond permanent damage to the affected area, the risk of severe dermatomycosis in immunocompromised people can be life-threatening. The potential risk of delayed or improper treatment highlights the need for a rapid and accurate diagnosis. However, with traditional methods of fungal diagnostics such as culture, a diagnosis can take several weeks. Alternative diagnostic technologies have been developed which allow for an appropriate and timely selection of an antifungal treatment, preventing nonspecific over-the-counter self-medication. Such techniques include molecular methods, such as polymerase chain reaction (PCR), real-time PCR, DNA microarray, next-generation sequencing, in addition to matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Molecular methods can help close the 'diagnostic gap' observed with traditional cultures and microscopy and allow for a rapid detection of dermatomycosis with increased sensitivity and specificity. In this review, advantages and disadvantages of traditional and molecular techniques are discussed, in addition to the importance of species-specific dermatophyte determination. Finally, we highlight the need for clinicians to adapt molecular techniques for the rapid and reliable detection of dermatomycosis infections and to reduce adverse events.

Dermatomycosis is one of the most common fungal infections worldwide. Traditional fungal diagnostics are limited and can take several weeks. Molecular techniques can detect dermatomycosis pathogens quickly and allow for species-specific identification which is important for treatment.

Two cases of bloodstream infections associated with opportunistic bacterial species (Enterococcus hirae and Enterobacter xiangfangensis) in companion animals.

BACKGROUND: Bloodstream infections are a matter of concern in small animal veterinary practice. Few reports are avaiable, especially regarding the role of opportunistic bacteria in becoming infectious. This report aims to add to the current veterinary literature on two opportunistic bacterial species (Enterococcus hirae and Enterobacter xiangfangensis) associated with bloodstream infections in small animals admitted to the Bologna University Veterinary Hospital. CASE PRESENTATION: In the first case, a 15-year-old, immunocompromised, cardiopathic dog was admitted to the hospital for anorexia and diarrhea. The patient had a history of previous surgery and hospitalization. After three days, hyperthermia, leukopenia and hyperlactatemia were recorded, and blood culture revealed positivity for Enterococcus hirae, identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The patient's general conditions progressively worsened, and the patient was euthanized. In the second case, a 2-year-old cat with chronic ocular herpesvirus infection and hypertrophic cardiomyopathy was admitted to the hospital for anorexia and hyperthermia. The cat was hospitalized one week before and received antimicrobial treatment for urinary tract infection by Staphylococcus felis. Hypokalemia and lymphopenia were also diagnosed. The patient progressively improved and was discharged after three days. On the same day, blood culture taken at admission revealed positivity for Enterobacter xiangfangensis, identified using MALDI-TOF MS. After five days, the patient returned with neurological symptoms, hypothermia and bradycardia, and was euthanized. CONCLUSIONS: In small animal veterinary practice, the impact of opportunistic bacterial agents (such as E.hirae and E.xiangfangensis) on bloodstream infections remains unclear. As in human medicine, they can be contracted in every healthcare setting and considered hospital-acquired infections. In this report, we highlighted the threat they pose especially in patients with multiple risk factors. Rapid and accurate diagnostic tools (such as MALDI-TOF MS) could be particularly important for reducing the severity of the infections.

Future Directions for Ruminant Diagnostics.

Diagnostic advances such as next-generation sequencing, highly multiplexed real-time PCR tests, and MALDI-TOF mass spectrometry have provided a tremendous increase in the amount of diagnostic information to clinicians. However, interpretation and application of these results to both individual and herd-level diagnostics still require the necessary skills in critical thinking and diagnostic interpretation to maximize benefit. This article provides a summary of advancements in diagnostic medicine and interpretation, as well as identifies gaps in knowledge that can be targeted to continue to build on best practices and application of diagnostic tools to improve ruminant health.

Current and Emerging Diagnostic Approaches to Bacterial Diseases of Ruminants.

