Previous Article | Next Article 
Journal of Clinical Microbiology, November 1999, p. 3722-3724, Vol. 37, No. 11
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Isolation of Rickettsia prowazekii from
Blood by Shell Vial Cell Culture
Marie-Laure
Birg,
Bernard
La Scola,
Véronique
Roux,
Philippe
Brouqui, and
Didier
Raoult*
Unité des Rickettsies, CNRS UPRESA
6020, Faculté de Médecine, 13385 Marseille Cedex 05, France
Received 1 April 1999/Returned for modification 13 May
1999/Accepted 23 July 1999
 |
ABSTRACT |
A blood sample from a patient who returned from Algeria with a
fever inoculated on human embryonic lung fibroblasts by the shell vial
cell culture technique led to the recovery of Rickettsia prowazekii. The last clinical strain was isolated 30 years ago. Shell vial cell culture is a versatile method that could replace the
classic animal and/or embryonated egg inoculation.
| |
TEXT |
Epidemic typhus is a body
louse-transmitted disease due to Rickettsia prowazekii.
Recent reports of cases occurring in Burundi (12), in Russia
(16), and in Peru (unpublished data) and the present case
from a patient returning from Algeria must remind us that epidemic
typhus is a reemerging disease. The laboratory diagnosis of epidemic
typhus is based on serology but is hampered by cross-reaction with
murine typhus (7). The shell vial assay (8),
originally devised for the isolation of viruses, has now been adapted
by members of our team for the isolation of spotted fever group
rickettsiae (7). In this report, we describe the first
application of this method to the isolation of R. prowazekii.
Case report.
A 65-year-old man was referred to our hospital
center in October 1998 for fever and diarrhea. This native Algerian,
who usually lives in France, returned to France by boat after a visit
to Algeria. On arrival in Marseille, he suffered fever, vomiting,
myalgias, and diarrhea. On examination he presented with a high fever
of 40.6°C and a dissociated pulse. The patient exhibited mild
confusion. A discrete rash and splenomegaly were noticed. A presumptive
diagnosis of typhoid fever was made, and treatment with ceftriaxone (3 g/day, administered intravenously) was started immediately. By day
three, blood and stool cultures were negative and the patient's
condition had worsened. He was still febrile, dyspnea was noted, the
rash became purpuric, and the patient was semicomatose. A diagnosis of
typhus (murine or epidemic) was suspected and doxycycline (200 mg/day)
was prescribed. His condition rapidly improved, as he was afebrile
within 3 days.
A blood sample from the patient was inoculated onto three shell vials
containing human embryonic lung (HEL) fibroblasts grown on coverslips,
as previously described (6), in a biosafety level 3 containment laboratory. Detection of growing bacteria was carried out
by cytocentrifugation of 100 µl of one shell vial supernatant for
further Gimenez staining and directly inside the shell vial by
immunofluorescence. After fixation with cold acetone, the vial was
washed twice with phosphate-buffered saline (PBS). One hundred
microliters of the patient's convalescent serum (taken 1 week after
admission), diluted 1:50 in PBS with 3% nonfat dry milk, was added,
and the vial was incubated at 37°C with 100 µl of a fluorescein
isothiocyanate-conjugated goat anti-human immunoglobulin (Ig) (Fluoline
H; Biomerieux, Marcy l'Etoile, France) diluted 1:200 in PBS
containing 0.2% Evans blue. After three washes with PBS, the
coverslip was mounted (cells face down) in phosphate-buffered glycerol
medium (pH 8) and examined at 400× with a Zeiss epifluorescence microscope. This detection procedure was performed on days 7 and 14. The supernatant of a positive shell vial was used for PCR-based identification and was inoculated on confluent layers of HEL cells in a
150-cm2 culture flask in order to establish the isolate.
DNA extracts suitable for use as the template in PCR assays were
prepared from one remaining shell vial. These DNA extracts
were
amplified by using PCR incorporating primers that allow amplification
of genes encoding the citrate synthase (
gltA) and the
rickettsial
outer membrane protein rOmpB (
ompB). Sequencing
reactions were
carried out by incorporating the same primers as those
used for
amplification, and sequence products were resolved in the ABI
PRISM 377 automatic sequencing system (Perkin-Elmer Applied Biosystems,
Warrington, United
Kingdom).
