Development and testing of a rapid diagnostic test for bubonic and

ARTlCLES
Development
pneumonic
and testing
of a rapid diagnostic
plague
Suzanne
Chanteau,
E/isabeth
Carnie/, Farida Nato
U/a Raha/ison,
La/ao Ra/afiarisoa,
Jeanine
Summary
Methods We developed a test that used monoclonal
antibodies to the F1 antigen of Yersinia pestis. Sensitivity and
specificity were assessed with a range of bacterial cultures and
clinical samples, and compared with findings from available
ELlSA and bacteriological tests for plague. Samples from
patients thought to have plague were tested with the RDTin the
laboratory and by health workers in 26 pilot sites in
Madagascar.
Findings The RDTdetected concentrations of F1 antigen as low
as 0.5 ngjmL in up to 15 min, and had a shelf life of 21 days
at 60°C. Its sensitivity and specificity were both 100%. RDT
detected 41.6% and 31% more positive clinical specimens
than did bactel;i°logical methods and EUSA, respectively. The
agreement rate between tests dane at remate centres and in
the laboratory was 89.8%. With the combination of
bacteriological methods and F1 ELlSA as reference standard,
the positive and negative predictive values of the RDT were
90.6% and 86.7%, respectively.
Interpretation Our RDTis a specific, sensitive, and reliable test
that can easily be dane by health workers at the patient's
bedside, for the rapid diagnosis of pneumonic and bubonic
plague. This test will be of key importance for the contrai of
plague in endemic countries.
Lancet 2003; 361: 211-16
See Commentary page 191
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Fou/on, Mahery Ratsitorahina,
La/a Ratsifasoamanana,
Introduction
Background Plague is often fatal without prompt and
appropriate treatment. It affects mainly poor and remate
populations. Late diagnosis is one of the major causes of
human death and spread of the disease, since it limits the
effectiveness of contrai measures. We aimed to develop and
assess a rapid diagnostic test (RDT)for plague.
i
I
test for bubonic and
.FI
:..
Rapid diagnostic tests (RDTs) for infectious diseases are
of much value in facilitation of major improvements in
disease management, especially in developing countries.
Some RDTs are based on immunochromatography,
with conjugated gold or latex particles used to detect
specific antigens from bacterial, viral, or parasitic
agents.'-3
Plague, a flea-borne rodent disease that is occasionally
transmitted
to man,' is still prevalent in more than
20 countries, mainly in Africa.' Madagascar is one of
the most active plague foci in the world.6-8 Prompt
diagnosis of this fulminant disease is of key importance
for control of the mortality rate and for effective controlo
The diagnosis of bubonic or pneumonic plague is still
based on clinical symptoms in most endemic areas.
However,
such diagnosis is not reliable. Biological
samples are usually assessed by microscopy in regional
hospitaIs, but in remote areas such tests are often
not done at alI or are unreliable. Resource-poor regions
do not have the technology
and staff needed for
bacteriological identification methods, ELISA, and PCR
for diagnosis of plague during the acute phase of the
disease.9,lO
In such endemic foci, the development of simple,
quick, and specific methods by which local health
workers could test biological samples from patients with
symptoms of plague, or dead rodents, is a key priority.
Indeed, such a test would eliminare doubt in suspected
cases of plague, facilitating
the immediate treatment
of patients
and contacts, and implementation
of
compulsory flea control measures. Such action is crucial
ifplague morbidity and lethality are to be reduced.
Very early diagnosis of plague would algo be essential
in cases of bioterrorism.
