Treatment of sputum specimens T h e s p e c i m e n s w e r e d i g e s t e d a n d d e c o n t a m i n a t e d u s i n g the m e t h o d of P e t r o f f , a n d then inoculated onto2 s l a n t s of L o w e n s t e i η - J e n s e n m e d i u m a n d 1 s

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M Y C O B A C T E R I A O T H E R T H A N T U B E R C L E B A C I L L I IN S P U T U M S P E C I M E N S F R O M P A T I E N T S IN M A N A U S (AMAZONIA, B R A S I L ) .

J u l i a I g n e z S a l e m (*) M a r i a d e F a t i m a M a r ó j a (*) F r a n c i s c o F a r i a s d e C a r v a l h o ( * ) M a r i O t s u k a d e L i m a (*) A n t o i n e t t e F e u i l l e t (**)

SUMMARY

Mycobacteria which are most commonly isolated In palmo nan y secretions o tf patients

with respiratory symptoms living in the. State ofi Amazonas are given. The high percentage o{f isolates [25,4) and the species obtained indicate a strong relatio nò hip o£ con tamination o fa man by the environment. Among the species isolated, òeven o& them axe con-sidered potentially pathogenic mycobacteria. S-tnce some o$ thc-be species may interfere with present and farther vaccination programo against Tuberculosis and Leprosy, these

investigations indicate a necessity ofi carrying oat studies ff$ population awareness

against specific PPV's prepared farom the same species.

I N T R O D U C T I O N

M y c o b a c t e r i a o t h e r t h a n t u b e r c l e bacilli (MOTT) , a r e i s o l a t e d from human clinical specimens a t f r e q u e n c i e s that d i f f e r w i d e l y a c c o r d i n g to the g e o g r a p h i c r e g i o n . T h e m a -jority o f t h e s e isolates lack clinical s i g n i f i c a n c e , h o w e v e r some o f t h e M O T T m a y cause severe a n d e v e n l i f e - t h r e a t e n i n g d i s e a s e s in humans ( W o l i n s k y , 1 9 7 9 ) · T h e M O T T s p e c i e s most frequently c a u s i n g d i s e a s e in humans a r e M . a v i u m a n d M . i n t r a c e l l u l a r e , b o t h in t h e general p o p u l a t i o n ( W o l i n s k y , 1979) a n d in p a t i e n t s w i t h t h e a c q u i r e d i m m u n o d e f i c i e n c y syndrome - A I D S ( C o l l i n s , 1 9 8 6 ) . O t h e r MOTT s p e c i e s o f t e n a s s o c i a t e d wi t h p u l m o n a r y di_s ease are M . k a n s a s i i (Anderson e t a l ., 1 9 7 5 ) , M . szulgai ( D a v i d s o n , 1 9 7 6 ) , M . m a l m o e n s e (Collins e t a l . , 1986) a n d M . xenopi ( W o l i n s k y , 1 9 7 9 ) . Some s p e c i e s a r e m o r e o f t e n a s -sociated w i t h e x t r a p u l m o n a r y d i s e a s e such a s M . s c r o f u l a c e u m (Lincoln ε G i l b e r t , 1972) a s a cause o f s u b m a n d i b u l a r a d e n o p a t h y in c h i l d r e e n , a n d M . marinuim, M . u l c e r a n s a n d M . h a e m o p h i l u m c a u s i n g s k i n lesions ( M a c C a l l u m e t a l . , 1 9 ^ 8 ; Linell ε N o r d e n , 195*»; Jolly ε Seabury, 1 9 7 2 ; F e l d m a n e t a l , , 1 9 7 ^ ; Davis e t a l . , 1 9 8 2 ) . M . f o r t u i t u m a n d M . c h e l o n a e

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have been found as the most frequent cause of postchi rurgi cal c o m p l i c a t i o n s (Hand & SaŁj f o r d , 1 9 7 0 ; F o s t e r 'e t a l ., 1 9 7 8 ; W a l l a c e e t al . , 1 9 8 3 ) . The m a j o r i t y o f these m y c o b a c -terial s p e c i e s are e n c o u n t e d in the e n v i r o n m e n t ( K u b i n , 1 9 8 4 ; T s u k a m u r a , 1984) a n d c o n -sequently k n o w l e d g e about the species m o s t f r e q u e n t l y isolated in a r e g i o n is o f great interest to d o c t o r s , clinical l a b o r a t o r i e s a n d e p i d e m i o l o g i s t s .