The diagnostic approaches and methods to detect bacterial pathogens in ruminants are discussed, with a focus on cattle. Conventional diagnostic methods using culture, isolation, and characterization are being replaced or supplemented with new methods. These include molecular diagnostics such as real-time polymerase chain reaction and whole-genome sequencing. In addition, methods such as matrix-assisted laser desorption ionization-time-of-flight mass spectrometry enable rapid identification and enhanced pathogen characterization. These emerging diagnostic tools can greatly enhance the ability to detect and characterize pathogens, but performance and interpretation vary greatly across sample and pathogen types, disease syndromes, assay performance, and other factors.

Fast and accurate identification by MALDI-TOF of the zoonotic serovar E of Vibrio vulnificus linked to eel culture.

Vibrio vulnificus is a zoonotic pathogen that can cause death by septicaemia in farmed fish (mainly eels) and humans. The zoonotic strains that have been isolated from diseased eels and humans after eel handling belong to clade E (or serovar E (SerE)), a clonal complex within the pathovar (pv.) piscis. The aim of this study was to evaluate the accuracy of MALDI-TOF mass spectrometry (MS) in the identification of SerE, using the other two main pv. piscis-serovars (SerA and SerI) from eels as controls. MALDI-TOF data were compared with known serologic and genetic data of five pv. piscis isolates or strains, and with the non pv. piscis reference strain. Based on multiple spectra analysis, we found serovar-specific peaks that were of ~3098 Da and ~ 4045 Da for SerE, of ~3085 Da and ~ 4037 Da for SerA, and of ~3085 Da and ~ 4044 Da for SerI. Therefore, our results demonstrate that MALDI-TOF can be used to identify SerE and could also help in the identification of the other serovars of the species. This means that zoonosis due to V. vulnificus could be prevented by using MALDI-TOF, as action can be taken immediately after the isolation of a possible zoonotic V. vulnificus strain.

Species identification by MALDI-TOF MS and gap PCR-RFLP of non-aureus Staphylococcus, Mammaliicoccus, and Streptococcus spp. associated with sheep and goat mastitis.

Staphylococci and streptococci are common causes of intramammary infection in small ruminants, and reliable species identification is crucial for understanding epidemiology and impact on animal health and welfare. We applied MALDI-TOF MS and gap PCR-RFLP to 204 non-aureus staphylococci (NAS) and mammaliicocci (NASM) and to 57 streptococci isolated from the milk of sheep and goats with mastitis. The top identified NAS was Staphylococcus epidermidis (28.9%) followed by Staph. chromogenes (27.9%), haemolyticus (15.7%), caprae, and simulans (6.4% each), according to both methods (agreement rate, AR, 100%). By MALDI-TOF MS, 13.2% were Staph. microti (2.9%), xylosus (2.0%), equorum, petrasii and warneri (1.5% each), Staph. sciuri (now Mammaliicoccus sciuri, 1.0%), arlettae, capitis, cohnii, lentus (now M. lentus), pseudintermedius, succinus (0.5% each), and 3 isolates (1.5%) were not identified. PCR-RFLP showed 100% AR for Staph. equorum, warneri, arlettae, capitis, and pseudintermedius, 50% for Staph. xylosus, and 0% for the remaining NASM. The top identified streptococcus was Streptococcus uberis (89.5%), followed by Strep. dysgalactiae and parauberis (3.5% each) and by Strep. gallolyticus (1.8%) according to both methods (AR 100%). Only one isolate was identified as a different species by MALDI-TOF MS and PCR-RFLP. In conclusion, MALDI-TOF MS and PCR-RFLP showed a high level of agreement in the identification of the most prevalent NAS and streptococci causing small ruminant mastitis. Therefore, gap PCR-RFLP can represent a good identification alternative when MALDI-TOF MS is not available. Nevertheless, some issues remain for Staph. haemolyticus, minor NAS species including Staph. microti, and species of the novel genus Mammaliicoccus.

