Brazilian Journal of Medical and Biological Research (2007) 40: 535-543 535

Diagnosis of malaria in endemic areas of Venezuela

ISSN 0100-879X

Comparison of the diagnosis of malaria

by microscopy, immunochromatography
and PCR in endemic areas of Venezuela

H. Rodulfo1, 1Laboratorio de Genética Molecular, Departamento de Biomedicina,

M. De Donato1, Instituto de Investigaciones en Biomedicina y Ciencias Aplicadas,
R. Mora2, Universidad de Oriente, Cumaná, Venezuela
L. González3 2Banco de Sangre, Hospital J.G. Hernández, Puerto Ayacucho,

and C.E. Contreras4 Venezuela

3Gerencia de Saneamiento Ambiental y Malariología, Region XI, Carúpano,

Venezuela
4Instituto de Inmunología, Universidad Central de Venezuela, Caracas, Venezuela

Abstract

Correspondence Whole blood samples (N = 295) were obtained from different loca- Key words
M. De Donato tions in Amazonas and Sucre States, in Venezuela. Malaria was • Malaria
Departamento de Biomedicina diagnosed by microscopy, OptiMAL™ and polymerase chain reac- • Molecular diagnosis of
Instituto de Investigaciones en malaria
tion (PCR), with Plasmodium vivax, P. falciparum, and P. malariae
Biomedicina y Ciencias Aplicadas • Microscopic diagnosis
(IIBCA), Universidad de Oriente
being detected when possible. We identified 93 infections, 66 of
of malaria
Cerro del Medio which were caused by P. vivax, 26 by P. falciparum, and 1 was a
• Polymerase chain reaction
Cumanás 6101 mixed infection. No infection caused by P. malariae was detected.
• OptiMAL
Venezuela The sensitivity and specificity of each diagnostic method were high:
Fax: +58-293-452-1297 95.7 and 97.9% for microscopy, 87.0 and 97.9% for OptiMAL, and
E-mail: [email protected] 98.0 and 100% for PCR, respectively. Most samples (72.2%) showed
more than 5000 parasites/µL blood. The sensitivity of the diagnosis by
Research supported by the Fondo
Nacional de Ciencia, Tecnología
microscopy and OptiMAL decreased with lower parasitemia. All
e Innovación (FONACIT, samples showing disagreement among the methods were reevaluated,
No. PEM-2001001621). but the first result was used for the calculations. Parasites were
detected in the 6 false-negative samples by microscopy after the
second examination. The mixed infection was only detected by PCR,
while the other methods diagnosed it as P. falciparum (microscopy) or
Received October 5, 2005
P. vivax (OptiMAL) infection. Most of the false results obtained with
Accepted January 9, 2007
the OptiMAL strip were related to the P. falciparum-specific band,
including 3 species misdiagnoses, which could be related to the test
itself or to genetic variation of the Venezuelan strains. The use of the
microscopic method for malaria detection is recommended for its low
cost but is very difficult to implement in large scale, population-based
studies; thus, we report here more efficient methods suitable for this
purpose.

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536 H. Rodulfo et al.