Yersinia pestis is one of the
bacteriological agents that could potentially be used for
biological warfare.11 If transmitted
in the forro of
dro?lets that are t?en inhaled ~r spread by ae~osols, the
bacIllus causes hlghly contaglous pneumornc plague,
which is always fatal if treatment is not started within
24 h óf onset of symptoms.'2
antigen is specific to Y pestis and is present in
large amounts
in blood and bubo samples from
Institut Pasteur de Madagascar,WHOCollaboratingCentre for
Plague,Antananarivo,Madagascar(S ChanteauPhO,
L RahalisonPhO,M RatsitorahinaMO);Plague National Contrai
Programme,Ministry of Health,Antananarivo,Madagascar
(L Ralafiarisoa,L RatsifasoamananaMO);and Institut Pasteur,
WHOCollaboratingCentre for Yers/nia,Paris, France
(JFoulon,E CarnielMO,F Nato PhD)
infected people.'~" It is stable in tropical climates, and
can be detected even after the patient has been treated
for several days.15,16An RDT for plague based on FI
antigen has been tested in the laboratory, and gave
promising
results.16 However, since this test is not
readily available, another rapid test is needed for use in
the plague foci of African, Asian, and North and South
American countries, and in non-endemic countries, in
cases of suspected imported plague or of biological
att~ck. T~ere!ore, we aime~ to d~velop a sensitive FI
Correspondenceto: Dr SuzanneChanteau,CERMES,
POBox10887, Niamey,Niger
antlgen dIPStlck, and to validare It both at the plague
reference
laboratory
and at 26 remote
gires in
(e-mail: [email protected])
Madagascar.
,
THE LANCET. Vol361 .January18,2003. www.thelancet.com
211
ARllCLES
Methods
Oevelapmentaf plague ROT
To develop monoclonal antibodies (Mab) against Fi
antigen, we immunised rnice (Biozzi BP/2 strain) with Fi
antigen that was purified as Chen and Meyer describedl7
from Y pestisstrain 185S/97 (Madagascar).The fusion and
screening methods used have been described.18
67 hybridomas with strong IgG responsesagainst F1were
obtained, 16 of which were cloned and characterised.For
development of the plague RDT, two monoclonal
antibodies (IgG1 and K chain isotype) that bound to two
difIerent epitopes of the FI antigen19
were selected on the
basisof their high affinity, and were purified by ammonium
sulphate precipitation and
fast protein liquid
chromatography (Pharmacia, Bois d'Arcy, France)
according to the column manufacturer's instructions. Mab
B18-1 (kD <5X1Q-1ImoI) was conjugatedto gold particles
as the mobile phase, and Mab G6-18 (kD=lX10~10 moI)
was deposited on nitrocellulose as the capture antibody.
The hybridomas Bl8-1 and G6-18 were stored at the
Collection National desMicroorganismes (Paris,France).
Our RDT was derived from a prototype developedby the
Naval Medical Research Institute (Bethesda, Maryland,
USA) and assessed
in Madagascar.16
For this new test, we
used a combination of B 18-1 and G6-18 produced at the
Institut Pasteur, whereas the original RDT combined
F1--O4-A-G1 Mab with a polyclonal rabbit antiserum, and
was based on one-step, vertical-tlow immunochromatography.'oThe colloidal gold particles (40 nrn diameter)
were conjugated to anti-F1 Mab B18-1 (British Biocell
Intemational CardifI, UK) and lyophilised (A54Qnrn=3)
onto
polyester release pads (Filtreri, Ambérieu en Bugey,
France). An automatic thin layer chromatography sampler
(CAMAG, Muttenz, Switzerland) was used to spray the
anti-F1 capture Mab G6-18 in a tine, at a concentrationof
2 ~cm, onto nitrocellulose membrane (Irnmunopore FP,
Wh}tman Intemational, Chateau Giron, France); together
with the contraI capture tine, which was affinity-purified
goat anti-mouse IgG (ICN Biomedicals, Aurora, Orno,
USA), sprayed as a liDe higher up the strip at a
concentration of 0,8 Ilg/cm. Cellulose filter paper was used
for the wicking pad and samplepads (Schull and Schleicher,
Ecquevilly, France).
The immunostrips were trimmed to a width of 5 mm and
stored in a waterproof bag at 4°C, or in 5 rnL disposable
plastic tubes at roam temperature at non-central sites.The
test was dane in the plastic tube with a sample volume of
about 200 j.l.L. After 10-15 min, a positive result appeared
as two pink tines (upper controltine and lower F1-positive
liDe), and a negative result as a single upper pink contraI
tine. Semiquantitative analysis was possible, with the
intensity afilie test tine scored from 0'5 to 4 (indicating the
concentration ofF1 antigen in the test sample). The cutof!
and the range of FI concentration that could be detected
was measured with tenfold dilutions (50 j.l.g/rnL to
0,05 ng/rnL) of purified FI antigen. The reliablility of the
RDT was assessedwith two concentrations of purified FI
antigen (0'5 ng/rnL and 1 j.l.g/rnL).