As this patient came from an area not known as a region of endemicity
for epidemic typhus, determination of antibody to
R. typhi
was initially performed by indirect fluorescent-antibody
assay, as
previously described (
12), on sera taken on admission
and 1 week later. Antibodies to
R. prowazekii were further
determined.
Gimenez staining performed on day 7 was negative. Gimenez staining
performed on day 14 yielded numerous red-stained bacteria
located in
the cytoplasm of HEL cells. The coverslip of the same
shell vial
allowed detection of numerous fluorescent bacteria
in intracellular
locations (Fig.
1). The supernatants of
positive
shell vials inoculated on confluent layers of HEL cells
allowed
establishment of the isolate. The nucleotidic sequence obtained
was compared to all previously reported sequences of these genes
by a
Gapped Blast 2.0 (National Center for Biotechnology Information)
search
of the GenBank database. The sequences derived from the
shell vial
isolate were found to share 100% sequence similarity
with those of
R. prowazekii already deposited in GenBank. By indirect
fluorescent-antibody assay seroconversion to
R. typhi was
first
demonstrated (in convalescent serum, an IgG titer of
1:2,048 and
an IgM titer of 1:128 were determined). In
convalescent serum,
an IgG antibody titer of 1:4,096 and an IgM
antibody titer of
1:128 against
R. prowazekii were
determined.
View larger version (163K):
[in this window]
[in a new window]
|
FIG. 1.
Immunofluorescence staining of R. prowazekii
within HEL cells on a coverslip from a shell vial inoculated with a
blood sample from the patient. Magnification, ×400. Bacteria are
isolated (label 1) or grouped in clusters (label 2).
|
|
The clinical isolation of
R. prowazekii, based on
inoculation of clinical samples on animals, has not been reported for
30
years (
3). The last reported isolates of
R. prowazekii were
isolated 20 years ago from flying squirrels by
using embryonated
hen's eggs (
1). Adult male guinea pigs
have long been the animal
of choice for primary isolation of
R. prowazekii, but isolation
can be obtained with mice. The use of
animals, especially guinea
pigs, for diagnosis is not convenient for
most laboratories because
it requires that animals be easily available
and specific equipment
be kept. As bacteria are shed by animals,
contamination of laboratory
workers or other experimental animals can
occur, so the animals
must be kept in specially designed cages equipped
with filters.
Given the ever-lessening tendency to use animals for
research
or diagnosis, efforts must be made to replace them whenever
possible
(
13). Culture on embryonated chicken egg yolk sacs
has also
been widely used (
2). This procedure requires
embryonated eggs
of 5 to 8 days of age from flocks fed an
antibiotic-free diet,
which are often difficult to obtain rapidly
without subscribing
to the services of an animal supplier and often
need several blind
passages to obtain an isolate. Furthermore, they are
easily contaminated.
The cell culture procedure described more than 60 years ago is
now the most widely used method for isolating rickettsiae
from
clinical samples (
10). The centrifugation-shell vial
system
(
8) is a versatile and rapid method which can be
applied to
many viruses as well as facultative or strictly
intracellular
bacteria. We have used this technique routinely and
successfully
to isolate spotted fever rickettsiae (
6),
Coxiella burnetii (
9),
Bartonella sp.
(
11),
Francisella tularensis (
4),
Mycobacterium tuberculosis (unpublished data), and
Legionella pneumophila (
5) from blood and tissue
biopsies. To avoid bacterial
contamination, antibiotics with no
activity against rickettsiae,
such as trimethoprim-sulfamethoxazole or
vancomycin, may be added.
The small surface area of the coverslip
containing cells enhances
the ratio of the number of rickettsiae to the
number of cells
and allows more efficient recovery. When used with HEL
cells (which
have the advantage that once a monolayer is established,
contact
inhibition prevents further division), as was done for this
report,
incubation may be prolonged. Furthermore, by comparison to
conventional
cell culture procedures, the centrifugation step after
inoculation
enhances rickettsial attachment to and penetration of cells
(
18).
