The Isolation of Pathogenic Fungi from Sodium
Hydroxide-processed Sputum Specimens from
Patients Suspected to Have Tuberculosis
RONALD P. NEIMEISTER, B.S., NORMA T. PATTERSON, B.A., JAMES H. COCKLIN,
AND GEORGE F. HARADA,
Pennsylvania
Department
B.S.,
B.S.
of Health, Division of Laboratories, 2100 West Girard
Philadelphia, Pennsylvania 19130
Avenue,
ABSTRACT
Neimeister, Ronald P., Patterson, Norma T., Cocklin, James H., and Harada,
George F. The isolation of pathogenic fungi from sodium hydroxide-processed sputum specimens from patients suspected to have tuberculosis. Amer.
J. Clin. Path. 56: 201-203, 1971. Specimens received for tuberculosis bacteriology from 1966 to 1969 were processed by the sodium hydroxide technic.
Occasionally, these specimens produced fungi on the Lowenstein-Jensen and
Middlebrook-Cohn 7H-10 media. Attempts to determine whether these isolates
had pathogenic potential revealed that 23 of the 860 fungal isolates were
known pathogens. Allescheria boydii, Aspergillus fumigatus and Nocardia
asteroides were among those isolated. Although the ratio of pathogenic to
saprophytic fungal isolates in this study was small, it is significant to learn
that it is possible to isolate and identify some pathogenic fungi when using
this relatively lethal digestion procedure for routine mycobacterial isolation.
of pathogenic fungi from
sputum of persons with clinical symptoms
simulating pulmonary tuberculosis is common.'.° These etiologic agents include organisms of the genera Allescheria,*."^
Aspergillus,1'. *° Nocardia,".» Mucor, and
astomyces. Nocardia asteroides is the causative agent
of nocardiosis. However, due to its rate of
growth, pigmentation, and acid-fast elements, it is often considered to be a saprophytic mycobactenum and discarded without further identification."
Unhke the reports of previous investigat o r s - . " - " who isolated fungi prior to sputurn digestion, the present study concerns
fungal isolation from 4% sodium hydroxide-digested sputum specimens.
Material and Methods
T H E ISOLATION
From
1966
tQ
1969j
72905
specimens
w e r e e x a m i n e d for t h e specific i s o i a t i o n
and
mycobacteria. These cons i s t e d o £ 7 2 6 0 6 spuU>
153 urines> m
gas.
t r k w a s h i n g s > a n d 2 9 other specimens from
patients suspected to have tuberculosis inf ef rions
E a c h ' s p e c i m e n was digested and decontamlnated by the 4 % sodium
hydroxide
t e c h n k A p p r o x i m a t e l y e q u a l quantities of
identification
4 %
sodium
mixed
in
a
of
hydroxide
20
by
110
and
m m
sputum
were
s c r e w . C apped
w a s a g i t a t e d o n a test
. until k was relatively h o m o .
g e n e o u s . Decontamination was accomplished
a [ r o Q m t e m p e r a t u r e f o r 15 m i n . T h e s o l u .
tufae
This mixture
tube mixer
* T h e test tube mixer used was a Vortex Jr.
Mixer, Model K-500-J, from Scientific Industries
Inc., Queens Village, N. Y.
Received August 24, 1970; accepted for publicacation September 21, 1970.
201
202
A.J.C.P.— Vol. 56
NEIMEISTER ET AL.
Table 1. Numbers of Pathogenic Fungal Isolates and Their Occurrence with Mycobacteria
Type of Mycobacterium
Mycobacterium tuberculosis
Group I
Group II
Group I I I
Group IV
None
TOTAL
Allescheria
boydii
Aspergillus
fumigatus
—
—
—
2t
It
—
3
7
4
6
1*
—
3
1*
Candida
albicans
1
—
—
—
—
1
2
Mucor
species
—
—
Nocardia
asteroides
1
—
—
—
—
—
3
5
2
3
1
1
* Patient had isolation of M. tuberculosis and Group III organisms,
t Patient had isolation of Group II and Group III organisms.
tion was centrifuged at 1,100 X g- for 10
min. The supernatant was decanted into a
5% phenol-containing splash-proof container. Using bromothymol blue as the pH
indicator, the sediment was neutralized
with 1 N hydrochloric acid and then seeded
onto two Lowenstein-Jensen 8 slants and
one Middlebrook-Cohn 7H-10 agar slant
(BBL formulation). 12 ' 13 The LowensteinJensen slants were incubated in an 8% to
10% C 0 2 atmosphere 23 for the initial 24
hr., after which they were transferred to
an air-atmosphere incubator for 8 weeks.
The 7H-10 agar slants were kept in the
C 0 2 atmosphere for 4 weeks.8
All cultures were incubated at 37 C. and
read at weekly intervals. Those with mycobacterial-like growth were studied and the
species of Mycobacterium determined according to the Runyon scheme of classification. 16
Eight hundred and sixty processed specimens which had fungal growth as determined by direct examination were subcultured onto Sabouraud dextrose and potato
dextrose agar slants and incubated at 25 C.
All cultures were examined macroscopically
at weekly intervals for pigmentation and
rate and type of growth. Periodically, teased
preparations were emulsified in lacto-phenol cotton blue and examined microscopically. When definitive characteristic structures were present, the cultures were identified by this direct examination. When definitive structures were not observed, subcultures were made onto special slide cul-
tures 1 to enhance the production of typical
structures. When this morphologic study
failed to identify the species, various biochemical reactions, such as, carbohydrate
fermentation or assimilation, or both, urea
utilization, and casein hydrolysis were employed.
Results
As shown in Table 1, 23 or 2.67% of
the 860 cultures contained growth of reported fungal pathogens. Of the 23 cultures, 10 (43.48%) were isolated from patients harboring clinically significant mycobacteria. The remaining 13 cultures
(56.52%) were isolated from specimens
which did not contain detectable mycobacteria.
Table 1 also indicates that no known
pathogenic fungi were demonstrable with
either Group I or Group IV Mycobacteria.
Discussion
The concurrent presence of pathogenic
fungi with mycobacteria has been documented by many authors. 0 ' 10 ' 19 The lack of
a concomitant isolation of the detected
fungi with Group I and Group IV organisms does not negate these possible combinations. In all probability, this is due to
the limited number of fungal isolates
which survived the near-lethal digestion
procedure.
Ajello and associates2 have reported that
N. asteroides will survive a digestion pro-
August 1971
FUNCl FROM N A O H - P R O C E S S E D SPUTUM SPECIMENS
cedure similar to the one used in this study
in 84.4% of the known specimens. Survival
rates of the other pathogens identified in
this present study are unknown. It appears,
however, from these results that either the
organisms identified are relatively resistant
to the 4% sodium hydroxide digestion procedure or the fungi occur in large enough
numbers that few organisms survive the
relatively lethal procedure.
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