Introduction as an antibody against the Plasmodium aldo-

lase (4), and OptiMAL (DiaMed, under li-
Malaria is the most important human cense from Flow, Inc., Portland, OR, USA)
parasitic disease throughout the tropical and which is based on the detection of the para-
subtropical regions of the world due to is site lactate dehydrogenase, an enzyme pro-
high prevalence and mortality rate. In Ven- duced only by live parasites, and not a re-
ezuela, malaria is also a great concern, with sidual metabolite such as parasite histidine-
46,244 cases being reported in 2004, mainly rich protein 2, which can thus detect active
from Sucre (10.26%), Bolivar (59.63%), and infection, being useful for the monitoring of
Amazonas (23.18%) States (1). Detailed antimalarial drug therapies (5,6).
knowledge of the incidence and transmis- Finally, the use of molecular biology for
sion dynamics of the Plasmodium species in the diagnosis of malaria has proved to be
our region is the key to design effective highly sensitive for the detection of the dis-
measures for malaria control and the treat- ease, but the equipment required is not widely
ment schemes to be used. Medical and mi- available in many of the endemic areas and
croscopic diagnoses are universally used for the protocol is more complex and needs
the collection of epidemiological data. The better trained technicians. The use of the
standard microscopic diagnosis of malaria polymerase chain reaction (PCR), however,
requires a relatively short time when the is extremely useful for decision making in
parasites are present in high numbers (≥1000 disease control and treatment, for example,
parasites/µL), but if parasitemia is very low, for the detection of mixed infections which
more than 10 min are needed to examine play a modulatory role in the severity of the
each slide, which is often the case in en- symptoms (2,7,8). The greatest advantage,
demic areas for malaria or when drugs are however, is the ability of PCR to detect
taken inappropriately (2). infections with parasitemia as low as 5 para-
On the other hand, the immunodiagnos- sites/µL blood (9,10). However, there are
tic tests used for the detection of Plasmo- some problems with false-negative results
dium are easy to use and can produce fast when the DNA isolation protocol is not ap-
results, but they usually have lower sensitiv- propriate (5,11); for example, not remov-
ity and specificity than conventional micro- ing the inhibitors from the sample and not
scopic examination. Many of them have been preventing the action of enzymes that de-
designed to specifically detect P. falciparum grade DNA.
and some of them can detect the other ma- Due to the increase of malaria cases in
laria species non-specifically. Among the the last years in the regions of Venezuela
most common immunodiagnostic tests avail- where malaria is endemic, we decided to
able are the Parasight F-test (Becton-Dick- carry out a study to evaluate three different
inson Advanced Diagnosis, Franklin Lakes, diagnostic methods applied to individuals
NJ, USA) which detects P. falciparum spe- from Sucre and Amazonas States, which
cifically using an IgG1 monoclonal anti- represent the extremes of the Southeastern
body that recognizes the parasite histidine- endemic region of the country, with the high-
rich protein 2 (3); the ICT Malaria Pf/Pv est prevalence values in Venezuela, after
(AMRAD, Frenchs Forest, New South Bolivar State.
Wales, Australia) and its new version NOW
ICT Malaria Pf/Pv (Binax, Inc., Portland, Material and Methods
ME, USA), which work according to the
same principle but use an IgM monoclonal We evaluated three diagnostic methods
antibody specific for P. falciparum as well for the detection of malaria infections. A

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Diagnosis of malaria in endemic areas of Venezuela 537

total of 295 samples were analyzed, 14 of a well of a microtiter plate previously hy-
which came from Cumaná, 41 from Yagua- drated with buffer. A nitrocellulose strip is
raparo, 100 from Cangua in Sucre State, a P. placed in the well, and the blood is drawn up
vivax endemic area, 17 from Puerto by capillary action. After the blood is taken
Ayacucho, 110 from San Fernando de up completely (about 10 min), the strip is
Atabapo, and 13 from Santa Barbara in transferred to the next well with buffer and
Amazonas State, a P. vivax/P. falciparum allowed to clear. The test contains a positive
endemic area (Figure 1). Samples from
Cangua, San Fernando de Atabapo, and Santa
Barbara were taken at random from indi-
viduals living in those communities; the rest
of the samples were obtained by active
searches for positive samples at different
places, except for the samples from Cumaná,
which were from blood bank donors known
to be free of malaria infection and were used
as negative controls. Blood samples were
taken intravenously from symptomatic and
asymptomatic individuals of both genders
and different age groups, who agreed to
participate in this study by signing a consent
form, and who received an immediate diag-
nostic result (both microscopic and OptiMAL
tests) and the corresponding treatment.
Samples to be used for PCR were stored
frozen with EDTA at -20ºC until use. Two
drops of blood were also taken from the ear
to prepare thick and thin smears for micro-
scopic diagnosis after standard Giemsa stain-
ing, by analyzing the slides under a 100X
objective. Samples with no visible parasites
after scoring 100 fields were considered to
be negative for this test. Parasitemia (para-
sites/µL) was determined by counting the
number of parasites present in the thick blood
smear in relation to 500 white blood cells
and using 8000 white cells per µL of blood
(5) as a reference.
Additionally, an immunochromatograph-
ic diagnosis was carried out using the
OptiMAL® strip (Flow Inc., Portland, OR,
USA) according to manufacturer instruc-
tions. This test is based on a membrane
coated with monoclonal antibodies specific
for parasite lactate dehydrogenase, an en-
zyme produced by metabolizing malaria Figure 1. Localization of the populations of Sucre and Amazonas States, Venezuela, where
parasites. Briefly, a drop of blood is added to the samples were obtained.