The shelf life of the strips in the laboratory was assessed
by testing twice per week for 3 weeksafter storageat 60°C,
4°C, -20°C, and :-80°C.'0The re~iabilityof the RDT was
assessedby repeanngthe test ten nInes on the samesample
and in ten difIerent seriesoftests.
Referencetestsand samples
We used two reference standard tests for the diagnosisof
plague. The first was gram staining and isolation of Y pestis,
either direcrly from the patients' samples or after mouse
inoculation. A specimen was judged to be confinned as
212
being plague when the culture was positive, presumptive
when culture was negativebut rnicroscopywas positive, and
negative when both tests were negative. Confinned and
presumptive cases were notified to WHO. The second
method was the immunocapture EUSA for detection of
FI antigen with F1--O4-A-G1 anti-F1 Mab.16The lowest
concentration of antigen detectable with this test was
2 ng/rnL.
To assessthe sensitivity of the plague RDT we used
85 strains of Y pestisthat had been cultured in brain-heart
broth at 37°C for 72 h. 55 of these were isolated in
Madagascar from 1996 to 2000 (33 from patients with
bubonic plague, four from those with pneumonic plague,
seven from rats [Rattus norvegicus], three from tleas
[Xenopsyllacheopis],seven from shrews [Suncus murinus],
and one from a hedgehog [Setifersetosus].30 strains carne
from 14 other countries (collection of the WHO
Collaborating Centre for Yersinia, Institut Pasteur, Paris,
France): tive of biotype Antiqua (four from Kenya, one
former USSR), tive ofbiotype Medievalis (Kurdistan [four],
Turkey [anel), and 20 of biotype Orientalis (Germany
[two], Morocco [anel, Turkey [anel, India [anel, Narnibia
[two], Vietnam [seven], Brazil [two], Argentina [anel,
Senegal [anel, South Africa [anel, Congo [anel).
Additionally, we used 198 clinical specimens from plague
patients that had tested positive by both bacteriology and
FI EUSA: 17 sputum samples,35 serum samples,43 urine
samples,and 103 bubo aspirates.
To assessthe specificity of the test we used 189 cultures
of yersinia and other enterobacteria grown in brain-heart
broth, at 37°C for 72 h. These sampleswere: 71 cultures of
enteropathogenicyersinia, consisting of Y pseudotuberculosis
(44 strains, serotypesI-V from 17 difIerent animal species
and environrnental samples isolated in 22 difIerent
countries), and Yenterocolitica(27 strains isolated in seven
countries and belonging to six biotypes [lA-5] and to
various serotypes); 52 non-pathogenic yersinia of various
serotypesbelonging to eight speciesand isolated in various
countries (Y kristenseni [nine), Y intermedia [ten],
Y mollaretii [ten], Y frederiksenii[ten], Y bercovieri[ten],
Y rhodei [anel, Y ruckeri [anel, and Yaldovae [anel; and
66 enterobacteriabelonging to 19 genera and 55 species
(Escherichiaspp [eight], Enterobacterspp [ten], Erwinia sp
[anel, Hafnia sp [anel, Serraria spp [ten], Klebsiellaspp
[ten], Salmonellaspp [four], Shigellaspp [four], Levinea sp
[anel, Citrobacterspp [two], Edwa~iella spp [two], Proteus
spp [two], Providencia spp [two], Morganella sp [anel,
Kluyvera spp [two], Budvicia sp [anel, Cedeceaspp [two],
Leminorella spp [two], and Obesumbacteriumsp [one]).
Additionally we used 137 clinical specimens that were
negative for plague: 47 sputum samples from suspected
tuberculosis patients (14 positive for Mycobacterium
tuberculosis),
66 normal serum samples diluted 1/10, and
24 undiluted normal urine samples. For ethical reasons,
bubo aspirateswere not taken from patients who were not
thoUghtto have plague.
