Development and optimization of quantitative PCR for the diagnosis of invasive aspergillosis with bronchoalveolar lavage fluid

Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
BMC Infectious Diseases (Impact Factor: 2.61). 02/2008; 8(1):73. DOI: 10.1186/1471-2334-8-73
Source: PubMed


The diagnosis of invasive pulmonary aspergillosis (IPA) remains challenging. Culture and histopathological examination of bronchoalveolar lavage (BAL) fluid are useful but have suboptimal sensitivity and in the case of culture may require several days for fungal growth to be evident. Detection of Aspergillus DNA in BAL fluid by quantitative PCR (qPCR) offers the potential for earlier diagnosis and higher sensitivity. It is important to adopt quality control measures in PCR assays to address false positives and negatives which can hinder accurate evaluation of diagnostic performance.
BAL fluid from 94 episodes of pneumonia in 81 patients was analyzed. Thirteen episodes were categorized as proven or probable IPA using Mycoses Study Group criteria. The pellet and the supernatant fractions of the BAL were separately assayed. A successful extraction was confirmed with a human 18S rRNA gene qPCR. Inhibition in each qPCR was measured using an exogenous DNA based internal amplification control (IAC). The presence of DNA from pathogens in the Aspergillus genus was detected using qPCR targeting fungal 18S rRNA gene.
Human 18S rRNA gene qPCR confirmed successful DNA extraction of all samples. IAC detected some degree of initial inhibition in 11 samples. When culture was used to diagnose IPA, the sensitivity and specificity were 84.5% and 100% respectively. Receiver-operating characteristic analysis of qPCR showed that a cutoff of 13 fg of Aspergillus genomic DNA generated a sensitivity, specificity, positive and negative predictive value of 77%, 88%, 50%, 96% respectively. BAL pellet and supernatant analyzed together resulted in sensitivity and specificity similar to BAL pellet alone. Some patients did not meet standard criteria for IPA, but had consistently high levels of Aspergillus DNA in BAL fluid by qPCR.
The Aspergillus qPCR assay detected Aspergillus DNA in 76.9% of subjects with proven or probable IPA when the concentrated BAL fluid pellet fraction was used for diagnosis. There was no benefit from analyzing the BAL supernatant fraction. Use of both extraction and amplification controls provided optimal quality control for interpreting qPCR results and therefore may increase our understanding of the true potential of qPCR for the diagnosis of IPA.

Download full-text


Available from: Robert Hackman,
  • Source
    • "The DNA amount in the samples was quantified using a Pico 100 Picodrop Microliter Spectrophotometer (Picodrop Ltd., Hinxton, UK) according to the manufacturer's instructions. DNA quality was assessed in all samples by a Taqman PCR targeting a 155-base-pair fragment of the human 18S rRNA gene as previously described [12] with minor modifications (Table 2). Plasmids containing phocid herpesvirus 1 (PhHV-1) sequences were added to each sample as internal controls to exclude sample inhibition. "

    BioMed Research International 03/2015; Volume 2015 (2015)(Article ID 938721). · 2.71 Impact Factor
  • Source
    • "In haematology patients, Aspergillus galactomannan (GM) enzymelinked immunosorbent assay (ELISA) and nucleic acid detection by PCR are rapid, sensitive diagnostic tests for IPA (Khot et al., 2008; Maertens et al., 2009). The clinical utility of GM and Aspergillus PCR testing of serum/blood samples has been confirmed in a randomised controlled trial (Morrissey et al., 2013). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Interpretation of Aspergillus galactomannan (GM) and PCR results in bronchoalveolar lavage (BAL) fluid for the diagnosis of invasive pulmonary aspergillosis (IPA) in patients with haematological malignancies requires clarification. A total of 116 patients underwent BAL for investigation of new lung infiltrates: 40% were neutropenic, 68% and 36% were receiving mould-active antifungal agents and β-lactam antibiotics. The diagnosis of proven IPA (n = 3), probable IPA (n = 15) and possible invasive fungal disease (IFD, n = 50) was made without inclusion of GM results. BAL GM (at cut-off of 0.8) had lower diagnostic sensitivity for IPA than PCR (61% versus 78%) but higher specificity (93% versus 79%). Both tests had excellent negative predictive values (85–90%), supporting their utility in excluding IPA. The use of BAL GM and PCR results increased the certainty of Aspergillus aetiology in seven probable IPA cases where fungal hyphae were detected in respiratory samples by microscopy, and upgraded 24 patients from possible IFD to probable IPA. Use of BAL GM and PCR improves the diagnosis of IPA.
    Diagnostic microbiology and infectious disease 07/2014; 79(3). DOI:10.1016/j.diagmicrobio.2014.03.020 · 2.46 Impact Factor
  • Source
    • "There are series of recommendations for performing BAL fluid, according to European Respiratory Society [22] [23]. Conventional mycological techniques like culture and histological examination of BAL fluid are the most commonly used ones for the diagnosis of these infections and the detection of the fungal cell wall antigen can be performed by galactomannan (GM) test [24] and PCR [25] in BAL samples. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Invasive fungal infections are a major cause of morbidity and mortality in immunocompromised patients. Because the etiologic agents of these infections are abundant in nature, their isolation from biopsy material or sterile body fluids is needed to document infection. This review evaluates and discusses different human body fluids used to diagnose fungal infections.
    08/2013; 2013(4):698325. DOI:10.1155/2013/698325
Show more