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Метод MALDI-TOF MS идентифицирует оомицет, вызывающий питиоз

By LabMedica International staff writers
Posted on 13 Dec 2018

Масс-спектрометр UltrafleXtreme MALDI-TOF/TOF (фото предоставлено Bruker Daltonics).

Питиоз является инвазивным, трудно поддающимся лечению и опасным для жизни инфекционным заболеванием, вызываемым Pythium insidiosum, членом уникальной группы грибоподобных микроорганизмов – оомицетов. О случаях заболевания питиозом во всем мире сообщается всё чаще.
В последнее десятилетие времяпролетная масс-спектрометрия с матрично-активированной лазерной десорбцией/ионизацией (MALDI-TOF MS) стала новым мощным диагностическим инструментом, облегчающим клиническую идентификацию многих патогенных микроорганизмов, включая бактерии и грибы.

Ученые из Университета Махидола (Бангкок, Таиланд) выделили в общей сложности 13 штаммов P. Insidiosum от образцов, полученных от 8 человек и 5 животных с питиозом из разных географических мест. Все организмы выдерживали на декстрозном агаре Сабуро при 25 °С. Несколько небольших порций колонии каждого организма были перенесены в 50-миллилитровую колбу, содержащую 10 мл бульона Сабуро с декстрозой, и инкубировались при 37 °С в течение одной недели перед сбором грибкового материала для экстракции белка.

Извлеченный из собранных организмов белок был нанесен на чистую матовую стальную пластину-мишень в 40 копиях (для создания базы данных MS MALDI-TOF для P. insidiosum) или в 5 копиях (для оценки MALDI-TOF MS при идентификации P. insidiosum) и высушен на воздухе при комнатной температуре перед обработкой. После сушки матричного раствора при комнатной температуре образец был немедленно проанализирован с использованием масс-спектрометра Bruker ultrafleXtreme. Матрицы геномной ДНК (gDNA) были извлечены из организмов и подвергнуты мультиплексной полимеразной цепной реакции для определения однонуклеотидного полиморфизма. (PCR).

Команда сообщила, что метод MALDI-TOF MS точно идентифицировал все 13 протестированных штаммов P. insidiosum на уровне видов. Масс-спектры P. insidiosum не соответствовали никаким другим микроорганизмам, включая грибы (т.е. виды Aspergillus, Fusarium и виды грибов класса Zygomycetes), которые имеют сходную микроскопическую морфологию с этим оомицетом. Методы биотипирования на основе MALDI-TOF MS и рДНК последовательно разделили P. insidiosum на три группы: Clade-I (американские штаммы), II (азиатские и австралийские штаммы) и III (в основном тайские штаммы).

Авторы пришли к выводу, что метод MALDI-TOF MS был успешно использован для идентификации и биотипирования P. insidiosum. Полученная база масс-спектральных данных позволит клиническим микробиологическим лабораториям, которые в достаточной мере оборудованы масс-спектрометрами MALDI-TOF, надежно идентифицировать P. Insidiosum без каких-либо специфических для патогена реагентов (антигенов, антител или праймеров). Исследование было опубликовано в декабрьском выпуске International Journal of Infectious Diseases за 2018 год.

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