Comparison of two MALDI-TOF MS systems for theidentification of clinically relevant anaerobic bacteriain Argentina

Abstract The aim of this study was to compare the performance of two MALDI-TOF MS systems in the identification of clinically relevant strict anaerobic bacteria. The 16S rRNA gene sequencing was the gold standard method when discrepancies or inconsistencies were observed between platforms. A total of 333 isolates were recovered from clinical samples of different centers in Buenos Aires City between 2016 and 2021. The isolates were identified in duplicate using two MALDI-TOF MS systems, BD Bruker Biotyper (Bruker Daltonics, Bremen, Germany) and Vitek MS (bioMèrieux, Marcy-l'Etoile, France). Using the Vitek MS system, the identification of anaerobic isolates yielded the following percentages: 65.5% (n: 218) at the species or species-complex level, 71.2% (n: 237) at the genus level, 29.4% (n: 98) with no identification and 5.1% (n: 17) with misidentification. Using the Bruker Biotyper system, the identification rates were as follows: 85.3% (n: 284) at the species or species-complex level, 89.7% (n: 299) at the genus level, 14.1% (n: 47) with no identification and 0.6% (n: 2) with misidentification. Differences in the performance of both methods were statistically significant (p-values <0.0001). In conclusion, MALDI-TOF MS systems speed up microbial identification and are particularly effective for slow-growing microorganisms, such as anaerobic bacteria, which are difficult to identify by traditional methods. In this study, the Bruker system showed greater accuracy than the Vitek system. In order to be truly effective, it is essential to update the databases of both systems by increasing the number of each main spectrum profile within the platforms.

Resumen El objetivo de este estudio fue comparar el desempeño de dos sistemas MALDI-TOF MS en la identificación de bacterias anaerobias estrictas de interés clínico. La secuenciación del gen 16S ARNr fue el método de referencia utilizado cuando se observaron discrepancias o inconsistencias entre plataformas. Se recuperaron 333 aislados de muestras clínicas de diferentes centros de la Ciudad Autónoma de Buenos Aires entre 2016 y 2021. Los aislados se identificaron por duplicado mediante dos sistemas MALDI-TOF MS: el BD Bruker Biotyper (Bruker Daltonics, Bremen, Alemania) y el Vitek MS (bioMèrieux, Marcy-l'Etoile, Francia). A través del sistema Vitek MS, los mismos fueron identificados a nivel de especie o complejo de especies en un 65,5% (n: 218) y de género en un 71,2% (n: 237), mientras que no se identificaron en un 29,4% (n: 98) y fue incorrecta en el 5,1% (n: 17). Mediante el sistema Bruker Biotyper, dichos valores fueron del 85,3% (n: 284), del 89,7% (n: 299), del 14,1% (n: 47) y del 0,6% (n: 2), respectivamente. La diferencia entre ambos métodos fue estadísticamente significativa (p<0,0001). En conclusión, los sistemas MALDI-TOF MS aceleran la identificación microbiana. Son especialmente útiles para los microorganismos de crecimiento lento, como las bacterias anaerobias, que son difíciles de identificar con los métodos tradicionales. El sistema Bruker demostró ser más preciso que el Vitek MS. Para que estos métodos sean realmente efectivos es fundamental actualizar las bases de datos de ambos sistemas e incrementar el número de espectros de referencia dentro de las plataformas.

Diagnostic value of nucleic acid matrix-assisted laser desorption ionization-time of flight mass spectrometry in sputum smear-negative patients with nontuberculous Mycobacterial pulmonary disease / 中华临床感染病杂志

