Introduction

• Invasive fungal infections caused by Aspergillus are one of the main

causes of morbidity and mortality in immunocompromised patients
• Examples: bone marrow transplant recipients and those undergoing
treatment for haematological malignancies.  The patients at the
highest risk are those with prolonged neutropenia.

• The mortality rate aspergilosis is very high  80%–95%, Difficulties

of diagnosing (IA)  nonspecific and late clinical signs & insensitivity
of conventional laboratory diagnostic.
• Early diagnosis is important, and sensitive molecular assays have the
potential to improve diagnosis.

• Diagnostic procedures include blood cultures for certain molds;

cultures, histopathological, and microscopic examinations of
appropriate diagnostic specimens; and imaging studies guided by
clinical findings.  But the problem is  the difficulties of obtaining
an adequate specimen, long-term cultures, and negative or delayed
results limit efficient and rapid microbiological diagnosis.
2 laboratorys test before molecular testing:
- Modern imaging techniques such as high-resolution computed
tomography (HRCT) can detect early signs that are consistent with
pulmonary aspergillosis (‘halo sign’ and macronodules)  Not
specific.
- Galactomannan (GM) antigen test  more than specific for detection
of circulating fungal cell wall components and fungal nucleic acids in
the blood and other body fluids
Objective
• In two decades, molecular diagnostic techniques have been
developed for IA, including polymerase chain reaction (PCR)

• this studywill bw evaluate the diagnostic potential of GM and real-

time PCR assay for IA diagnosis.
Material and method

Study design

- January 2011 and January 2012; febrile neutropenic patients with

haematologic malignancies were included in the present study.
• Criteria of neurtopenic: fever >38.5 °C and absolute neutrophil count
of < 0,5 x 10 −9  Serum samples were obtained twice weekly from
patients at increased risk of developing IA.
• Samples obtained from each patient with febrile neutropenic episode
at a risk of IA were tested by realtime PCR, both MAP and IHP tests, to
investigate the presence of Aspergillus DNA, and by ELISA to detect
the GM antigen.
DNA extraction from serum specimens

• What the steps?

- Performed in a Class II laminar flow cabinet .

- DNA extraction from serum samples was performed using a QIAamp
DNA mini kit (Qiagen, Hilden, Germany).
- One negative control was included in each extraction procedure.
- The extraction performance was monitored by testing both blinded
and known simulated positive control specimens.
- DNA samples were stored at 20 °C until PCR analysis.
DNA amplification and detection

• Use In vitro diagnostic test, MAP (Myconostica Ltd, Manchester, UK)

 based on molecular beacon real time PCR technology that targets
the multi-copy 18S rRNA gene included an internal amplification
control.

• PCR positivity was determined using a threshold of 39 cycles that

corresponded to a target sensitivity of ≤50 DNA copies, which was
approximately equivalent to one A. fumigatus genome.
In-house real-time PCR

• The IHP test was performed using a real-time PCR Mastermix

synthesized by Primer design (Southampton, UK).

• Used for IHP, which targeted the 28S rRNA, with 10 ll of the DNA
template in a final reaction volume of 50 ll.

• The amplification was performed using the following conditions: a 10

s denaturation step at 95 °C followed by 45 cycles of denaturation at
95 °C (15 s), annealing at 60 °C (60 s), and polymerization at 72 °C (20
s).
GM antigent test

• Sandwich ELISA for serum GM detection (Platelia Aspergillus; Bio-Rad,

Marnes-la-Coquette, France). The results were considered positive if
the optical index value in serum samples was ≥0.5.
Statistic analysis

• The associations between baseline patient characteristics and the

assay results were analysed using Fisher’s exact test, Wilcoxon’s rank-
sum test, and the Mann–Whitney U-test. All tests were two-sided,
and a P value of<0.05 was considered statistically significant. Analyses
were performed using SPSS