The negative and positive predictive values (NPV and
PPV, respectively)of the FI dipstick were calculated by
Bayes' theorem, and 95% CI by Fleiss' quadratic
approximation method (Sp=sensitivity,Se=sensitivity):
Sp (l-prevalence)
NPV=
Sp (l-prevalence)
+ (l-Se)prevalence
Se (prevalence)
PPV= Se(prevalence)+
(l-Sp) (1- prevalence)
The agreementbetween RDT and reference tests results
(bacteriology or FI EUSA, and bacteriology combined
THELANCET.Vol361.January
18,2003'www.thelancet.com
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ARTICLES
O Plague
endemic
areas
Pilotsites
I
I
200 km
I
Locationof 26 pilot sites inplagueendemicdistricts of
Madagasc
ar
i
wirll FI ELISA) was assessedwirll rlle percentage of
concordant results and rlle Kappa coefficient wirll
confidence intervals. The correlation between FI ELISA
and RDT results was assessedby Kendall's correlation
coefficient. Finally, we used rlle RDT to test 283 bubo
aspirates,which were stored at rlle Institut Pasteur,rllat had
been isolated from patients who were suspectedon clínical
grounds to have plague but tested negative wirll rlle two
referencestandard assays.
We did PCR with any cultures of Y pestisrllat tested
negative on RDT, to find out wherller cafl gene, which
encodes for FI antigen, was present. The cafl gene was
amplified by PCR, wirll heat-killed bacteria as templates
and two oligonucleotide primers: forward 5'-CAGTTC
CGTTATCGCCATTGC-3',
and reverse 5/-TATTGG
TTAGATACGGTTACGGT-3'.
PCR amplification was
done in a final volume of 50 ILL, in a PTC 100 rllermal
cycler (MJ Research,Basel,Switzerland). We mixed 7 ILL
of boiled bacterial suspension(target DNA), 200 ltInol of
each deoxyribonucleotide triphosphate, 2 mmol MgCI.
0.1 ILmol of each primer, and 1 U of rllermostable DNÀ
polymerase (Roche, Meylan, France) in the corresponding
IX polymerase buffer. The gene was amplified over
25 cycles, each consisting of denaturation for 1 min at
95°C, annealing for 1 min at 55°C, and polymerisation for
2 min at 72°C; followed by one cycle at 55°C for 1 min and
72°C for 3 minoPlasmid DNA was extracted21
and digested
wirll EcoRV. PCR products and digested plasmid DNA
were electrophoresed in agarose gels and stained wirll
ethidium bromide.
THELANCET.Vol361.January
18,2003'www.thelancet.com
Assessmentof ROr
We undertook a prospectivestudy to assessrlle effectiveness
of rlle FI dipstick during rlle 200G-Ql plague season in
Madagascar, as part of rlle national plague control
programme. We obtained a biological sample (bubo
aspirate, sputum, or post-mortem organ puncture, as
appropriate) from patients wirll suspectedplague, and rlle
patients were treated wirll streptomycin. Sampleswere sent
on a swabin Cary Blair agar, at room temperature,to rlle
Central Laboratory at rlle Institut Pasteurde Madagascar.
On arrival, rlle specimenwas washed out of rlle swab by
incubation in 1 mL phosphate-buffered saline, and was
tested wirll rlle two referencemerllods and wirll RDT. The
mean transporttimes of rlle positive and negativesamplesto
rlle Central Laboratory were compared wirll rlle Kruska11Wallis non-parametric testo
To test rlle RDT in remote areas,a network of 26 pilot
sites was set up from Dec 1, 2000, to Jan 25, 2001,
consistingof six district hospitaIsand 20 healrll care centres
in remote areas (figure). After rlle efficiency of rlle plague
RDT had been assessedat rlle Central Laboratory, kitseachcomposed of one syringe,one needIe,PBS, one plague
RDT in a disposabletest tube wirll silica gel, one Cary Blair
tube, one swab,a user's manual, and a questionnaire-were
distributed to rllese field sites. The robustness of rlle test
under field conditions was assessed,based on several
criteria: reliability of rlle results obtained by non-biologist
staff, presentation of rlle kit, ease of manipulation and
interpretation, shelf life under real field conditions, lengili
and content ofrlle training programme, and intelligibility
rlle illustrated manual.
of
The RDTs were stored at room temperature during rlle
study (2o-30°C). 29 medical doctors, 19 nurses,and nine
healrll workers participated in rlle assessment.They were
trained onsite for 3 h as to how to obtain clinical samples
and to use, read, and archive rlle dipsticks. An illustrated
instruction guidebook, in French and Malagasy,was ~ven
to
eachcentre.