Objective:To investigate the diagnostic value of nucleic acid matrix-assisted laser desorption ionization-time of flight mass spectrometry（MALDI-TOF MS）in sputum smear-negative patients with nontuberculous Mycobacterial（NTM）pulmonary disease.Methods:Clinical data of 123 patients suspected of NTM pulmonary disease admitted in Public Health Clinical Center Affiliated to Shandong University between July 2022 and November 2023 were retrospectively analyzed. Bronchoalveolar lavage fluid（BALF）specimens were collected for MALDI-TOF MS assay and MGIT 960 culture. The diagnostic efficacy of MALDI-TOF MS for NTM pulmonary disease in patients with negative sputum smears for acid-fast bacilli was evaluated with receiver operating characteristic（ROC）curve. Statistical analysis was performed using SPSS 26.0 software and MedCalc statistical software.Results:Diagnosis of NTM pulmonary disease was finally confirmed in 66 out of the 123 suspected patients. It took 8 to 24 h for MALDI-TOF MS to identify NTM species and resistance. By MALDI-TOF MS，72 NTM strains were identified，with the Mycobacterium avium complex being the most prevalent（34 strains，47.22%），followed by the Mycobacterium abscessus complex（13 strains，18.06%）；resistance to macrolides was detected in 6 cases，while no resistance to aminoglycosides was found. It took 9 to 45 days for BALF MGIT 960 culture to identify NTM，and took 7 to 15 days for NTM typing and drug sensitivity testing. By BALF MGIT 960 culture，28 NTM strains were identified；and 1 case was found to be resistant to macrolides. Using confirmed diagnosis as the gold standard，MALDI-TOF MS demonstrated higher sensitivity，negative predictive value，and agreement rate compared to MGIT 960 culture（84.85% vs. 42.42%，81.13% vs. 56.32%，80.49% vs. 62.60%， χ2=25.667，8.998，9.664， P<0.05 or <0.01）. The area under ROC curve（AUC）for MALDI-TOF MS was significantly higher than that of MGIT 960 culture（0.801 vs. 0.642， Z=3.300， P=0.001）. Conclusion:Compared to MGIT 960 culture，MALDI-TOF MS exhibits superior diagnostic efficiency in detecting NTM pulmonary disease in patients with acid-fast bacilli smear-negative sputum，with advantage of rapidly identifying NTM species and resistance.

EGFR Methylation in Peripheral Blood of Driver Gene-Positive Lung Cancer and its Clinical Correlation / 医学研究杂志

Objective To explore the methylation status of EGFR promoter region in peripheral blood of EGFR driver gene-positive lung adenocarcinoma patients and its clinical significance.Methods The methylation of EGFR promoter region in peripheral blood DNA of 32 EGFR driver gene-positive lung adenocarcinoma patients and 24healthy controls were detected by(matrix-assisted laser desorp-tion/ionisation,time-of-flight mass spectrometry,MALDI-TOF MS)technique to screen the differential methylation of CpG sites with lung cancer and analyze its correlation with clinicopathological characteristics and EGFR mutant subtype.Results The methylation levels of EGFR were significantly lower in the lung cancer group than in the normal control,EGFR#29 CpG_2,EGFR#30 CpG_8.9.10.11,CpG_25,CpG_40.41.42methylation of lung cancer group(25.7％±2.1％,4.6％±0.4％,2.8％±0.7％,4.40±0.4％)were low-er than the normal control(36.5％±4.6％,6.0％±0.6％,4.8％±0.9％,7.1％±0.4％),the difference was statistically signifi-cant(P＜0.05).No significant differences in the methylation levels within EGFR CpGs were associated with age,cancer differentiation,disease location,TNM stage with lung cancer(P＞0.05).The methylation level of EGFR#30 CpG_25 in the female group was signifi-cantly lower than that in the male group(P＜0.05),and the methylation level of EGFR#30 CpG_25 in the smoking group was higher than that in the non-smoking group(P＜0.05),the methylation level of EGFR#30 CpG_8.9.10.11 in the lymph node metastasis group was significantly lower than that in the non-lymph node metastasis(P＜0.01).There was no significant difference in methylation of EGFR between EGFR 19del and 21 L858R mutant subtypes.Conclusion Our findings suggest that EGFR methylation may be associated with the occurrence,development,gender,and smoking history of patients with EGFR driving gene-positive lung cancer,which may serve as a biomarker for the diagnosis and prognosis prediction of lung cancer.

