Zastosowanie dwustopniowego algorytmu w diagnostyce chorych z niskim poziomem toksyn A/B Clostridium difficile w kale potwierdzonym testem immunoenzymatycznym
[The use of two-stage algorithm in the diagnosis of patients with low levels of Clostridium difficile toxins A/B in feces confirmed by using enzyme immunoassay]

Streszczenie

Sześćdziesiąt dziewięć próbek kału pobranych od chorych z biegunką szpitalną, w których nie wykryto toksyn C. difficile testem EIA lub uzyskano wynik wątpliwy stosując rutynową diagnostykę, poddano dalszej analizie. Powodem wykonania dodatkowych oznaczeń był stan kliniczny pacjentów. Zastosowano metodę hodowlaną oraz RT-PCR. Zdiagnozowano 55 (78%) pacjentów z niskim poziomem toksyn w kale, zakażonych toksynotwórczym szczepem C. difficile, w tym 34 (62%) pacjentów zakażonych szczepem NAP1/BI/027 lub genetycznie pokrewnym. Zastosowanie dodatkowo dwustopniowego algorytmu diagnostycznego zwiększa szansę prawidłowego rozpoznania zakażenia C.difficile u pacjentów z niskim poziomem toksyn A/B w kale.

Abstract

Introduction: Clostridium difficile infection (CDI) is a serious problem in hospitalized patients. Rapid and accurate laboratory diagnosis is the key to reducing of CDI. The suboptimal sensitivity and specificity of many commercial enzyme immunoassays have limited their utility. The aim of this study was analysis of faecal samples obtained from patients with clinical evidence of CDI, with non-detectable or questionable result of toxins A/B C. difficile recognized by toxins A/B EIA test. Methods: A two-step algorithm for diagnostics of C. difficile infection (CDI) in patients with non-detectable or questionable result of toxins A/B C. difficile confirmed by C. difficile enzyme immunoassay (EIA) (Wampole, TOX A/B II, TechLab, USA) was used. Sixty nine faecal samples obtained from patients with nosocomial diarrhea were retested. All faecal samples were cultured on selective medium CLO C.difficile (BioMérieux, Francja). The positive samples on selective medium were tested by using Real Time-PCR (Xpert CD assay, Cepheid, Sunnyvale, CA, USA). Xpert CD assay is a real time multiplex PCR that can be used to detect toxigenic C. difficile strains and differentiate the C. difficile presumptive NAP1/BI/027 strain. All results when faecal samples were negative in culture growth on selective medium and result of EIA test were questionable was confirmed by use a RT-PCR test.Results: Among 69 faecal samples 56 were negative for toxins A/B using EIA test and 13 gave questionable results. By anaerobic culture 60 of 69 specimens yielded C. difficile isolates. Among 69 faecal samples 55 were positive using RT-PCR. Thirty four (62%) of patients was infected by presumptive C. difficile NAP1/BI/027. Conclusions: C. difficile testing by use of culture and Real Time PCR (RT-PCR) increases diagnostic yield in a hospital patients with non-detectable or low level of toxins A/B in stool samples of patients infected by toxigenic C. difficile strains including presumptive C. difficile NAP1/BI/027.