TITLE PAGE Title: Comparison of Sedimentation and Flotation ...

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TITLE PAGE

Title:
Comparison of Sedimentation and Flotation Techniques for Identification of
Cryptosporidium
sp. o
ocysts from stool specimens

of humans.

Authors:

1.

Shaista Masarat; (Ph.D to be awarded)


Sr. Assistant Professor,
D
epartment of Z
oology, S.P.College, M.A.Road, Srinagar

2.

Dr. Fayaz Ahmad (Ph.D)

Associate Professor, P.G.Department of Zoology, University of Kashmir

3.

Bashir Ahmad Sofi (M.Sc Microbiology)

Tutor, Department of Microbiology, SKIMS, Hospital, Srinagar.


Correspondence

Shaista

Masarat

Department of Zoology, S.P.College, Srinagar
-
190001

Email:
Shaistamasarat@gmail.com

Contact No
-

9906522533


Efficiency of

Sheathers flotation and Formalin
-
ethyl acetate techniques

for Identification of

Cryptosporidium
sp. Oocysts

in routine parasitological examination





2


Title:
Comparison of Sedimentation and Flotation Techniques for Identification of
Cryptosporidium
sp. oocysts from stool specimens of humans.

M.
Shaista
1
;
A.
Fayaz

2
and
S
.A.

Bashir
3
.

1
Department of Zoology, S.P.College, M.A.Road, Srinagar
;
2
P.G.Department of Zoology,
University of Kashmir
;
3
Department of Microbiology, SKIMS, Hospital, Srinagar

Correspondence

Shaista Masarat

Department of Zoology, S.P.College, Srinagar
-
190001

Email:
Shaistamasarat@gmail.com


______________________________________________________________________________

Abstract

Due to increasing numbers of patients with
Cryptosporidium
, it is important for the physician
and
clinical laboratory to be aware of the appropriate diagnostic techniques necessary for
recovery an
d identification of organism.
Cryptosporidium

is found in
gastrointestinal tract,
the
exa
mination of stool specimens is

noninvasive procedure and will provide

better opportunities
for recovery of organism.

We compared two procedures for the demonstration of the organism in
stool specimens. Of 456 stool specimens

tested by both techniques, Sheather sucrose flotation
(SSF)
identified 89

(19.5%) as positive for

Cr
yptosporidium

sp. oocysts. Formalin
-
ethyl acetate
sedimenta
tion (F/EA) plus
modified cold Kiny
oun acid
-
fast stain (MCK) of

sediment
identified
93

(
20.3
%) as positive for
Cryptosporidium
sp. oocysts. Of 456 samples, 182 were positive for
oocysts by both te
chniques and 274 samples were negative by both techniques. A total of 182
(39.9 %) were
positive by one technique or

other
; 107

(23.4 %) were positive by both
techniques.
37

specimens were positive by SSF but n
egative by F
EA plus MCK,
and 38

3


specimens were

positive by F
EA

plus MCK but negative by SSF. The discrepancies between the
two techniques occurred in stool specimens that

contained rare to
f
ew oocysts. We concluded
that F
EA plus MCK of
sediment

was as effective in the concentration and identification
of
Cryptosporidium

sp. oocysts as SSF. F/EA plus MCK
may be advantageous as

single
concentration method for general parasitology when
Cryptosporidium
sp. is to be identified.

Key Words
:
Cryptosporidium
; Sheathers flo
tation; Formalin
-
ethyl acetate.


Introdu
ction

Cryptosporidium
, an intracellular protozoan has been reported as an important cause of
diarrhea in animals and humans worldwide and the potential for signif
icant morbidity and
mortality (
Meisel
et al
.
,

1976
). Until recently, human infections by the c
occidian parasite
Cryptosporidium

sp. have been reported infrequently. The first two documented cases of
cryptosporidiosis were reported
in 1976 (
Meisel
et al.
,

1976;

Nime
et al.
,

1976
)
.

Case studies of
this disease link its occurrence with exposu
re
to inf
ected animals (
Current
et al
.,
1983; Reese
et

al
.
,
1982
).

The diagnosis of cryptosporidiosis was based originally on finding the organism in
histological sections of intestinal tissue obtained by biopsy. Unstained and iodine
-
stained wet
mounts of directly
prepared or concentrated fecal material or permanent staining techniques (e.g.
trichrome or iron hematoxylin) failed to demonstrate the organism.
Cryptosporidium

sp. oocysts
are small and may easily be confused with yeasts. In 1980 oocysts were fo
und in hu
man stool
specimens (
Tzipori
,

1980
). In 1981 the acid
-
fast nature of the organism was demonstrated
(
Heniksen and Pohlez 1981
). Since this time, new and varied concentration
and staining
techniques (
Bronsdon
,
1984
) for demonstrating the organism in stools ha
ve been
reported;

4


without
tissue biopsy techniques and allowing routine diagnosis by examination of freshly
passed or preserved feces.