Clinical application of MALDI-TOF MS for homology analysis of Acineto-bacter baumannii / 中国感染控制杂志

Objective To evaluate the clinical application value of matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS)in analyzing the homology of Acinetobacter baumannii(AB).Methods After excluding repetitive strains from multiple specimens of the same patient or environment,a total of 46 AB strains isolated from patients'sputum and environmental specimens of neurological intensive care unit(ICU)in a tertiary first-class general hospital from May 2020 to February 2021 were collected.Strains were detected by VITEK-MS mass spectrometer.Cluster analysis was performed by SARAMIS Premium software,and verified by multilocus sequence typing(MLST).Results Cluster analysis and comparison of MALDI-TOF MS and MLST found that among the 46 AB strains,39 were the type MS-a of MALDI-TOF MS,of which 22 strains were the clus-ter MT-A of MLST,including ST208(n=3),ST540(n=3),ST195(n=8),ST369(n=5),ST136(n=1),ST436(n=1)and ST1893(n=1);16 strains were MT-B,including type ST381(n=4),type ST469(n=11),and type ST938(n=1);one strain was cluster MT-C(ST1821);one strain of type MS-b was ST381;two strains of type MS-c were ST369;one strain of type MS-d was ST195;two strains of type MS-e were ST540 and ST369,respectively;one strain of type MS-f was STN1.Conclusion As a homology analysis method,MALDI-TOF MS still has certain limitations such as low consistency with MLST results,low resolution and specificity,thus cannot replace MLST technology.

The influence of growth time on the identification of Bartonella henselae strains by MALDI-TOF mass spectrometry

ABSTRACT Bartonella spp. are bacteria responsible for neglected diseases worldwide. Bartonella henselae is the species most associated with human infections. It is associated with a large spectrum of clinical manifestations and is potentially fatal. The identification of Bartonella spp. is considered a challenge in clinical routine. These bacteria are fastidious, and the time required to isolate them varies from one to six weeks. MALDI-TOF mass spectrometry has emerged as an application for research on Bartonella spp. , and has still been little explored. We investigated whether three different B. henselae strains with different growth times—14 and 28 days—could be correctly identified by MALDI-TOF mass spectra fingerprint comparison and matching. We found that the spectra from strains with different growth times do not match each other, leading to misidentification. We suggest creating database entries with multiple spectra from strains with different growth times to increase the chances of accurate identification of Bartonella spp. by MALD-TOF MS.

Coinfección por Aggregatibacter aphrophilus y SARS-CoV-2 con desenlace fatal / Fatal outcome in coinfection by Aggregatibacter aphrophilus and SARS-CoV-2 / Coinfecção por Aggregatibacter aphrophilus e SARSCoV- 2 com desfecho fatal

Resumen Aggregatibacter aphrophilus es un cocobacilo gram negativo de requerimientos nutricionales exigentes, que obliga al microbiólogo a utilizar métodos de identificación no convencionales. Se comunica el caso de una paciente que fue internada con un cuadro clínico caracterizado por fiebre, cefalea y dificultad respiratoria por COVID-19. De las muestras de hemocultivos se rescataron colonias cocobacilares gram negativas de lento crecimiento, las cuales fueron finalmente identificadas como A. aphrophilus mediante MALDI-TOF MS (Matrix-assisted laser desorption ionization time-of-flight mass spectrometry). Dado que en nuestro medio la identificación de este tipo de gérmenes es dificultosa, es fundamental y se recomienda formar una red de laboratorios que den respuesta a las necesidades diagnósticas y tecnológicas.

Abstract Aggregatibacter aphrophilus is a gram-negative coccobacillus with demanding nutritional requirements, which forces the microbiologist to use unconventional typification methods. The case of a patient who was admitted with a clinical history characterised by fever, headache and respiratory distress due to COVID-19 was reported. Slow-growing gram-negative coccobacillary colonies were recovered from the blood culture samples, which were finally typed as A. aphrophilus using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Identifying this organism is difficult in our setting. As it is considered essential, building a network of laboratories that give response to diagnostic and technological needs is highly recommended.

