T~~~srrcrm~s OF THE ROYAL SOCIETYOF TROPICALMEDICINE AND HYGIENE(1989) 83, 119-120
Rapid laboratory diagnosis of
cholera in the field
Nathan Shaffer’ Epiphanio Silva do Santos2, Per-
Ake Andreaaonl’ and J. J. Farmer III’ *Enter%
DiseasesBranch, Division of Bacterial Diseases,Center
for Infectious Diseases, Centersfor Disease Control,
Atlanta, Georgia, USA; 2National Public Health
Laboratory, B&au, Guinea-Bissau
The seventh cholera pandemic has now affected 35
countries, including 19 in Africa (WHO, 1984), and
continues to spread to new areas where laboratory
diagnosis is difficult. Mortality rates as high as 30%
have been reported (TAUXE et al., 1988), but early
treatment can limit this to less than 1% (LEWIS et al.,
1972). While Vibrio doZerae 01 can be cultured
within 18-48 h, laboratory confirmation in remote or
unprepared areas may take up to l-2 weeks, if
available.
During an epidemic of cholera in Guinea-Bissau in
October 1987, we evaluated a new laboratory test kit
for the detection of V. duderae01, ‘Vibrio chokrae0 1
AD Seiken’ (De&a Seiken Company, Japan), to seeif
it could co&m the diagnosis of cholera at the
bedside*. The kit had been previously tested only
with laboratory cultures and enriched clinical speci-
mens; each kit contains 3 reagents and a negative
control. Reagent a is a suspension of latex particles
coated with a monoclonal antibody to an antigen in
the cell wall of V. cholerae serogroup 01 strains
(SAKAZAKI i? TAMURA,1971). In laboratory tests, the
manufacturers state that it reacts with lo6 organisms
per ml but not with non-01 V. choleraestrains or
other enteric organisms. Reagentsb and c can be used
for subtyping the 01 serogroup. We used only
reagent a, since it is the most sensitive and reacts
strongly with Inaba, Ogawa, and Hikojima serotypes.
Over a 3-week period, rectal swabs from 33 new
patients with cholera-like illness from one urban and
one rural hospital were collected in Cary Blair
transport medium. Throughout the epidemic culture
specimenswere similarly obtained and transported in
either Cary Blair medium or alkaline peptone water.
The urban area had a large number of culture-
confirmed cholera cases;the rural area, 5 h travelling
time from the capital, was visited to evaluate a report
of a new outbreak. Approximately 20% of patients at
both sites had empirically been treated with an initial
dose of tetracycline before testing.
Within 30 min after collection, the swab was
applied to one of the kit’s cardboard slides or to a
standard microscope glass slide. The residue was
mixed with a drop of saline and one drop of reagent a
Please address reprint requests to Nathan Shaffer, Centers
for DiseaseControl, CID/DBD/EDB l-5428, Mailstop C-09,
Atlanta, GA 30333, USA.
*Use of trade names is for identification only and does not
imply endorsement by the Public Health Service or by the
US Department of Health and Human Services.
119
Figure. Illustration of latex agglutination reactions: on the left, 2
positive reactions; on the right, 2 negative reactions.
and observed for 2 min for signs of agglutination
(Figure). The cotton-tipped swabs were returned to
their Gary Blair tubes and transported within 24 h to
the national laboratory where .they were plated on
thiosulphate-citrate-bile salt-sugar agar (TCBS), sub-
cultured on heart infusion agar, and tested with V.
chobme 01 polyvalent and type-specific antisera.
Latex agglutination results obtained at the bedside
were compared with culture results available 2-5 d
later.
Table. Latex agglutination results compared with
culture results of rectal swab specimens from
patients with suspected cholera, Guinea-Bissau,
1987
V. chokrae 01 culture
Latex agglutination Positive Negative
Positive 10 2
Negativea 6 15
16 17
Quivocal latex agglutination reactions considered
negative.
The epidemic strain was toxigenic V. cholerae01
biotype El Tor, serotype Ogawa, contirmed by
culture in Guinea-Bissau and at the Centers for
Disease Control. The latex agglutination test was
positive for 63% of culture-positive patients and
negative for 88% of culture-negative specimens
(Table). 8 agglutination wells showed persistent
clouding but did not develop a clearly positive
reaction. These equivocal reactions were classified as
negative before the culture results were known.
This new kit can be used to screenfaecal specimens
for V. cholerae01 directly at the bedside. The test is
simple, easy to interpret, and requires only a fresh
specimen, refrigerated latex reagent and control, and
a toothpick or wire loop for mixing. Our preliminary
experience suggests that this method may be a
significant advance over earlier attempts at rapid
cholera diagnosis using dark-field and phase contrast
microscopy or fluorescent antibody techniques.
Further testing may improve diagnostic accuracy.
The sensitivity could probably be improved if speci-
mens were obtained directly from liquid stool, since
more antigen would be available and rectal swabs
often contain solid particles which can interfere by
making antigen inaccessible, or by clouding the well.