Little data are available as to the most effective routine techniques for the recovery and
identification of
Cryptospori
dium

sp.
We compared two concentration techniques currently
recommended for use in the diagnosis of
Cryptosporidium

sp. infect
ions: (
i
) The Formalin
-
ethyl
acetate (F
EA)

(

Garcia and Shimizer 1981
) concentration procedure combined with a modified

cold Kinyo
un acid
-
fast stain (MCK) of the sediment and (ii)

Shea
ther sucrose flotation (SSF)
(
Sheather 1923
), a specialized procedure

adapted from veterinary science to clinical
microbiology
laboratories (
Reese et al.1982
) and currently recommended for the di
agnosis

of
cryptosporidiosis (
Ma and Soave 1983
).

Materials and Methods

Specimen collection
. 456 stool samples were chosen to evaluate the two methods. The fresh
stool specimens were placed into 10% buffered Formalin preservative. The specimens in
Formalin were d
ivided into two par
ts. One part was processed by F
EA sedimentation, and the
other part was processed by SSF. The two concentration procedures and examination of slides
were performed independent.
T
he fresh specimens
were permanently stained and
examined
un
der oil immersion magnification for the presence of other parasites.

F
EA and MCK
. The F
EA concentration procedure was performed, 4 to 5 ml of the Formalin
-
treated stool specimen was washed in 10% For
malin
-
saline, and the sediment
collected by
centrifugatio
n at 650 x g for 5 min, was suspended in 8 ml of Formalin
-
saline
-
3 ml of ethyl
acetate. This mixture was vortexed for 3 min and centrifuged at 500 x g for 5 min, resulting in
four layers: a layer of ethyl acetate, a plug of debris, a layer of Formalin
-
sali
ne, and sediment.

The plug was rimmed with an applicator stick, and the top three layers were decanted. One
5


portion of the sediment was placed on a microscope slide and dried for the acid
-
fast stain.

The
remainder of the sediment was examined at 100 and 45
0x for eggs and cysts in saline and iodine
wet mounts.

The
smear was stained by MCK following the
protocol for MC
K as

described
previously (6
).

Each MCK
-
stained slide was examined under oil immersion power for 15 min.
The r
esults

were recorded as the avera
ge number of oocysts observed

per oil immersion field.

S
heathers
S
ugar
F
lo
tation
. Approximately 2 ml of the Formalin
-
treated stool suspension was
strained through two layers of gauze into a
conical tube. Eight ml

of Sheather sugar solution was
added, and t
he suspension was mixed
thoroughly by

inversion.

The tube was capped and
centrifuged at 500 x g for 15 min. After centrifugation, material at the surface of the suspension
was removed with a loop
,
placed on a slide and covered with a cover slip. Each slide

was
examined for approximately 5 min for pink, oocysts by microscopy
.
Results were recorded

as the
average number of oocysts observed per high
-
power

dry field.

Data analysis
:

Statistical analysis was done by the chi
-
square test. Probability level of p < 0
.05
was considered significant.

RESULTS

A total of 456 stool specimens were examined by the two

procedures
and the prevalence
of
Cryptosporidium

infection is shown in
Table 1
.
Cryptosporidium

sp. oocysts were

identified
in 182 (39.9%) specimens. Using SSF,

we identified

ooc
ysts in 89 (19.5%) specimens. When
F
EA sedimentation

plus MCK
was employed only 93 (20.3%) specimens were positive. There
was no significant difference (
p > 0.05) in number of positive samples between SSF and FEA.
The calculated sensitivi
ties of

FEA and SSF

were

51
%

(93/182)
and 49
%

(89/182) respectively
under the assumption that there were no false negatives.
A

total of 274 specimens were negative
for
Cryptosporidium

sp. oocysts by both techniques. A total of 37 specimens that

were po
sit
ive
6


by SSF but negative by F
EA plus
MCK
contained less than one oocyst per high
-
power field
. In
38 specimens oocysts were recovered

by only F
EA plus MCK
.

Table 2

shows the distribution of
the number of oocysts

detected using different techniques.


Table
1. Comparison of positivity among two different fecal examination techniques

Method

No of specimens

No of positive

Prevalence

FES


456

93

20.3%

SSF

456

89

19.5%

Total

456

182

39.9%

FES: formalin

ether sedimentation technique, SSF: Sheathers Sucrose flo
tation technique.




Table 2
.

Distribution of number of oocysts

detected by the two techniques

No. of oocysts found


Number of oocysts detected by (%)



FEA


SSF

1

38

(41)

37
(
42)

2
-
10

19

(20)

18
(20)

11
-
50

16
(17)

14
(16)

51
-
100

11
(12)

9
(10)

>100

9
(10)

11
(12)

No. of specimens

93 (100)

89(100)


7


DISCUSSION

T
he
F
EA concentration procedure

which is generally used for recovery of
parasites

was
compared with the more classical method (SSF) for the detection
of
Cryptosporidium
sp.