Identification of bacterial species in milk by MALDI-TOF and assessment of some oxidant-antioxidant parameters in blood and milk from cows with different health status of the udder.

This study aimed to identify bacterial pathogens in milk samples from dairy cows with subclinical and clinical mastitis as well as to assess the concentrations of oxidant-antioxidant parameters [malondialdehyde (MDA), reduced glutathione (GSH), and total GSH levels] in both blood and milk samples. From a total of 200 dairy cows in 8 farms, 800 quarter milk samples obtained from each udder were tested in the laboratory for the presence of udder pathogens. Cultivated bacteria causing intramammary infection from milk samples were identified by Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF). In addition, from tested animals 60 cows were selected including 20 healthy cows that were CMT negative, 20 cows with subclinical mastitis (SM), and 20 cows with clinical mastitis (CM) for detection of MDA, GSH, and total GSH levels in blood and milk samples. Three hundred and eighty (47.5%; 380/800), 300 (37.5%; 300/800), and 120 (15%; 120/800) of milk samples, respectively were CMT positive or SM and CM, and those positives were cows from different farms. We observed that 87.4% (332/380), 25.3% (76/300), and 34.2% (41/120) of cows with CMT positive, CMT negative, and CM had bacterial growth. The most predominantly identified bacteria were Staphylococcus chromogenes (18.7%) obtained mainly from SM and Staphylococcus aureus (16.7%) as the most frequent cause of CM. According to our results, dairy cows with CM had the highest MDA levels, the lowest GSH, and total GSH levels in both blood and milk samples however, high MDA levels and low GSH levels in milk samples with SM were observed. Based on our results, lipid oxidant MDA and antioxidant GSH could be excellent biomarkers of cow's milk for developing inflammation of the mammary gland. In addition, there was no link between nutrition and MDA and GSH levels.

Identification of Algerian field-caught mosquito vectors by MALDI-TOF MS.

Vector-borne diseases represent a real threats worldwide, in reason of the lack of vaccine and cure for some diseases. Among arthropod vectors, mosquitoes are described to be the most dangerous animal on earth, resulting in an estimated 725,000 deaths per year due to their borne diseases. Geographical position of Algeria makes this country a high risk area for emerging and re-emerging diseases, such as dengue coming from north (Europe) and malaria from south (Africa). To prevent these threats, rapid and continuous surveillance of mosquito vectors is essential. For this purpose we aimed in this study to create a mosquito vectors locale database using MALDI-TOF mass spectrometry technology for rapid identification of these arthropods. This methodology was validated by testing 211 mosquitoes, including four species (Aedes albopictus, Culex pipiens, Culex quinquefasciatus, and Culiseta longiareolata), in two northern wilayahs of Algeria (Algiers and Bejaia). Species determination by MALDI TOF MS was highly concordant with reference phenotypic and genetic methods. Using this MALDI-TOF MS tool will allow better surveillance of mosquito species able to transmit mosquito borne diseases in Algeria.

Use of MALDI-TOF MS technology to evaluate adulteration of small ruminant milk with raw bovine milk.

Detection of adulteration of small ruminant milk is very important for health and commercial reasons. New analytical and cost-effective methods need to be developed to detect new adulteration practices. In this work, we aimed to explore the ability of the MALDI-TOF mass spectrometry to detect bovine milk in caprine and ovine milk using samples from 18 dairy farms. Different levels of adulteration (0.5, 1, 5, 10, 20, 40, 60, and 80%) were analyzed during the lactation period of goat and sheep (in May, from 60 to 90 d in milk, and in August, from 150 to 180 d in milk). Two different ranges of peptide-protein spectra (500-4,000 Da; 4-20 kDa) were used to establish a calibration model for predicting the concentration of adulterant using partial least squares and generalized linear model with lasso regularization. The low molecular weight part of the spectra together with the generalized linear model with lasso regularization regression model appeared to have greater potential for our aim of detection of adulteration of small ruminants' milk. The subsequent prediction model was able to predict the concentration of bovine milk in caprine milk with a root mean square error of 11.4 and 17.0% in ovine milk. The results offer compelling evidence that MALDI-TOF can detect the adulteration of small ruminants' milk. However, the method is severely limited by (1) the complexity of the milk proteome resulting from the adulteration technique, (2) the potential degradation of thermolabile proteins, and (3) the genetic variability of tested samples. Additionally, the root mean square error of prediction based only on one individual sample adulteration series can drop down to 6.34% for quantification of adulterated caprine milk and 6.28% for adulterated ovine milk for the full set of concentrations or down to 2.33 and 4.00%, respectively, if we restrict only to low concentrations of adulteration (0, 0.5, 1, 5, 10%).