P r e v i o u s l y w e r e p o r t e d the isolation of 8 mycobacterial s p e c i e s from 29,05ľ of hand and f o r e a r m w a s h i n g s of 241 v o l u n t a r e e s in the c i t y of M a n a u s A m a z o n i a (Salem e t al . , 1 9 8 8 a ) . In p u r s u i n g our i n v e s t i g a t i o ns o n the d i s t r i b u t i o n of m y c o b a c t e r i a l s p e c i e s in the A m a z o n i a n r e g i o n , w e h e r e i n report d a t a In M O T T isolates from s p u t u m s p e c i m e n s .

M A T E R I A L A N D M E T H O D S

P o p u l a t i o n s t u d i e d

Sputum s p e c i m e n s from 516 individuals w i t h clinical a n d / o r radiological e v i d e n c e of p u l m o n a r y t u b e r c u l o s i s were s t u d i e d . A m o n g these s u b j e c t s 144 h a d a p r e v i o u s history of t u b e r c u l o s i s w h i l e 372 w e r e first time c a s e s .

T r e a t m e n t o f s p u t u m s p e c i m e n s

T h e s p e c i m e n s w e r e d i g e s t e d a n d d e c o n t a m i n a t e d u s i n g the m e t h o d of P e t r o f f , a n d then inoculated o n t o 2 s l a n t s of L o w e n s t e i η - J e n s e n m e d i u m a n d 1 slant of L o w e n s t e i n­ J e r i sen p y r u v a t e , a s d e s c r i b e d by D a v i d e t a l ., 1 9 8 9 · B r i e f l y , the specimens w e r e treated w i t h an equal v o l u m e of 1.ON s o d i u m h y d r o x i d e for 20 m i n u t e s at 37°C In a cent r i fuge tube, An equal v o l u m e o f s t e r i l e d e s t i l l e d w a t e r is a d d e d . T h e tube is s t i r r e d , and t h e n it is c e n t r i f u g e d at 2 . 0 0 D χ g for 20 m i n u t e s . T h e sediment is n e u t r a l i z e d w i t h 4.0ľ s u l f u r i c a c i d using bromothymol blue a s a pH i n d i c a t o r . T h e n e u t r a l i z e d s e d i m e n t w a s then Inocula_ ted o n 2 L o w e n s t e i n - J e n s e n a n d 1 L o w e n s t e i η - J e n s e n p y r u v a t e s l a n t s ( 0 , 2 ml per s l a n t ) . T h e inoculated s l a n t s w e r e incubated at 37°C for 2 m o n t h s before c u l t u r e s w e r e d i s -c a r d e d as n e g a t i v e . P o s i t i v e -c u l t u r e s w e r e -che-cked for p u r i t y u s i n g the K i n y o u n a -c i d fast staining p r o c e d u r e (David e t a l . , I 9 8 9 ) , and w e r e identified a s d e s c r i b e d below.