After inoculation and incubation in shell vials,
detection of
bacteria can be assessed by the use of acridine orange,
Gimenez,
and Giemsa stainings of the shell vial supernatant or by
immunofluorescence
staining of the cell monolayer by using the patient
serum, if
suitable, or sera from immune animals as the primary
antibody.
When bacterial growth is detected, identification can be
achieved
by PCR amplification and then sequencing of universal genes
such
as the 16S rRNA gene or of specific genes, such as
gltA,
ompB or
ompA, for rickettsiae
(
14,
15). PCR assay performed on
blood has been proposed as
an efficient technique to detect rickettsiae,
but in our experience and
that of others it has proven less sensitive
than the shell vial assay
(
6,
17).
As the use of the shell vial technique for isolation of rickettsiae is
restricted to specialized research and public health
laboratories that
have biosafety level 3 containment, it is not
suitable for use in most
clinical microbiology laboratories, especially
in poor countries where
epidemic typhus is more likely to reemerge.
However, in its favor, this
procedure provides a means for the
isolation of a wide range of
intracellular bacteria that are usually
isolated by inoculation of
animals, and so may help save animals,
and is well adapted to isolation
of such bacteria in developed
countries for travelers returning from
areas of endemicity. Furthermore,
as shown in this case, it allows
determination of the infecting
bacterial species, whereas sera
cross-react extensively with closely
related
species.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unité des
Rickettsies, CNRS UPRESA 6020, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France. Phone:
33.4.91.38.55.17. Fax: 33.4.91.83.03.90. E-mail:
Didier.Raoult{at}medecine.univ-mrs.fr.
| |
REFERENCES |
| 1.
|
Bozeman, F. M.,
S. A. Masiello,
M. S. Williams, and B. L. Elisberg.
1975.
Epidemic typhus rickettsiae isolated from flying squirrels.
Nature
255:545-547[Medline].
|
| 2.
|
Cox, H. R.
1938.
Use of yolk sac of developing chick embryo as medium for growing rickettsiae of Rocky Mountain spotted fever and typhus group.
Public Health Rep.
53:2241-2247.
|
| 3.
|
Eremeeva, M. E.,
V. F. Ignatovich,
G. A. Dasch,
D. Raoult, and N. M. Balayeva.
1996.
Genetic, biological and serological differentiation of Rickettsia prowazekii and Rickettsia typhi, p. 43-50.
In
J. Kazar, and R. Toman (ed.), Rickettsia and rickettsial diseases. Veda, Bratislava, Slovakia
|
| 4.
|
Fournier, P. E.,
L. Bernabeu,
B. Schubert,
M. Mutillod,
V. Roux, and D. Raoult.
1998.
Isolation of Francisella tularensis by centrifugation of shell vial cell culture from an inoculation eschar.
J. Clin. Microbiol.
36:2782-2783[Abstract/Free Full Text].
|
| 5.
|
La Scola, B.,
G. Michel, and D. Raoult.
1999.
Isolation of Legionella pneumophila by centrifugation of shell vial cell cultures from multiple liver and lung abscesses.
J. Clin. Microbiol.
37:785-787[Abstract/Free Full Text].
|
| 6.
|
La Scola, B., and D. Raoult.
1996.
Diagnosis of Mediterranean spotted fever by cultivation of Rickettsia conorii from blood and skin samples using the centrifugation-shell vial technique and by detection of R. conorii in circulating endothelial cells: a 6-year follow-up.
J. Clin. Microbiol.
34:2722-2727[Abstract].
|
| 7.
|
La Scola, B., and D. Raoult.
1997.
Laboratory diagnosis of rickettsioses: current approaches to diagnosis of old and new rickettsial diseases.
J. Clin. Microbiol.
35:2715-2727[Medline].
|
| 8.
|
Marrero, M., and D. Raoult.
1989.
Centrifugation-shell vial technique for rapid detection of Mediterranean spotted fever rickettsia in blood culture.
Am. J. Trop. Med. Hyg.
40:197-199.
|
| 9.
|
Musso, D., and D. Raoult.
1995.