Resumo Aggregatibacter aphrophilus é um cocobacilo gram-negativo com pedidos nutricionais exigentes, o que obriga o microbiologista a utilizar métodos de tipagem não convencionais. É relatado o caso de um paciente que deu entrada com quadro clínico caracterizado por febre, cefaleia e desconforto respiratório devido à COVID-19. Colônias de cocobacilos gram-negativas de crescimento lento foram recuperadas das amostras de hemoculturas, que foram finalmente identificadas como A. aphrophilus usando MALDI-TOF MS (Matrix-assisted laser desorption ionization time-of-flight mass spectrometry). Devido a que a identificação deste tipo de germes é difícil no nosso meio, considera-se essencial e se recomenda construir uma rede de laboratórios que respondam às necessidades diagnósticas e tecnológicas.

Identificación directa con MALDI-TOF desde el frasco de hemocultivo a partir de subcultivos con incubación corta. Estudio multicéntrico / Direct identification with MALDI-TOF from the blood culture bottle from subcultures with short incubation. Multicenter study / Identificação direta com MALDI-TOF do frasco de hemocultura de subculturas com incubação curta. Estudo multicêntrico

Resumen El objetivo de este trabajo fue evaluar el rendimiento de la identificación realizada con MALDI-TOF a partir de la incubación de 3-5 h de subcultivos de hemocultivos positivos monomicrobianos que se comparó con la obtenida con la incubación de 24 h de los mismos. En dos hospitales se utilizó el sistema Vitek-MS (bioMérieux, Francia) y en uno el sistema Micro- Flex LT (Bruker, Daltonics). A partir de la incubación corta, MALDI-TOF identificó correctamente a 5/5 de las levaduras, a 91,1% (153/168) de las bacterias gram positivas, a 96,7% (119/123) de los bacilos gram negativos y a 93,6% (277/296) del total de cepas. La identificación por medio de MALDI-TOF a partir de una corta incubación de los subcultivos de los hemocultivos en medio sólidos es un método práctico, sencillo y confiable.

Abstract The objective of this work was to evaluate the performance of the identification carried out with MALDI-TOF from the 3-5 h incubation of subcultures of monomicrobial positive blood cultures that was compared with that obtained with the 24 h incubation of the same subcultures. The Vitek-MS system (bioMérieux, France) was used in two hospitals and the Micro-Flex LT system (Bruker, Daltonics) in one. With a short incubation, MALDI-TOF correctly identified 5/5 of the yeasts, 91.1% (153/168) of the gram-positive bacteria, 96.7% (119/123) of the gram-negative bacilli and 93.6% (277/296) of the total strains. Identification by means of MALDI-TOF with a short incubation of subcultures of blood cultures in solid media is a practical, simple and reliable method.

Resumo O objetivo deste trabalho foi avaliar o desempenho da identificação realizada com MALDI-TOF a partir de 3 a 5 h de incubação de subculturas de hemoculturas positivas monomicrobianas que foi comparada com a obtida com a incubação de 24 h das mesmas. O sistema Vitek-MS (bioMérieux, França) foi utilizado em dois hospitais e o sistema Micro-Flex LT (Bruker, Daltonics) em um. A partir da incubação curta, o MALDI-TOF identificou corretamente 5/5 das leveduras, 91,1% (153/168) das bactérias gram positivas, 96,7% (119/123) dos bacilos gram-negativos e 93,6% (277/296) das cepas totais. A identificação por meio de MALDI-TOF a partir de uma incubação curta das subculturas das hemoculturas em meio sólido é um método prático, simples e confiável.