There
was no significant difference between these two methods in rates of recovery of

Crypt
osporidium

sp. oocysts. All disparate results were associated with the

presence of very few
oocysts. The use of both techniques

resulted in a
39.9
% positivity rate, as compared with rates of

approximately
20
% for either technique alone. In view of the

large number of specimens
included in this study, this

difference in
positivity rates may appear to demand

the

recommendation that both methods be

used for the optimal

detection of
Crypto
sporidium

sp.
However, an examination

of multiple specimens by either technique may

provide a similar
increase in sensitivity. As the
F
EA concentration

procedure is already performed routinely in
most

clinical micro
biology laboratories, preparation of the acid

fast

smear for detecting
Cryptosporidium

sp. in the same

sediment may reduce cost and technical time. The acid
-
fast

smear provides a permanent record of the results, but the

preparation and reading of the smear

require some technical

expertise. SSF concentration is easy to perform, and the

pink,
refractile
oocysts are easily recognized under high
-
power

magnification, but the wet mounts should be
examined

within 15 min after preparation or the oocysts may

collaps
e.

We examined each MCK
-
stained slide for 15 min under oil

immersion magnification to
cover approximately 100 fields.

This amount was roughly equal to the area covered when we

examined each SSF wet mount for 5 min under high
-
power

dry magnification. We cho
se these
two
parameters to standardize

our procedures and make our data available for

comparison.
Although there are several reports on the prevalence

of
C
ryptosporidium

in human patients, very
few studies used

SS
F technique
.


8


We concluded that F
EA plus MC
K of the sediment

was as effective in the concentration
and identification of
Cryptosporidium

sp. oocysts as SSF.

F
EA plus MCK may be advantageous
as single concentration method for general parasitology when
Cryptosporidium
sp. is to be
identified.

Referen
ces

1.


Bronsdon, M. A. 1984. Rapid dimethyl sulfoxide
-
modified acid

fast stain of
Cryptosporidium oocysts in stool specimens. J. Clin. Microbiol. 19:952
-
953.

2.

Current, W. L., N. C. Reese, J. V. Ernst, W. S. Bailey, M. B. Heyman, and W. M.
Weinstein. 1983. Hum
an cryptosporidiosis in immunocompetent and
immunodeficient persons. N. Engl. J. Med. 308:1252
-
1257.

3.

Garcia, L. S., and R. Shimizer. 1981. Comparison of clinical results for the use of ethyl
acetate and diethyl ether in the Formalin
-
ether sedimentation tec
hnique performed
on polyvinyl alcohol
-
preserved specimens. J. Clin. Microbiol. 13: 709
-
713.

4.

Garcia, L. S., D. A. Bruckner, T. C. Brewer, and R. Y. Shimizer.1983. Techniques for the
recovery and identification of Cryptosporidium oocysts from stool specimens
. J.
Clin. Microbiol. 18:185
-
190.

5.


Henriksen, S. A., and J. IF. L. Pohlenz. 1981. Staining of Cryptosporidia by a modified
Ziehl
-
Neelsen technique. Acta Vet. Scand. 22:594
-
596.

6.


Ma, P., and R. Soave. 1983. Three
-
step stool examination for cryptosporidiosis

in 10
homosexual men with protracted watery diarrhea. J. Infect. Dis. 147:824
-
828.

7.

Meisel, J. L., D. R. Perera, C. Meligro, and C. E. Rubin. 1976. Overwhelming watery
diarrhea associated with Cryptosporidium in an immunosuppressed patient.
Gastroenterolo
gy 70
:
1156
-
1160.

9


8.

Melvin, D. M., and M. M. Brooke. 1982. Centrifugal sedimentation
-
ether method, p. 103
-
109. In Laboratory procedures for the diagnosis of intestinal parasites. Health and
Human Services publication no. 82
-
8282. U.S. Government Printing Offi
ce,
Washington, D.C.

9.

Nime, F. A., J. D. Burek, D. L. Page, M. A. Holsher, and J. H. Yardley. 1976. Acute
enterocolitis in a human being infected with the protozoan Cryptosporidium.
Gastroenterology 70:592
-
598.

10.


Reese, N. C., W. L. Current, J. V. Ernst, and

W. S. Bailey. 1982. Cryptosporidiosis of
man and calf: a case report and results of experimental infections in mice and rats.
Am. J. Trop. Med. Hyg. 31:226
-
229.

11.

Sheather, A. L. 1923. The detection of intestinal protozoa and mange parasites by a
flotation
technique. J. Comp. Pathol. 36:266
-
275.

12.

Tzipori, S., K. W. Angus, E. W. Gray, and I. Campbell. 1980. Vomiting and diarrhea
associated with cryptosporidial infection. N. Engl. J. Med. 303:818.