Coxiella burnetii blood cultures from acute and chronic Q-fever patients.
J. Clin. Microbiol.
33:3129-3132[Abstract].
|
| 10.
|
Nigg, C., and K. Landsteiner.
1932.
Studies on the cultivation of the typhus fever rickettsia in the presence of live tissue.
J. Exp. Med.
55:563-576[Abstract].
|
| 11.
|
Raoult, D.,
P. E. Fournier,
M. Drancourt,
T. J. Marrie,
J. Etienne,
J. Cosserat,
P. Cacoub,
Y. Poinsignon,
P. Leclercq, and A. Sefton.
1996.
Diagnosis of 22 new cases of Bartonella endocarditis.
Ann. Intern. Med.
125:646-652[Abstract/Free Full Text].
|
| 12.
|
Raoult, D.,
J. B. Ndihokubwayo,
H. Tissot-Dupont,
V. Roux,
B. Faugere,
R. Abegbinni, and R. J. Birtles.
1998.
Outbreak of epidemic typhus associated with trench fever in Burundi.
Lancet
352:353-358[Medline].
|
| 13.
|
Roush, W.
1996.
Hunting for animal alternatives.
Science
274:168-171[Medline].
|
| 14.
|
Roux, V.,
P. E. Fournier, and D. Raoult.
1996.
Differentiation of spotted fever group rickettsiae by sequencing and analysis of restriction fragment length polymorphism of PCR-amplified DNA of the gene encoding the protein rOmpA.
J. Clin. Microbiol.
34:2058-2065[Abstract].
|
| 15.
|
Roux, V.,
E. Rydkina,
M. Eremeeva, and D. Raoult.
1997.
Citrate synthase gene comparison, a new tool for phylogenetic analysis, and its application for the rickettsiae.
Int. J. Syst. Bact.
47:252-261[Abstract/Free Full Text].
|
| 16.
|
Tarasevich, I.,
E. Rydkina, and D. Raoult.
1998.
Epidemic typhus in Russia.
Lancet
352:1151[Medline].
|
| 17.
|
Tzianabos, T.,
B. E. Anderson, and J. E. McDade.
1989.
Detection of Rickettsia rickettsii DNA in clinical specimens by using polymerase chain reaction technology.
J. Clin. Microbiol.
27:2866-2868[Abstract/Free Full Text].
|
| 18.
|
Weiss, E., and H. R. Dressler.
1960.
Centrifugation of rickettsiae and viruses onto cells and its effect on infection.
Proc. Soc. Exp. Biol. Med.
103:691-695.
|
Journal of Clinical Microbiology, November 1999, p. 3722-3724, Vol. 37, No. 11
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Gouriet, F., Fenollar, F., Patrice, J.-Y., Drancourt, M., Raoult, D.
(2005). Use of Shell-Vial Cell Culture Assay for Isolation of Bacteria from Clinical Specimens: 13 Years of Experience. J. Clin. Microbiol.
43: 4993-5002
[Abstract]
[Full Text]
-
Zhu, Y., Fournier, P.-E., Ogata, H., Raoult, D.
(2005). Multispacer Typing of Rickettsia prowazekii Enabling Epidemiological Studies of Epidemic Typhus. J. Clin. Microbiol.
43: 4708-4712
[Abstract]
[Full Text]
-
Drevets, D. A., Leenen, P. J. M., Greenfield, R. A.
(2004). Invasion of the Central Nervous System by Intracellular Bacteria. Clin. Microbiol. Rev.
17: 323-347
[Abstract]
[Full Text]
-
Fenollar, F., Birg, M.-L., Gauduchon, V., Raoult, D.
(2003). Culture of Tropheryma whipplei from Human Samples: a 3-Year Experience (1999 to 2002). J. Clin. Microbiol.
41: 3816-3822
[Abstract]
[Full Text]
-
La Scola, B., Rydkina, L., Ndihokubwayo, J.-B., Vene, S., Raoult, D.
(2000). Serological Differentiation of Murine Typhus and Epidemic Typhus Using Cross-Adsorption and Western Blotting. CVI
7: 612-616
[Abstract]
[Full Text]
Top
Abstract
Text