Retroperitoneal abscess and splenitis with Clostridium spp. in a dog.

An 11-year-old spayed female Basset Hound was presented to the Colorado State University Veterinary Teaching Hospital for evaluation of a 7-week history of intermittent collapse, waxing and waning lethargy, and hyporexia. Abdominal ultrasound revealed a 6-mm hypoechoic splenic nodule that, on cytologic evaluation, revealed marked neutrophilic inflammation with intracellular and extracellular bacterial rods frequently producing oval subterminal to terminal endospores, suggestive of Clostridium. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) performed on bacteria isolated from this nodule initially identified a Clostridium species, which was eventually confirmed with 16 s rDNA sequencing. Computed tomography (CT) and exploratory laparotomy subsequently identified a 2.5-cm diameter tubular structure beginning at the caudal aspect of the right kidney and coursing caudally containing gas and fluid, consistent with a retroperitoneal abscess, which was resected and also cultured Clostridium spp. The dog was discharged 3 days postoperatively and was alive at the time of writing, 7 months after discharge. This case highlights a previously unrecognized bacterial agent in a retroperitoneal abscess. The use of cytologic evaluation yielded a diagnosis of endospore-forming bacteria suggestive of Clostridium sooner than culture and histopathology, which allowed for adjustment in the antibiotic protocol.

Evaluation of the Vitek Ms system for the identification of filamentous fungi.

The implementation of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of fungal isolates remains challenging and has been limited to experienced laboratories in sample preparation and in-house libraries construction. However, the development of commercial kits for standardized fungal sample preparation and updated reference libraries can fill this gap. This study aimed to evaluate the performance of the commercial VITEK MS Mould Kit (bioMérieux, Marcy l'Etoile, France) and the VITEK MS system (bioMérieux) for identification using a panel of fungal species of clinical interest. Overall, 200 isolates belonging to 13 genera and 43 fungal species were analyzed with the VITEK MS system equipped with the v3.2 IVD database. Overall, 89.0% of the isolates were correctly identified, 41.5 and 43.5% at species and complex level, respectively. For an additional 4.0% of the identifications, correlation at the genus level was reported. The remaining 21 isolates (10.5%) could not be identified among which 85.0% (18/21) were species not claimed in the database. One Syncephalastrum isolate was misidentified as Rhizopus microsporus complex. Specifically, 100% of the Scedosporium/Lomentospora, 97.1% of the Fusarium, 65.7% of the Mucorales and 86.4% of the Aspergillus isolates were correctly identified at the species and complex level. The methodology described allows for an easy implementation of MALDI-TOF MS for routine identification of fungal species in a fast and reliable manner. Although further improvement in the databases is still required, an important number of fungal species can be correctly identified at the species level using this method. LAY SUMMARY: The use of MALDI-TOF for fungal identification remains a challenge. In this study, using a commercial protein extraction kit and updated database, VITEK MS system was able to identify up to 89.0% of a diverse collection of 200 filamentous fungi representing 43 fungal species.

