PCR, Invasive Aspergillosis, Simon N Hanung, Mikro UI, 2019

Simon Yosonegoro Liem
Wasista Hanung Pujangga

PPDS MIKROBIOLOGI KLINIK
FKUI 2019
Semester 2
Dosen Pengampu:
Dr. dr. Yeva Rosana, MS, SpMK(K)
RESULT
PATIENT DETAILS
• 99 patients  161 febrile neutropenic episodes
• 78 febrile neutropenic episodes in patient with IA (Invasive
Aspergillosis)o:
• 1 proven,
• 17 probable
• 60 possible
• 83 febrile neutropenic episodes in patient no IA (according to
the EORTC/MSG criteria).
• 29 negative control patients
• Total: 358 sera obtained from febrile neutropenic episodes
and 29 sera from negative controls.
• Antifungal administration was present in 106 of these
episodes.
Aslan, Muge, et al. "Potential of polymerase chain reaction and galactomannan for the diagnosis of invasive aspergillosis in patients with febrile neutropenia." Mycoses 58.6 (2015): 343-349.
MOLECULAR ASSAYS AND
THE GM ANTIGEN TEST
• Positive :
• GM = 26 (16.14%)  galactomannan (GM) antigen test
• MAP = 125 (34.91%)  MycAssay Aspergillus PCR
• IHP = 22 (6.14%)  in-house real-time PCR
• Negative :
• Both GM and MAP = NO false positive.
• IHP = 1 false positive.
• The characteristics of the patients with proven and
probable IA (n = 18) and MAP, IHP, and GM antigen
test results of them  Table 1.
MOLECULAR ASSAYS AND THE GM ANTIGEN TEST
• The performance of these tests  Table 2.

• MAP = higher sensitivity than IHP.
• Using a Ct (Cycles threshold) cutoff of ≤39 
9/18 patients with proven or probable IA had
a positive PCR result, and 4 of these patients
were also positive by IHP.
• Per test sensitivity of MAP was higher in episodes with

possible IA than in patients with proven-probable IA;
however, that difference did not reach statistically
significance [70% (42/60) vs. 50% (9/18); P = 0.135].
• To analyse the temporal relationship between the different
molecular assays, in each assay, the results that were positive
first for a proven or probable case were compared to the
definitive diagnosis on a CT scan (time point ‘zero’).
• Episodes of proven or probable cases who had a CT scan
prior to PCR and ELISA screening (n = 9) were excluded from
this particular analysis.
• The diagnostic means (molecular assays, GM, and the HRCT
scan) were compared to determine the earliest signs of IA in
each patient.
• In 6 episodes, MAP was positive before the other tests (mean
= 25.4 days; range = 23–30 days), but in three episodes, GM
was the first assay to be positive (mean = 7 days; range = 3–
12 days).
• PCR detected the earliest signs
of IA before diagnosis on HRCT.
• There was no significant
difference between the two
screening assays (P > 0.05).
COMPARISON OF MAP AND IHP
• According to the EORTC criteria :
(EORTC = European Organization for Research and Treatment of Cancer)
• seven IA episodes = positive by 2 molecular assays (MAC, IHP)
• 44 IA episodes = positive by MAP but negative by IHP (Table 4)
 no significant difference between 2 screening assays (P > 0.05).
• MAP detect of Aspergillus DNA  9 IA patients.
• The mean Ct values were calculated for the samples that were
positive by MAP (34.66 2.62 cycles) and by IHP (39.69 2.61
cycles; P < 0.05), and there was a significant difference
between the two PCR assay Ct values.
DISCUSSION
DISCUSSION
• The traditional diagnostic methods and the isolation of
fungal agents from clinical samples are the gold
standard, but blood cultures are almost always
negative for Aspergillus spp.
• Clinical sampling such as BAL or deep tissue biopsy is
generally not available because it requires invasive
procedures that are not feasible, particularly in
neutropenic patients.
• Thus, sensitive non-invasive diagnostic methods that
allow early diagnosis of IA required facilitating a better
prognosis.
• Aim of study : to compare and evaluate the GM
antigen test and molecular assays using a cohort of
patients at high risk of IA.
• Single positive results were considered positive for PCR
as recommended previously.
DISCUSSION
• The GM sensitivity rate was 23.07%, and the primary

variables that may have affected the false negative
rate included the presence of anti-Aspergillus
antibodies and the prophylactic or empiric use of
mould-active antifungal agents.
• It is a widely accepted belief that immunosuppressed
patients are unable to mount a significant antibody
response.
• Antifungal administration was present in 65.83% of these
episodes and GM performance may have been
negatively affected by this case.
DISCUSSION
• Molecular methods vs GM antigen test for detecting IA 
NO consistent results as to which test is the best.
• However, Suarez et al.  PCR highly sensitive and specific for
early diagnosis of IA in high-risk patients with haematological
disorders.
• In this study, the sensitivity of real-time PCR > GM test for IA
diagnosis, but a combination of the three could improve the
IA diagnosis.
DISCUSSION
• Study results suggest: combination of both methods
(GM and real-time PCR) could maximize performance,
but high costs.
• GM assay is a part of the EORTC criteria :
• GM (+) = probable cases of IA
• GM (-) = possible IA.
• This study : possible IA (70%) > proven-probable IA (50%)
by real-time PCR.
• It could be because of proven-probable episodes
number less than possible episodes.
DISCUSSION
• Better diagnostic strategies are required to facilitate
accurate diagnosis  TIMING IS IMPORTANT.
• In the present study, real-time PCR produced a positive
result:
• before HRCT at a mean of 25.4 days
• before GM at 7 days.
CONCLUSION
CONCLUSION
• The aim of the study  evaluate and compare the
performance of commercial real-time PCR, in-house real-
time PCR, and GM antigen tests using serum specimens.
• The use of real-time PCR assays with serum samples appears
to be a promising method for the diagnosis of IA, particularly
when used in association with other diagnostic tests such as
the GM antigen test and HRCT.
• Regular screening for GM and Aspergillus DNA could be
useful in high-risk patients to develop early treatment
strategies such as pre-emptive antifungal therapy.
• In all conditions, theGM and PCR results must be compared
with clinical, radiological, and laboratory findings.
CONCLUSION
• In the future, new molecular tests are expected to
become available for targeting several molds.
• A combination of biomarker tests will have the highest
diagnostic utility and would significantly improve the
early and accurate diagnosis of IA.
• However, large-scale evaluations of MAP are required
to determine its actual potential for IA diagnosis in high-
risk patients.
REFERENSI
1. Aslan, Muge, et al. "Potential of polymerase chain
reaction and galactomannan for the diagnosis of
invasive aspergillosis in patients with febrile
neutropenia." Mycoses 58.6 (2015): 343-349.
2. https://www.eortc.org/guidelines/
TERIMA KASIH
THANK YOU

PCR, Invasive Aspergillosis, Simon N Hanung, Mikro UI, 2019

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Simon Yosonegoro Liem

Wasista Hanung Pujangga

• The performance of these tests  Table 2.

• Per test sensitivity of MAP was higher in episodes with

• The GM sensitivity rate was 23.07%, and the primary

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