B e c t e r i o l o g y s t u d y m

All strains w e r e e x a m i n e d in respect to their g r o w t h - r a t e , p i g m e n t p r o d u c t i o n a n d a b i l i t y to g r o w o n L o w e n s t e i n - J e n s e n m e d i u m at 3 0 ° , 3 8 ° a n d 42°C- G r o w t h inhibition in L o w e n s t e I n - J e n s e n m e d i u m s u p p l e m e n t e d w i t h 5ľ sodium c h l o r i d e (NaCL) , 500 yg/ml of p-ni t r o b e n z o i c a c i d (PNB) 3 0 . 0 yg/ml of D - c y c l o s e r i n e ( D C S ) , 2 5 0 . 0 yg/ml o f hydroxy lami ne ( N H ^ O H ) , tO.O yg/ml of t h i o s e m i c a r b a z o n e ( T b j ) , a n d 2 . 0 yg/ml of t i o p h e n c a r b o x i1i c a c i d hydrazlde (TCH) w e r e a l s o e x a m i n e d as r e c o m m e n d e d by CDC (Kent 6 K u b i c a , 1985) and the Pasteur Institute (David e t a l ., 1 9 8 9 ) . T h e biochemical p r o p e r t i e s s t u d i e d w e r e the pro duct ion o f n i a c i n , reduction o f n i t r a t e , h y d r o l i s i s o f tween 8 0 , room t e m p e r a t u r e a n d heated c a t a l a s e test (Kent S K u b i c a , 1985) a r y s u l f a t a s e , u r e a s e , Bgl ucosi d a e , a n d p e n i -cilinase (Lιvy-Frιbaul t e t al . , 1 982) . W h e n 11 w a s consi d e r e d necessary , the m y c o l i c acid c o m p o s i t i o n w a s studied as d e s c r Í b e d before , Daffι e t a l .( 1 9 O 3 ) a n d David e t a l . ( 1 9 8 9 ) .

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R E S U L T S

F r o m the p u l m o n a r y s e c r e t i o n s of the 5 1 6 p a t i e n t s s t u d i e d , 125 s t r a ν n s o f M . t u b e Ł culosis a n d 1 3 9 MOTT s t r a i n s w e r e i s o l a t e d . T h r e e o f t h e M . t u b e r c u l o s i s isolates w e r e mixed c u l t u r e s 1 w i t h M . a v i u m - i n t r a c e l l u l a r e a n d 2 w i t h H . t e r r a e . Eight o f the Mott

isolates w e r e m i x e d c u l t u r e s : Ί w i t h M . a v i u m - i n t r a c e l1u l a r e a n d M . t e r r a e ; 2 w i t h M . terrae and M . g o r d o n a e ; 1 w i t h M . f o r t u i t u m a n d M . t e r r a e , a n d 1 m i x e d M.avium-intracej_ lulare a n d H . g o r d o n a e .

In this series o f p a t i e n t s , t h e f r e q u e n c y of isolation of MOTT w a s thus 26.16Ύ ( 1 3 9 / 5 1 6 ) a n d their d i s t r i b u t i o n is d e p i c t e d in T a b l e 1. T h e s p e c i e s most frequently i so-lated w e r e M . a v i u m - i n t r a c e l l u l a r e a n d M . f o r t u i t u m (7.36ľ a n d 6.20ľ o f all specimens c u l t u r e d ) .

Among the 10 s p e c i e s i d e n t i f i e d , 7 w e r e o f p o t e n t i a l l y p a t h o g e n i c (opportunistic) m y c o b a c t e r i a ; M . avi urn- intracel 1 u l a r e (38 st ra Ϊ n s­ 7 . 3 6 % ); M . fortui t u m ( 3 2 strai n s­ 6. 2 0 ^ ; M. a s i a t i c u m (8 s t r a i n s - 1 . 5 5 % ); M . s c r o f u l a c e u m (7 s t r a i n s -1 . 3 6 ľ ) ; H . c h e l o n a e (2 strains-0. 3 9 ľ ) ; M . szulgai (1 strain-0.19%) ; a n d M . malimoense (I st ra i n-0 ,1 9ľ) · Nine of the i so lates c o u l d n o t be i d e n t i f i e d t o the s p e c i e s l e v e l , a n d their study is under w a y .