Evaluation of formic acid sandwich (FA-sandwich): A pretreatment method for filamentous fungi, for the identification of clinically relevant filamentous fungi by two MALDI-TOF MS systems.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been successfully applied to identify microorganisms. However, unlike bacteria and yeast where identification results can be obtained rapidly and accurately by using a simple direct-coating pretreatment method, the traditional pretreatment methods for filamentous fungi are more complex, involving ethanol, formic acid, acetonitrile, and a protein extraction process by centrifugation, i.e., the EtOH-FA full extraction. This cumbersome pretreatment for filamentous fungi is a major reason for the lack of widespread use of MALDI-TOF MS for the identification of filamentous fungi in clinical settings. The present study describes an alternative method, the FA-sandwich, and demonstrates that the approach is efficient and effective. 148 clinical filamentous fungal isolates collected from three large general hospitals in Hubei Province, China, were processed by the FA-sandwich method and identified by two MALDI-TOF MS platforms, Autof ms and Vitek MS. The FA-sandwich allowed a 93.9% species-level identification with Autof ms, and 97.3% species-level identification rates were found for Vitek MS when the IVD, the RUO and in-house databases are used in combination. Further comparison of the ease of FA-sandwich with the EtOH-FA full extraction showed that the FA-sandwich is a more convenient, time- and reagent-saving, and sensitive pretreatment method. These findings indicate that the FA-sandwich method is suitable for pretreating filamentous fungi followed by MALDI-TOF MS identification in clinical microbiology laboratories. LAY SUMMARY: The FA-sandwich method improves the efficiency of identification of filamentous fungi using MALDI-TOF MS while ensuring identification accuracy. The method is easy to perform and very suitable for detecting mold in the microbiology laboratory with the goal of promoting timely and accurate therapies.

Assessment of the main pathogens associated with clinical and subclinical endometritis in cows by culture and MALDI-TOF mass spectrometry identification.

Clinical endometritis (CE) and subclinical endometritis (SCE) are diseases that affect dairy cows during the puerperium, causing negative effects on the animals' milk production and fertility. The objective of this study was to assess the main bacteria related to cases of CE and SCE from uterine samples of dairy cows in Brazilian herds. Selective and differential media were used for isolation of aerobic and anaerobic bacteria and further MALDI-TOF mass spectrometry (MS) identification. A total of 279 lactating dairy cows with 28 to 33 d in milk from 6 commercial farms were evaluated. Initially, cows were classified in 3 groups: cytologic healthy cows (n = 161), cows with CE (n = 83), and cows with SCE (n = 35). Healthy animals presented 97 species, followed by the CE group with 53 identified species, and SCE cows presented only 21 bacterial species. We found a significantly higher isolation rate of Trueperella pyogenes in CE (26.5%) cows compared with healthy and SCE cows. Some anaerobic species were exclusively isolated from the CE group, even though they presented lower frequency. Interestingly, 18.1% of samples from CE cows and 40% of SCE cows were negative to bacterial isolation. Despite the use of culture-dependent methods instead of molecular methods, the present study enabled the identification of a complex community of 127 different species from 48 genera, composed of aerobic and anaerobic bacterial species among the 3 different animal groups. The method of sample collection, culture, and identification by MALDI-TOF MS were essential for the success of the analyses.

Limitations of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of Aspergillus species.