The plague
RDTwere
wasrllen
doneasked
at rlletopatlent's
bedside.
The trained
personnel
send a
sample of rlle specimen to rlle Central Laboratory for a
dipstick test wirll rlle same batch of strips, bacteriological
identification, and FI ELISA assay.The technicians who
did rlle testsat rlle Central Laboratory were not awareof rlle
results obtained in rlle pilot centres.
From May to June, 2001 (end of rlle plague season),rlle
26 sites were visited again, RDT results were compared,
and rlle registerswere checked.The healrll personnelwere
askedto fil1 in an individual user's questionnaire,to provide
criticisms, comments, and observations about rlle plague
RDT and rlle instruction book.
Role of the fundingsource
The sponsorsofrlle study had no role in study design,data
collection, data analysis,data interpretation, or writing of
rlle reporto
Results
The lower detection threshold of rlle newly developed
plague RDT was 0'5 ng/mL, and rlle range of FI
concentrations extended from 0'5 ng/mL to 50 lLg/mL
(5 log), wirllout any hook effect (in which no signal is
detected for high concentrations). Results obtained were
much rlle same after storageof RDT for 21 days at 60°C,
4°C, -20°C, and -80°C.
The RDT had a sensitivityof 100% on fresh1yisolated Y
pestis strains and positive control clínical samples: alI 55
bacterial cultures and all198 positive specimenstestedwere
positive wirll rlle RDT. Positive RDT results were obtained
for 28 of 30 Y pestisstrains from various continents. PCR
213
ARTlCLES
Plague
RDT
Bacteriology
Confirmed Presumptive Negative
Fi ELlSA
Positive Negative
Comblnatlon
of reference
tests
Positive
Negative
Positive
Negative
279(40%)
412(60%)
151(83%) 12(80%)
31 (17%) 3 (20%)
116(24%)
378(77%)
212(100%)67 (14%)
0(0%) 412(86%)
232(87%)
34 (13%)
47 (11%)
378(89%)
Total
691
182
494
212
266
425
Number (%) samples
15
479
Table1: ComparisonbetweenplagueRDT,bacteriology,and Fj. ELISAfor specimenstested at CentralLaboratory
assaysdone on the two RDT -negativestrains with primers
internal to the cafl gene yielded no amplification product,
indicating that this gene (or the entire pFra plasmid) was
deleted in these two strains. Comparison of the plasmid
restriction profiles of these strains with that of wild-type
strain 6/69 from Madagascarconfirmed the absenceof the
restriction fragments corresponding to pFra (data not
shown). The specificity of the plague RDT was algO100%
for alI bacterial cultures and alI negative control clinical
specimens.Of the 283 bubo aspiratesfrom suspectedplague
patients thar testednegativein bacteriologyand EUSA, 256
(90.5%) algo tested negative by RDT, but 27 (9'5%) gave
weak positive resultswith this test (scoreof 0'5).
From Dec 1, 2000, to May 30, 2001, 671 cases of
suspectedplague were declared to the national surveillance
system:598 (89%) survived,61 (9%)died, 12 (2%) had an
unknown outcome. 642 (96%) patientshad bubonic plague,
20 (3%) had pneumonic plague, and nine (1 %) had an
unknown clipical status. In total, 691 clinical specimens
were sentto the Central Laboratory in Cary Blair agar: 643
(93%) were bubo aspirates,13 (12%) sputa, and 35 (5%)
post mortem lung or tiver puncture samples.The median
transport time to the Central Laboratory of the 691 samples
was 8 days, ranging from O to 66 days (25th percentile
5 days, 75th percentile 14 days). The mean transport time
was 10.6 days (SD 9.6) for bacteriologically confirmed
specimens and 12.1 days (11'9) for negative specimens
(non-significant difference, Kruskall-Wallis non-parametric
test). ~
After recovery of lhe samples from Cary Blair media,
Y pestiswas cultured from 182 (26%) samples (classified
as confinned specimens),whereas 15 (2%) were negative
by culture but positive by microscopy (classified as
presumptive specimens; table 1). Thus, the total number
of plague patients identified by bacteriology was 197 of
691 suspected. With RDT, 279 (40%) specimens were
positive for plague, 116 (42%) of which tested negative
both by microscopy and culture. The concordance
between lhe results of bacteriological and plague RDT
results was 78,3% (K=0'52, 95% CI 0.50-0'54; moderate
agreement). Therefore, use of plague RDT alone led to
identification of 41.6% more cases than when only
bacteriology was used (279 vs 197 positive tests).