The best specimen is likely to be a freshly passed,
watery stool obtained before the patient has received
antibiotics (BARUA, 1974).
120
If rectal swab snecimens are used. the sensitivitv
might be increased by incubating *the residue in
alkaline peptone water for several hours. The manu-
facturer renorted 1000/osensitivitv when testing such
specimens~Future studies should compare &sults
from fresh watery stool, rectal swab specimens, and
snecimens incubated 2-12 h in alkaline nentone
\;ater. In addition, the stability of the reagenis heeds
to be evaluated; they have a l-year shelf-life if
refrigerated, but their durability otherwise is not
known.
The observed specificity of 88% was based upon a
comtinrison with V. choZerae01 culture results. The
specificity may have been lowered by false-negative
cultures resulting from inadequate specimen collec-
tion, improper culture technique, or prior antibiotic
exposure; optimally, cholera cultures can be 90%
sensitive (BARUA, 1974).
A practical test for rapidly diagnosing cholera in the
field, though not needed to determine treatment for
an individual patient, could provide provisional con-
T~NSA~~IONSor THE ROYAL8ocmrv or TROPICAL
MED~INEAND HYGIENE(1989)
1 Short Report 1
Antibiotic-multiresistant
Salmonella typhi in Egypt*
I. A. B, R. L. Haberberger, Z. Farid, N. I.
Girgia and J. N. Woody US Naval Medical Re-
search Unit No. 3, Cairo, Egypt
Salmonella typhi resistant to one or more antibiotics
has been reported by ROWE& TIU&LFALL (1984) and
GOLDSTEIN et al. (1986). In 1981 we rewrted the
appearanceof a chl&am~henicol-resistant &rain of S.
lrphi isolated from the stool of a patient with typhoid
fever in Upper Egypt (SIPPEL et al., 1981).
During the past 10 years, we have isolated over a
thousand strains of S. typhi from blood and other
specimens from patients with typhoid fever and
meningitis. All strains were susceptible to ampicillin,
*This work was supportedby the Naval Medical Research
and Development Command, NMC, NCR, Bethesda, Mary-
land, USA, Work Units nos 3M162770A870.AQ.320 and
3M464758D849.BH.341. The opinions and assertions con-
tained herein are the rivate ones of the authors and are not
to be construed as o& cial or as refIecting the views of the
Department of the Navy or the naval service at large.
kmation of a suspected cholera outbreak and help
monitor its extent, course, and termination.
References
Barua, D. (1974). Laboratory diagnosis of cholera. In:
Cholsra~ Barua, D. & Burrows, W. (editors), Phi-
ladelphta: W. B. Saunders, pp. 85-126.
Lewis, E. A., Francis, T. I., Monteliore, D., Okubadejo, 0.
A., Oyediran, A. B. 0. O., Ayoola, E. A., Mohammed,
I., Onyewotu, I. I., Vincent, R., McSweeney, P. L.,
Molloy, M. R., Sheehan,J. C., Cooke, A. R. & Wright,
E. A. (1972). Cholera in Ibadan, 1971.American3oumal
of Tropical Medicine and Hygiene, 21, 307-314.
Sskazaki, R. & Tamura, K. (1971). Somatic antigen
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Tauxe, R. V., Hohnberg, S. D., Dodin, A., Wells, J. V. &
Blake, P. A. (1988). Epidemic cholera in Mali: high
mortality and multiple routes of transmission in a famine
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WHO (1985). Weekly Epidemiological Record, 60, 149.
$xked 24 May 1988; accepted for publication 5 July
83, 120
chloramphenicol, trimethoprimkulfamethoxaxole and
other antibiotics as determined bv the disc diffusion
method.
On 7 June 1988we isolated S. typhi from the blood
of a natient with tvnhoid fever. This strain was
identikd by the APi-2OE commercial system and
confumed by serotyping using MinESS antisera
(Difco): it demonstrated the oresenceof 0. H. and Vi
intigeii. The strain was resistant to chlo&nphenicol,
ampicillin, and trimethoprimkulfamethoxaxole but
susceptible to axtreonam, ceftriaxone, amikacin and
to the quinolones, cinoxacin and notioxacin.
This is the first report of a strain of S. tvphi isolated
from an Egyptian patient which was resistant to all 3
drugs of choice for typhoid treatment.
References
F. W., Chumpitaz, J. C., Guevara, J. M.,
Goldstein,
Papadopoulou, B., Acar, J. F. & Vieu, J. F. (1986).
Plasmid-mediated resistance to multiple antibiotics in
Salmonella mhi. Jownal of Infectious Diseases, 153,
261-266.
Rowe, B. 81 Threlfall, E. J. (1984). Drug resistance in
Gram-negative aerobic bacilli. British Medical Bulletin,
40. 68-76.
Sipp& J.-E., Diab, A. S.?M&hail, I. A. & Iskandar, S. H.
(1981). Chloramphemcol resistant Salnwnella mvphiin
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Received 10 August 1988; accepted for publication 25
October 1988