Actinomyces europaeus (Gleimia europaea) asociado con absceso cerebral: comunicación de tres casos / Actinomyces europaeus (Gleimia europaea) associated with brain abscess: A reportof three cases

Resumen El absceso cerebral es una infección focal caracterizada por acumulación de pus enel parénquima cerebral; su diagnóstico es de urgencia debido a la alta mortalidad que acarrea.Presentamos tres casos de pacientes con abscesos cerebrales con foco otogénico de origen poli-microbiano, que presentaron en común el aislamiento de Actinomyces europaeus, agente nodescrito hasta el momento en esta localización. A. europaeus fue identificado por la metodo-logía convencional, por espectrometría de masas por desorción/ionización asistida por matriz(MALDI-TOF MS) y por secuenciación del gen ARNr 16S. La sensibilidad antibiótica se evaluó porel método epsilométrico. Todos los aislados presentaron sensibilidad a penicilina, vancomicinay linezolid, mientras que la sensibilidad a clindamicina y eritromicina fue variable. La iden-tificación por MALDI-TOF MS permitió arribar a nivel de especie de forma rápida y confiabley dar una respuesta oportuna y efectiva, evitando el retraso en el tratamiento, lo que sueleincrementar la morbimortalidad del cuadro clínico.

Orquiepididimitis por Haemophilus parainfluenzae: caso clínico y revisión de la literatura / Orchiepididymitis due to Haemophilus parainfluenzae: case report and review of the literature / Orquiepididimite por Haemophilus parainfluenzae: caso clínico e revisão da literatura

Resumen Haemophilus parainfluenzae forma parte de la microbiota normal de la cavidad oral y del tracto respiratorio superior. Es un reconocido agente causal de endocarditis y, con menor frecuencia, de enfermedades como neumonía, sepsis, osteomielitis, celulitis, meningitis y gastroenteritis aguda. Aquí se presenta un caso de orquiepididimitis en un joven adulto donde H. parainfluenzae, confirmado por espectrometría de masas (MALDI-TOF MS), fue el único patógeno detectado. Este caso contribuye a valorar el rol de H. parainfluenzae como patógeno humano, aislado a partir de sitios diferentes del torrente sanguíneo y las vías respiratorias.

Abstract Haemophilus parainfluenzae is part of the normal microbiota of the oral cavity and the upper respiratory tract. It is a recognised causal agent of endocarditis and, less frequently, of diseases such as pneumonia, sepsis, osteomyelitis, cellulitis, meningitis, and acute gastroenteritis. A case of orchiepididymitis in a young adult is reported, where H. parainfluenzae, confirmed by mass spectrometry (MALDI-TOF MS), was the only pathogen detected. This case contributes to assess the role of H. parainfluenzae as a human pathogen, isolated from sites other than the bloodstream and the respiratory tract.

Resumo Haemophilus parainfluenzae faz parte da microbiota normal da cavidade oral e do trato respiratório superior. É um reconhecido agente causal de endocardite e, menos frequentemente, de doenças como pneumonia, sepse, osteomielite, celulite, meningite e gastroenterite aguda. Aqui é relatado um caso de orquiepididimite em um adulto jovem onde H. parainfluenzae, confirmado por espectrometria de massa (MALDI-TOF MS), foi o único patógeno detectado. Este caso contribui para avaliar o papel do H. parainfluenzae como patógeno humano, isolado de outros locais que não sejam a corrente sanguínea e o trato respiratório.

Microascus gracilis y otomicosis / Microascus gracilis and otomycosis

Microascus spp, teleomorfo de Scopulariopsis, es un hongo saprofito encontrado normalmente en suelo, alimentos, vegetales e incluso en ambientes interiores. Considerado un contaminante ambiental, se caracteriza por la resistencia intrínseca a los antifúngicos disponibles. Existen escasas referencias de infecciones por Microascus gracilis, asociándose como causa de eumicetoma o enfermedad diseminada en pacientes sometidos a trasplante pulmonar. Presentamos un caso de otomicosis por M. gracilis con el fin de considerar la búsqueda de hongos en los cultivos óticos y poner de relevancia el poder patógeno y colonizador de este agente.

Microascus spp, a teleomorph of Scopulariopsis, is a saprophytic fungus normally found in soil, food, vegetables, and even indoors. Considered an environmental pollutant, it is characterized by its intrinsic resistance to available antifungals. There are few references to infections by Microascus gracilis, associating it as a cause of eumycetoma or disseminated disease in lung transplant recipients. We present a case of otomycosis caused by M. gracilis, to consider the search for fungi in ear cultures and highlight the pathogenic and colonizing power of this agent.