However, 31 (17%) samples tested negative with the
plague RDT were positive by bacteriology.
When FI EUSA was used as the reference method,
results obtained with this technique and with RDT were
highly concordant (concordance 90'3%, results ofthe two
tests agreed for 624 of691 specimens tested)and strongly
correlated (Kendall's correlation coefficient r=0.834,
p<10-3).K was 0.79 (95% CI 0'77-0.81; good agreement).
AlI EUSA-positive samples algo tested positive on RDT.
Moreover, RDT detected 67 weak1y positive samples
(score of 0'5) among lhe 479 samples that tested negative
by EUSA (table 1). Therefore, use of RDT resulted in
31 % (67 of212) more positive results than were obtained
with FI EUSA alone. When we used a combination of
bacteriological tests and FI EUSA as lhe reference
standard (table 1), 266 of the 691 specimens tested
positive, whereas with RDT alone, 279 tested positive
(K=0.75, 95% CI 0.73-0'77).
At the 26 remote sites, 128 patients (122 survivors and
six patients who died) were suspected, on clinical
grounds, to have plague (123 bubonic and tive
pneumonic fonns) and were treated. Results of RDTs
done by health workers at lhe patient's bedside, and by
skilled technicians at the Central Laboratory, on the
garoespecimens (table 2) were concordant in 115 of 128
patients (90%). Four test~ done at remote sites (3%)
were not valid since the controlline was absent (table 3).
With bacteriology and FI EUSA as reference standards,
58 samples were identified as positive among the 128
tested, whereas RDTs done at remote sites and at lhe
Central Laboratory identified 53 and 55 positive samples,
respectively. Agreement between results from RDTs
done at the field sites and bacteriology was moderate
(K=0.62, 95% CI 0'57-0.66), but agreement was very
good when RDT in the field was compared with FI
EUSA (0.83, 0'78-0.87). The false negatives with RDT
corresponded with samplesthat tested positive in culture
after amplification in two mice (low bacterial load) but
negative with FI EUSA. Five patients who were
identified at remote sites as plague-infected were negative
for plague with the combination of reference tests.
On the assumption that the combination of lhe two
reference tests is lhe gold standard (which is not
lhe case), lhe calculated PPV and NPV would be
90.6% (95% CI 78.6-96.5) and 86.7% (76.4-93.1),
respectively, at lhe pilot sites, and 92.7% (81.6-97.6)
and 90.4% (80.7-95.7), respectively, at lhe Central
Laboratory. There was 85% concordance between lhe
results of the combined reference methods and those for
the plague RDT in the field (K=0'76, 95% CI
0'71-0.80), and 91 % concordance between lhe
combined and RDT methods at lhe Central Laboratory
(0.82, 0'77-0.86).
The 57 individual user's questionnaires gave a mean
overall satisfaction score of 17'6 of 20 [range 14--20}.
Most (51 of 57,90%) ofthe health staffthought that lhe
RDT would help to reduce plague morbidity and
mortality in Madagascar.
Sputum obtained from 16 patients with suspected
pneumonic plague were tested with lhe plague RDT as
well as with lhe two reference tests for comparison
(table 4): 13 sputa were tested only at lhe Central
Laboratory; lhe remaining three sputum samples were
algo tested at lhe field sites. The eight patients for whom
the diagnosis of plague was confinned by bacteriology
algo tested positive by RDT (one tested negative by
ELISA). However, two patients who tested negative by
bacteriological methods gave a weak positive result with
lhe plague RDT, one of whom algo tested positive by
EUSA.