D I S C U S S I O N

This study s h o w e d that M O T T w e r e often isolated from s p u t u m s p e c i m e n s s i n c e these organisms w e r e found in 131 o u t of 5 1 6 s p e c i m e n s e x a m i n e d ( 2 5 ­ 3 9 ľ ) . That MOTT may b e widely d i s t r i b u t e d in the region w a s reported b e f o r e in a study of hand a n d forearm washings w h e r e the f r e q u e n c y o f isolates w a s 29-05Ύ (Salem, ) 9 8 8 ) . In b o t h studies M .

a v i u m - i n t r a c e l l u l a r e w a s t h e s p e c i e s m o s t f r e q u e n t l y e n c o u n t e r e d .

The overall f r e q u e n c y of M O T T isolates in M a n a u s w a s s i g n i f i c a n t l y h i g h e r than re ported in F r a n c e w i t h 3, 7 ^ ľ of all c u l t u r e s ( D a v i d , I986) a n d e l s e w h e r e i n B r a s i l - R i o d e J a n e i r o , w i t h 6.5Ύ o f all c u l t u r e s ( A n d r a d e , 1 9 7 6 ) . In the s o u t h e r n part of Brasil MOTT has been s e l d o m r e p o r t e d ( G o n t i j o e t al ., 1 9 7 1 ; C o r r e a , 1 9 7 1 ; F o n s e c a & G o n t i j o , 1971*;

B a r r e t o , I S 8 0 ) .

T h e f r e q u e n t isolation of t h e s e MOTT in M a n a u s m a y b e r e l a t e d to environmental con d i t i o n s a s t h e city is l o c a t e d in the A m a z o n i a n forest next to the A m a z o n i a n r i v e r , at 03 d e g r e e s of latitude a n d 60 d e g r e e s o f l o n g i t u d e . T h e c l i n i c a l a n d immunological signifi cance of e x p o s u r e to these m y c o b a c t e r i a w a s n o t e s t a b l i s h e d d u r i n g th i s s t u d y , however skin infection c a u s e d by M . a v i u m - i n t r a c e l l u l a r e a n d M . f o r t u i t u m w e r e reported before (Salem e t a l . , 1988b}. C o n s e q u e n t l y , this a n d o u r p r e v i o u s s t u d i e s ( S a l e m e t a J . , 1988a and 1 9 8 8 b ) ) , indicate that f u r t h e r i n v e s t i g a t i o n s o n t h e d i s t r i b u t i o n o f MOTT in the en v i r o n m e n t , a n d o f their clinical a n d immunological sigi ificance in m e n a r e necessary a s they m a y induce immunity r e a c t i o n s interfering w i t h c u "rent a n d further vaccination

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C o n s i d e r i n g t h e r e l a t i v e l y h i g h e r n u m b e r o f s p u t u m s p e c i m e n s e x a m i n e d , it is w o r t h m e n t i o n i n g that M . b o v i s a n d M . a f r i c a n u m w e r e n o t i s o l a t e d .

A C K H O W L B D G E M E N T S

W e t h a n k H u g o D a v i d (Unite d e la T u b e r c u l o s e e t M y c o b a c t é r i e s , Institut P a s t e u r , Paris) f o r his i n t e r e s t , c r i t i c i s m , a n d s u p p o r t d u r i n g t h e s e i n v e s t i g a t i o n s .

R E S U M O

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T a b l e 1. M y c o b a c t e r i a l s p e c i e s , a n d their d i st r i but ion a m o n g 1 31 i so lates f r o m 5 1 6 s p u t u m s p e c i m e n s .

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a s i a t i c u m a v i u m i n t r a -cel lulare c h e l o n a e f l a v e s c e n s f o r t u i t u m g o r d e n a e m a l n o e n s e s c r o f u l a c e u m szulgai t e r r a e

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1 27

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Total 38 2 1 32 12 1 7 1 20 131

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(Aceito para publicação em 14.04.1989)

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