An overview of mycetoma and its diagnostic dilemma: Time to move on to advanced techniques

The neglected tropical disease mycetoma can become extremely devastating, and can be caused both by fungi and bacteria; these are popularly known as eumycetoma and actinomycetoma respectively. The classical triad of the disease is subcutaneous swelling, multiple discharging sinuses and the presence of macroscopic granules. The present study aims to highlight the existing diagnostic modalities and the need to incorporate newer and more advanced laboratory techniques like pan fungal/pan bacterial 16S rRNA gene polymerase chain reaction (PCR) and sequencing, Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). It is important for the medical team to be aware of the various diagnostic options (both existing and future), so that diagnosis of such a debilitating disease is never missed, both by clinicians and microbiologists/pathologists. The newer diagnostic methods discussed in this article will help in rapid, accurate diagnosis thus facilitating early treatment initiation, and decreasing the overall morbidity of the disease. In the Indian context, newer technologies need to be made available more widely. Making clinicians aware and promoting research and development in mycetoma diagnostics is the need of the hour.

Etiologic identification and drug susceptibility analysis of a Citrobacter freundii food poisoning event / 中国热带医学

@#Abstract: Objective　In order to provide reference for emergency treatment of a sudden food poisoning incident, pathogen detection and drug resistance analysis were carried out. Methods　Diarrheal stool and surplus food samples were detected by GB 4789 and the isolates were identified by VITEK2 and matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), at the same time, the bacterial drug sensitivity test was carried out by using the method of microbroth dilution, and the isolates from different sources were molecularly classified by pulsed field gel electrophoresis (PFGE), and the correlation between the strains was analyzed by BioNumerics software. Results　Totaly 13 leftovers and 3 diarrhea patients were isolated and identified, The total number of colonies and coliforms in 7 leftovers samples all exceeded the standard, and Citrobacter freundii was detected in 5 leftovers and 2 stools. The results of drug sensitivity test showed that seven strains of Citrobacter freundii were sensitive to ciprofloxacin, tetracycline, chloramphenicol, gentamicin, amikacin, cefotaxime and meropenem, but completely resistant to ampicillin, and there was no multiple drug resistance. The results of pulsed field gel electrophoresis (PFGE) showed that 7 strains of Citrobacter freundii had the same PFGE bands and 100% homology, showing the same clone. Conclusions　This food poisoning incident was caused by Citrobacter freundii. The pathogen of food poisoning can be quickly and accurately determined by MALDI-TOF MS, which is beneficial to the early diagnosis and treatment of infectious diseases. It is suggested to strengthen the corresponding management, improve food safety awareness and prevent similar incidents.

Progress in protein extraction methods for fungal identification with MALDI-TOF MS / 中华微生物学和免疫学杂志

With the development of techniques for rapid microbial identification, MALDI-TOF MS has become an important tool for clinical identification of fungi. Problems such as the applicability and standardization of protein extraction methods have hindered the development of MALDI-TOF MS technology in the fungal field. This paper analyzed the complex structure of fungal cell walls, introduced the protein extraction methods recommended by MALDI-TOF MS commercial mass spectrometry systems, discussed the protein extraction methods for the identification of various genera of yeast-like fungi and filamentous fungi by MALDI-TOF MS, such as direct smear method, formic acid acetonitrile extraction method and magnetic bead grinding method, and summarized the current status and drawbacks of protein extraction methods in fungal identification by MALDI-TOF MS with a view to providing theoretical reference for subsequent research.