ROT,rematesites
Tot~1
Positive Negative Invalid patlents
RDT,CentralLaboratory
Positive
(n=55) 49 (92.5%%)5 (7%) 1 (25%) 55 (43%)
Negative
(n=73)
4 (7'5%) 66 (93%) 3 (75%) 73 (57%)
Totalpatients
(%) 53
71
4
128
Table2: ComparisonbetweenplagueRDTsdone at remote
sites and at CentralLaboratoryon the samespecimens
214
THELANCET'Vo1361'January
18,2003'www.thelancet.com
~
AR11CLES
Total patients(%)
Combination
Positive
Negative
Total
RDT,remotesites
RDT,CentralLaboratory
Positive
Negative
Invalid
Positive
Negative
48 (83%)
5 (7%)
10 (17%)
61 (87%)
O (0%)
4 (6%)
51 (88%)
4 (6%)
7 (12%)
66 (94%)
53 (41%)
71 (56%)
4 (11%)
55 (43%)
73 (57%)
bacteriology/ELlSA
58 (100%)
70 (100%)
128 (100%)
Table 3: Comparison between plague RDTdone at remote sites and at Central Laboratory and combination of bacteriology and
Fi ELiSA
Discussion
We have shown that our RDT is as specific as, and at
least as sensitive as, the two available standard methods.
The excellent specificity of the RDT, its low detection
threshold, and the higher number of positive specimens
detected among samples from patients with suspected
plague, suggest a greater sensitivity than bacteriology and
ELISA. Thus, in the operational conditions ofthe plague
control programme
at the Central Laboratory,
we
recommend a combination of bacteriological tests and
RDT
for diagnosis and surveillance
of plague in
Madagascar.
Our new test detected FI antigen at a wider range of
concentrations than other tests, in 10-15 min, without
prozone effect. Absence of prozone phenomenon is
essential to avoid false-negative results, especially in postmortem samples or sputum samples that contain very
high concentrations
of FI antigen. The highest FI
concentration
ever noted
in clinical samples was
30 J.lg/mL (S Chanteau, unpublished).
The existing reference tests, which can only be done in
the laboratory-the
isolation of Y pestis and the detection
of FI antigen by ELISA-are
specific, but lack sensitivity
in ali countries in which plague is endemic. The failure of
these standard assays is due to the deterioration,
contaminati~,
or both, of specimens during their long
transport time to the laboratory, antibiotic treatment of
patients before sampling, and the diffusion of FI antigen
in the Cary Blair transport medium. The difference
between the numbers of suspected plague samples found
positive by RDT and culture could algo be accounted for
by diffusion ofthe FI antigen in the Cary Blair transport
medium, a difficulty that is not encountered if these tests
are directly done on clinical samples by health staff.
Furthermore, other investigators have shown that some
patients who were originally diagnosed as negative with
(degree
PlagueRDT
of positivity) F:1
(ngjmL)
ELlSA
I
Bacteriology
Patlent'snumber
230/01
Neg
Neg
Neg
307/01
Neg
Neg
Neg
369/01
Neg
Neg
Neg
382/01
Neg
Neg
Neg
967/00
Neg
Neg
Neg
1058/00*
Neg
Neg
Neg
23:1/01
Pos(0.5+)
Pos(5)
Neg
:1052/00
Pos(0.5+)
Neg
Neg
969/00
Pos(0.5+)
Pos(:13)
Confirmed
:1027/00
Pos(1+)
Pos(44)
Confirmed
999/00
Pos(2+)
Neg
Confirmed
1157/00
Pos(3+)
Pos(125) Confirmed
:1:157/00*
Pos(3+)
Pos(125) Confirmed
974/00
Pos(4+)
Pos(>:125) Confirmed
1:158/00
Pos(4+)
Pos(>125) Confirmed
1:158/00*
Fios(4+)
Pos(>125) Confirmed
Totalpositive/tested 10/16
8/16
8/16
Confirmed;Y
pestisisolated.Neg;negative.
Pos;positive.
0.5+to 10+;score
(seetext). 'Patientalgotestedat remotesites.