This study aimed to detect the identification limitations for Aspergillus species from patients or the environment based on MALDI-TOF MS analysis. A total of 209 Aspergillus isolates were selected in this study. One hundred and sixty-eight of the strains were selected as challenge strains for MALDI-TOF MS analysis, while the remaining 41 strains were used to construct a supplementary database. The 168 challenge strains were identified by the Bruker Filamentous Fungi Library v1.0 (the Bruker Library) and identified again using the Bruker Library combined with the supplementary database (the combined database). The sensitivity of MALDI-TOF MS with the Bruker Library alone and with the combined database in identifying the challenge strains at the species level was 64.3 and 85.7%, respectively. With the combined database, the sensitivity of MALDI-TOF MS in identifying strains in Aspergillus sections Fumigati, Flavi, Nigri, Terrei, and Nidulantes was 100, 86.5, 76.1, 100, and 80%, respectively, and the sensitivity in identifying strains of other Aspergillus species was 71.4%. The specificity of MALDI-TOF MS in identifying strains in all Aspergillus sections at the species level was 100%. Even when using the combined database, MALDI-TOF MS analysis showed some misidentification for the species A. niger, A. welwitschiae, A. luchuensis, A. flavus and A. sydowii. In conclusion, with the combined database, MALDI-TOF MS showed good performance in identifying the species in Aspergillus sections Fumigati and Terrei but limited performance in distinguishing some closely related species in sections Nigri, Flavi and Nidulantes. LAY SUMMARY: MALDI-TOF MS showed good performance in identifying Aspergillus species in sections Fumigati and Terrei but limited performance in distinguishing some closely related species in sections Nigri, Flavi and Nidulantes.

Initial steps on mapping differentially expressed proteins in bovine preantral follicles and ovarian tissue: An approach using single-follicle MALDI-MS and mass spectrometry imaging (MSI) analysis.

The molecular mechanisms regulating follicular development and ensuring primordial follicle activation remain undefined. To help elucidate these mechanisms, this proteomic study of bovine ovarian tissue identified the differential molecular profiles of preantral follicles together with the spatial distribution of the most abundant molecular components in the tissue. Isolated primordial, primary and secondary follicles were individually placed on a MALDI target plate for mass spectral acquisitions, with detection of different m/z ranges. Ovarian tissue was sectioned and analysed in the m/z 400-2,000 range. Results of the first analysis indicated a similarity pattern in the molecular protein profile among different follicular classes in the m/z ranges of 100-1000 and 25,000-200,000, but in the m/z ranges of 800-4000, 4000-20,000 and 15,000-70,000, primary and secondary follicles shared similar clustering profiles which were different from primordial follicles (p < .05). In the second analysis, it was possible to correlate some intense molecular components in the tissue from global mass spectrum with the ions detected in the first analysis. Molecular components at m/z 11,325 (±230) were also detected in primary and secondary follicles in the experiment with isolated follicles, in addition to ions at m/z 4,029 (±120), 13,799 (±70), 5,547 (±9), 15,313 (±200), 7,018 (±40) and 7,663 (±90) which were also intensely detected in primary and secondary follicles. The present proteomic approaches evaluated different mass ranges of preantral follicles in bovine ovarian tissue and also indicated the spatial distribution of the most abundant molecular components. This study hopes to pave the way for future research identifying and characterizing specific proteins involved in follicle activation in bovine follicles, in order to better understand folliculogenesis and potentially improve mammalian follicle culture systems.

Sequence determination of phosphorothioated oligonucleotides using MALDI-TOF mass spectrometry for controlling gene doping in equestrian sports.

In human and equestrian sporting events, one method of gene doping is the illegal use of therapeutic oligonucleotides to alter gene expression. In this study, we aimed to identify therapeutic oligonucleotides via sequencing using matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). As a model of therapeutic oligonucleotides, 22 bp-long phosphorothioated oligonucleotides (PSOs) were used. By using a Clarity OTX kit for extracting short-length oligonucleotides, a spectrum of singly charged PSO with a mean intensity of 6.08 × 104 (standard deviation: 4.34 × 103 ) was detected from 500 pmol PSO in 1 ml horse plasma using the linear negative mode of MALDI-TOF MS. In addition, a 17 bp sequence was determined using in-source decay (ISD) mode, indicating that 500 pmol of a PSO in 1 ml plasma is the detection limit for sequencing. Using the determined sequences (17 bp), a targeted gene for PSO was singly identified on the horse reference genome, EquCab2.0, via a GGGenome search. These procedures can be potentially used to identify therapeutic oligonucleotides, whose nucleotides are unknown, for gene doping control.