Identification and cluster analysis of Streptococcus suis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry / 公共卫生与预防医学

Objective To evaluate the feasibility of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for identification and cluster analysis of Streptococcus suis. Methods Eighteen clinical isolates biochemically identified as Streptococcus suis were pre-treated by smearing formic acid method and formic acid-acetonitrile extraction method. The identification results and protein profiles of MALDI-TOF-MS method were analyzed and compared. A self-constructed database of profiling with better pretreatment method was established, and cluster analysis was performed on the 18 strains of Streptococcus suis. At the same time, PFGE homology analysis was performed to compare the results of the two genotyping. Results Both pretreatment methods could accurately identify Streptococcus suis with scores above 2.1. The protein fingerprint of formic acid and acetonitrile extraction method had a smoother baseline, fewer miscellaneous peaks and more identifiable ion peaks. Comparison of the results of homology typing showed that the homology results of MALDI-TOF-MS were significantly different from those of PFGE. Conclusion MALDI-TOF-MS can accurately identify Streptococcus suis for the strains pre-treated with formic acid method or formic acid-acetonitrile extraction method, and the formic acid-acetonitrile extraction method can obtain a better protein mapping. MALDI-TOF-MS can play a certain role in typing, but it still has some limitations and cannot completely replace the PFGE.

Acid-fast bacteria as causative agents of skin and soft tissue infections: case presentations and literature review

ABSTRACT Acid-fast bacteria can be implicated in skin and soft tissue infections. Diagnostic identification can be challenging or not feasible by routine laboratory techniques, especially if there is no access to the Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) technology. Here, we present two cases of skin and soft tissue infections caused by two different acid-fast bacteria, Nocardia brasiliensis and Mycobacterium marinum. They both grew on Löwenstein-Jensen medium, Sabouraud agar medium and blood agar medium. Both bacteria appeared acid-fast by Ziehl-Neelsen stain and Gram-positive by Gram stain. The identification was performed by MALDI-TOF MS and gene analysis. N. brasiliensis and nontuberculous mycobacterium M. marinum represent rare pathogens that cause severe skin and soft tissue infections. Failure to identify the causative agent and subsequent inappropriate or inadequate treatment may lead to severe complications or even disseminated disease, especially in immunocompromised individuals.

Rapid bacterial identification by MALDI-TOF MS directly from blood cultures and rapid susceptibility testing: A simple approach to reduce the turnaround time of blood cultures

Abstract Antimicrobial treatment of patients with bloodstream infections (BSI) is time-sensitive. In an era of increasing antimicrobial resistance, rapid detection and identification of bacteria with antimicrobial susceptibility are critical for targeted therapy early in the disease course. This study describes the performance of a rapid method for identifying and testing antimicrobial susceptibility of Gram-negative bacteria performed directly from blood culture bottles in a routine microbiology laboratory. A total of 284, 120, and 24 samples were analyzed by rapid identification (Rid), rapid susceptibility testing (RAST), and rapid broth microdilution for polymyxin B (rMIC), respectively, and compared with standard methods. Our protocol was able to identify 93% of isolates at the species level using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We obtained 100% agreement for RAST compared to the standard method and 96% agreement for rMIC. Our protocol has proven to be an excellent tool for rapid identification of Gram-negative bacilli causing BSIs. It can also be used in microbiology laboratory routine along with RAST and faster polymyxin microdilution, especially for carbapenemase-producing bacteria, allowing for rapid, simple, accurate, and cost-effective diagnosis.

Application of MALDI-TOF Mass Spectrometry for Non-invasive Diagnostics of Mucopolysaccharidosis IIIA

Abstract Mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disorder (LSD) caused by deficiency of lysosomal N-sulphoglucosamine sulphohydrolase, which is one of four enzymes involved in heparan sulfate degradation. Traditional methods used for MPS IIIA diagnostics usually constitute of selective screening, based on the analysis of urinary glycosaminoglycans, further enzymatic assays in leukocytes, and mutation analysis. Nowadays, some LSDs, including mucopolysaccharidoses, can be precisely diagnosed by mass spectrometry-based techniques. Up to this date, there are no comprehensive studies of MPS IIIA diagnostics by MALDI-TOF analysis of free oligosaccharides in urine published. In the presented work, MALDI-TOF/TOF analysis of permethylated oligosaccharides was performed to obtain the set of glyco-biomarkers that together form the specific fingerprint of this disease. Early and accurate diagnostics of MPS IIIA is crucial to stabilize the progressive cellular damage and improve the overall well-being of patients.

Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry azole susceptibility assessment in Candida and Aspergillus species

BACKGROUND Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) allows rapid pathogen identification and potentially can be used for antifungal susceptibility testing (AFST). OBJECTIVES We evaluated the performance of the MALDI-TOF MS in assessing azole susceptibility, with reduced incubation time, by comparing the results with the reference method Broth Microdilution. METHODS Resistant and susceptible strains of Candida (n = 15) were evaluated against fluconazole and Aspergillus (n = 15) against itraconazole and voriconazole. Strains were exposed to serial dilutions of the antifungals for 15 h. Microorganisms' protein spectra against all drug concentrations were acquired and used to generate a composite correlation index (CCI) matrix. The comparison of autocorrelations and cross-correlations between spectra facilitated by CCI was used as a similarity parameter between them, enabling the inference of a minimum profile change concentration breakpoint. Results obtained with the different AFST methods were then compared. FINDINGS The overall agreement between methods was 91.11%. Full agreement (100%) was reached for Aspergillus against voriconazole and Candida against fluconazole, and 73.33% of agreement was obtained for Aspergillus against itraconazole. MAIN CONCLUSIONS This study demonstrates MALDI-TOF MS' potential as a reliable and faster alternative for AFST. More studies are necessary for method optimisation and standardisation for clinical routine application.

Descripción de un caso de sepsis abdominal por Desulfovibrio desulfuricans / Description of a case of abdominal sepsis due to Desulfovibrio desulfuricans

Resumen Desulfovibrio spp. son bacterias anaerobias estrictas, ubicuas en la naturaleza, quepueden formar parte del tracto gastrointestinal humano o animal, pero también son bacteriasambientales presentes en el suelo y en el agua. Pueden persistir de manera asintomática enel intestino o comportarse como patógenos oportunistas, asociados con bacteriemia primariae infecciones intraabdominales. El número de infecciones por Desulfovibrio spp. puede estarsubestimado debido a su lenta velocidad de crecimiento y a que muchos laboratorios no realizancultivos en anaerobiosis de manera rutinaria. Pruebas sencillas, como el examen de la movilidaden fresco y de la morfología celular en la coloración de Gram, sumadas a la presencia de SH2en agar SIM y a la observación de una fluorescencia roja a pH alcalino bajo luz UV, seríanindicativas de Desulfovibrio spp. Se describe el caso de una bacteriemia por Desulfovibriodesulfuricans en una mujer con cuadro clínico de sepsis abdominal por apendicitis gangrenosacon fallo multiorgánico.

Abstract Desulfovibrio spp. are strict anaerobes that are ubiquitous in nature. They can reside in the human or animal gastrointestinal tract and, as they are also environmental bacteria, may be present in soil and water. They can persist asymptomatically in the intestine or behave as opportunistic pathogens associated with primary bacteremia and intraabdominal infections. Several Desulfovibrio spp. infections may be underestimated due to their slow growth rate and because many laboratories do not routinely perform anaerobic cultures. Simple tests such as motility detection on a fresh subculture, Gram stain to confirm cell morphology, presence of H2S in SIM agar and production of a red fluorescence in alkaline pH under UV light would be indicative of Desulfovibrio spp.

Budding yeast in a child with acute leukemia: Nothing CRYPT(O)IC about it

A 13-year-old girl child with B-cell precursor acute lymphoblastic leukemia presented with complaints of fever, fatigue and left-sided iliac mass of 20 days duration. Preliminary blood culture from the peripherally inserted central catheter (PICC) demonstrated the presence of budding yeast cells. This is a rare form of “Disseminated cryptococcosis”. Budding yeast cells emphasizes the significance of various differentials of yeast in positive blood cultures bottles, as identifying Cryptococcus from gram stain can be complicated. This manuscript also highlights the presence of crystalloid geometric appearance like “Buckminsterfullerene”, which is derived from the mucopolysaccharide capsule in Cryptococcus. These structures are rarely observed, and in this case, are exceptionally remarkable.