Table 4: Diagnosis of pneumonic plague in sputum with plague
RDT, Fi ELiSA, and bacteriology
TOELANCET. Vol361 .January18,2003. www.thelancet.com
the reference tests (and positive with the initial plague
dipstick) subsequently underwent FI antibody seroconversion.16
Conversely, 116 samples that tested positive by plague
RDT gave negative results in bacteriological
tests,
probably for the reasons given above. These reasons
explain the fair concordance
between RDT
and
bacteriology. Similarly, 67 samples that tested negative
with FI ELISA gave weak positive results with RDT,
probably because the detection threshold of RDT was
lower than that of EilSA.
Finally, 27 of the 283 clinical
specimens classified as negative with the combined
bacteriological and ELISA tests tested positive by RDT.
Since the RDT was highly specific and had a low
detection threshold, we suggest that these 27 specimens
were true plague-positive specimens, and that the plague
RDT is thus more sensitive than the two reference
assays.
Of the 30 Y pestis strains of the three biotypes and
from different continents, most tested positive by RDT,
indicating that this test is potentially applicable to plague
foci worldwide. The two strains that tested negative
proved to have total or part deletions of the cal] gene;
this deficiency seems to be due to the loss of the 110 kb
plasmid, probably during the long storage time of the
strains in the laboratory.
The invalid tests in the conditions of the remote sites
(no control liDe in 3% of the specimens) were due to
moistening of the dipsticks, caused by the humidity of
the air. This drawback can be overcome by improvement
of the RDT packaging, with waterproof bags instead of a
disposable test tube. We expect the shelflife ofthe RDT
in such conditions
to be about 2 years at room
temperature, on the basis of stability after 21 days at
60°C.21 The test kit should therefore tolerate storage at
room temperature for long periods in plague endemic
zones. The other discordant results were due either to
weak positive bands that were not seen in the field, or to
a mlsmterpretatlon ofthe control and pOSltlve lIDes. ThlS
difficulty could be solved by a second training session
focusing on interpretation of the test results.
...
Dunng non-central evaluatlon of the RDT, thlS test
was applied to the diagnosis of pneumonic plague as well
as bubonic plague. Although
sputum contains high
concentrations of FI antigen, we thought that its density
and viscosity might inhibit antigen-antibody interactions
.
and obstruct the flow of lmmune complexes through the
dipstick. This difficulty was overcome by diluting sputum
samples with saline or PBS (1 in 2 to 1 in 10 dilution).
Since the sensitivity and specificity of the test with
samples from patients with confirmed and suspected
.
1
h.
pneumornc pague was ~gh, the RDT seems ~o b~ a
useful method
for rapld,
easy, and non-mvaSlve
confirmation of this disease. The availability of a reliable
onsite diagnostic test should result in rapid treatment of
patients and efficient administration of chemoprophylaxis
to the contact population; these measures should, in turn,
help to prevent the spread of pneumonic plague in
endemic countries or in cases of bioterrorist use.
215
ARTlCLES
Assessment
of the RDT
results
and of the users'
questionnaires
showed that the technical
transfer of the
test to remote pilot sites was successful and allowed the
reliable
diagnosis
of bubonic
and pneumonic
plague.
Th
find'
I d
I
fr om hea Ith sta ff
ese
mgs e to a genera request
k
I
fi
fi
li .
b
d
. 1 bl
wor mg m pague
OCl or
lS test to
e ma e aval a e at
their centres. Bacteriological
and EUSA tests are between
..
.
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Contrioutors
S Chanteau designedme study, developedanti-F1 Mabs and RDT,
planned me user's guide book, trained healm staff in me field, analysed
data, and wrote me manuscript. L Rahalison supervisedassaysat Central
Laboratory, trained healm staffin me field, and made me user's guide
book (French and Ma)agasylanguages). L Ralafiarisoaproduced RDT
kits, trained healm staffin me field, and did RDT and EUSA tests at me
Central Laboratory.] Foulon assessedRDT wim bacterial cultures and
did PCR. L Ratsifasoamananacoordinated me plague national contrai
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study, supervised o.fme assessment~f RDT wim b~cterial cultures, and
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pro)ect, supervlsedme ~evelopment and producnon afilie ann-F1 Mabs,
and wrote me manuscnpt.
Confli~ of interest statement
None declared.
Acknowledgments
We mank me US Naval Medical ResearchInstitute (Bemesda-MDUSA) which sharedwim us meir experiencein me development ofRDTs
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