Aspergillus fumigatus detection by PCR in broncho-alveolar fluid

D2114

Aspergillus fumigatus

détection

by

PCR

in

Bronchoalveolar Lavag

e Flui

d

M.P. HAYETTE

, P

. BOLLAND

, B

. EVRAR

D an

d P

. D

E MOL,

*

Univ. Hosp

. Liège

, Belgiu

m

Introduction

Aspergillus fumigatus is an opportunisme nosocomial pathoge n whic h causes sévère to fatal infectio n i n neutropeni c patients . Th e ris k o f invasive aspergillosis (IA) increase s wit h th é duratio n o f neutropeni a and can reach 70% after 5 weeks of neutropeni a ( 1 ) . The diagnostic of I A is one of th é mos t difficul t an f th é gol d standar d i s still base d o n cultur e and/o r histolog y result s whic h sensitivft y i s ver y low. Recently , molecula r biolog y method s wer e developpe d t o diagnose aspergillosis in urines, sérum , bloo d an d BAL . We used a neste d PCR-base d amplificatio n o f A . fumigatus DNA in BAL fluids that target s th é genes-encodin g alkalin e protease s o f th é fungus to evaluate thé usefullness of PC R t o diagnos e IA .

Abstract

The usefullness of a nested PCR for détectio n o f Aspergillus fumigatus DN A wa s evaluated in bronchoalveolar lavag e (BAL ) flui d durin g a period of tw o year s (1996 -1998). Th e ai m o f th é stud y wa s t o asses s th é rôl e o f PC R i n diagnosin g invasiv e pulmonarry aspergillosis (IPA). Methods : a nested PCR-based amplplification of fragments of genes-encodin g alkalin e protease s fro m Aspergillu s fumigatu s wa s used to testt 16 7 BA L samples . A H sample s wer e checke d fo r th é absenc e o f amplification inhibitors. Madical , radiological , microbiologica l record s an d autops y findings were reviewed for assessin g invasiv e aspergrllosis . Ai l successiv e patient s investigated by BAL were included in thé study. The y wer e distribute d i n thre e groups: A : prove n o r probabl e aspergillosi s (n=11), ; B , colonizatio n (n=é) ; C : n o évidence of IP A (n=154) . PC R result s wer e compare d t o cultur e détectio n a s gol d standard and to clinical data . Results : BA L fluid s fro m 1 0 patient s o f grou p A were PCR positive. On e cas e wa s falsel y négative . Amon g grou p B , on e cas e wa s PC R positive, an d th é secon d on e PC R négativ e bu t ha d négativ e BA L culture s (onl y culture positive sputum). N o fals e positiv e wa s detecte d amon g grou p C . Comparing to culture, sensitivit y wa s 91% , specificity , 100% , positiv e prédictiv e value, 100% , an d négativ e prédictiv e value , 99% . Conclusions : Aspergillu s fumigatus PCR in BAL fluid was an accurate test t o diagnos e cultur e négativ e patients with IPA and to confirm culture positive samples; howeve r i t does' t mak e différence between infection and colonization. 2 . I t is an appropriate test t o exclud e Aspergillus infectio n in patients at ris k o f invasiv e illness .

Bibliographv

•LRibaud P., Esperou-Bourdea u H. , Devergi e A. , Gluckma n E . Aspergillose invasive et allogreff e d e moelle . Path . Biol. , 1994 , 42 ,

652-655.

•2.Tang C., Holde n D. , Aufauvre-Brown , Cohe n J . Th e détectio n o f Aspergillus spp. b y th é polymeras e chai n reactio n an d i l s évaluation in bronchoalveolar lavag e fluid . Am . Rev . Resp . Dis. , 1993 , 148 , 1313-1317. Marie-Pierre Hayette Microbidogy department, Universit y Hospita l 4000 Liege.Belgium. mphayette@ulg.ac.b e

Methods

•Clinical data : ai l patient s undergoin g bronchoscop y a t th é universit y hospita l of Lièg e betwee n 199 6 an d 199 8 wer e include d i n th é stud y an d distribute d i n three groups: •A: prove n o r probabl e aspergillosi s ( n = 1 1) •Bicolonization (n=2) •C: n o évidenc e o f aspergillosi s (n=154 ) •BAL fluid spécimens: ai l lavage s collecte d b y th é microbiologica l laborator y were included in thé study and stored at -20° C befor e processing . Microbiological examinatio n wa s directl y performe d o n ever y sampl e an d results recorded. •DNA extraction: adapte d fro m Tan g e t coll . (2) . •BAL fluid (250/ul) + extraction buffer (250/j\) containing proteinase K (10 0 //g): 65° C fo r 6 0 m n •extraction once with phenol:chloroform:isoamylalcool (25:24:1 ) an d the n with chlorofomrisoamylalcool (24:1) . •Précipitation of DN A b y ethano l •Nested PCR: Th e targe t DN A correspond s t o genes-encodin g alkalin e proteases from A. fumigatus (2). External primers : alp l 1 and alp12 (2 ). Internai primers : alp l 3 (5'-CTGGCATACAACGCCGCT-3') alp 14 (5'-TTGTTGATCGCAACC-3') Product lengt h afte r amplification : 52 7 bas e pairs . Mix, (50> l volume) : 10m M TRIS-C I a t p H 8,3 , 5 0 m M KC I an d 1. 5 m M MgCI2 with 100 pmol o f bot h primer s an d 1.2 5 U Taq polymerase (Takara Taq, Japan) . Thermal cyclin g conditions : 3 0 cycle s a t 94° C fo r 3 0 sec. , 63° C fo r 45sec., an d 72° C fo r 2 min. Products of PC R : analyzed on 1.5% agarose gels, staine d wit h ethidium bromide and visuallized by UV transillumination. case UNDERLYiNG DISEASE GROUP A: AP I o r probabl e AP I fn=11 i Hepatic transplantatio n COPB corticoids dépenden t Hématologie malignancy Cardiac transplantatio n Arthritis COPB cortcoids dépendent

Results

COPB corticoids dépendent Bone marrow transplantation Hématologie malignancy Hématologie malignancy o histolog y ye s es ye s io histolog y ye s GROUP B: colonisatio n (n=2 J case UNDERLYING DISEASE j 1 Mitra l valvulopath y i 2 COP B (n o corticoids } i COPB: Chroni c obstructiv e pulmonar y hronchitis . GROUP C: n o évidenc e o t asperoillosi a fn=154 ) n UNDERLYIN G DISEAS E JP A 154 Respiratory disease 9 10111 2 1 3

Sensitivitv

and

soecificitv

of PC

R

IPA NO IPA

PCR-1

155 156 PCR+ 1 0 11 1 15 6 1 1 16 7 Sensitivity: 91% Specificity: 100 % Prédictive values négative 99% positive 100% Figure 1. Amplification products on ethidium stained agarose gel. Above : tane s 1-5,7,8 : négative BAL. Lane s G , 9 : patien t 7 from group A. Lane:10,11: A . fumigatus DNA. Lane s 12,13 : négative sample (water). Betow : beta-globin e amplification of BA L sample s to assess thé lack of amplificatio n inhibitors .

Conclusions

1 .A. fumigatus PC R in BAL fluid was an accurate test t o diagnos e cultur e négativ e patient s wit h IP A an d t o confirm culture positive samples. However i t doesn' t mak e différenc e betwee n infectio n an d colonization . 2. Thi s PC R is an appropriate method to exclude Aspergillus infectio n i n patient s a t ris k o f invasiv e illness .