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538 H. Rodulfo et al.

control line that should be present at the top concentration of 175 nM. The program used
of the strip to demonstrate that it is func- for the amplification included a modification
tional. A second line, adjacent to the positive of the original program (2), with the first 10
control line, indicates the presence of a Plas- cycles containing one step of denaturation at
modium parasite (P. falciparum, P. vivax, P. 94ºC for 1 min, one step of annealing at 54ºC
ovalae, or P. malariae). A third line indi- for 2 min and one step of polymerization at
cates a positive infection with P. falciparum, 72ºC for 2 min. Next, 35 cycles were used with
containing specific antigens. denaturation at 94ºC for 45 s, annealing at
Finally, molecular biology diagnosis was 56ºC for 90 s and polymerization at 72ºC for 1
carried out using the amplification of the min. A final extension at 72ºC for 10 min was
18S rRNA genes with primers specific for P. then carried out. This modification improved
falciparum, P. vivax, and P. malariae (2). the intensity of the signal while decreasing the
For this, total genomic DNA was extracted background noise of the amplification.
from blood samples using the Wizard Ge- Because no method was used as a gold
nomic DNA extraction kit (Promega Corp., standard, the sensitivity and specificity of
Madison, WI, USA) according to manufac- each test were estimated indirectly using
turer instructions. Briefly, 150 µL of blood maximum likelihood calculations, obtained
was mixed with 450 µL of cell lysis solution by Newton-Raphson and Expectation-Maxi-
and incubated for 10 min at room tempera- mization algorithms available in the Web-
ture. The white blood cells and parasites based software TAGS (http:/www.epi.ucdavis.
were isolated by centrifugation and then edu/diagnostictests/tags.html), which was de-
mixed with 150 µL nucleus lysis solution. signed for the evaluation of test accuracy in
Fifty microliters of protein precipitation so- the absence of a gold standard (12). To run
lution was added and vortexed for 20 s. The this program, the total number of individuals
protein pellet was eliminated by centrifuga- was divided into three populations: indi-
tion and the DNA was precipitated with viduals with unknown infection status (Can-
isopropanol, washed with 70% ethanol and gua, San Fernando de Atabapo, and Santa
then dried and hydrated with Tris-EDTA Barbara), those known to be infected and
buffer. We used a DNA sample from a pa- those known to be uninfected. The estimates
tient from Yaguaraparo as the PCR-positive of the prevalences used for input in the soft-
control for the amplification of the P. vivax ware were those calculated with the actual
fragment, as well as DNA from the cultured data and the estimates of the sensitivities and
strain FCB (Instituto de Inmunología, Uni- specificities of each method were the diag-
versidad Central de Venezuela, Caracas, nostic agreements calculated as sensitivities
Venezuela) of P. falciparum as the PCR- and specificities using the true-positive and
positive control for the amplification of this negative results. True results were identified
species fragment. by the agreement of all three methods, since
Amplification was done in a 25-µL vol- the false-negative and positive results were
ume using Taq polymerase buffer (10 mM found to be in agreement with the other two
Tris-HCl, pH 9.0, 50 mM KCl, 0.1% Triton X- diagnostic tests after a second test/examina-
100), 200 µM of each dNTP, 0.75 U Taq tion of all three methods. Mixed infections
polymerase (Promega Corp.), either 1 mM were scored as two separate infections to
MgCl2 (for P. vivax) or 3 mM MgCl2 (for P. facilitate the calculations, and the misdiag-
falciparum and P. malariae) and 2 µL of noses were considered to be false-negative
diluted DNA (about 100 ng). PCR was per- results since treatment for the wrong species
formed individually for each species using the will not eliminate the infection due to the
previously published oligonucleotides at a differences in parasite resistance (13).

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Diagnosis of malaria in endemic areas of Venezuela 539

Results
Table 1. Characteristics of the diagnostic methods for malaria in 295 blood samples.

We detected 93 cases of true malaria Method Positive results Negative results Sensitivity Specificity
infection among the 295 samples evaluated,
Malaria
66 of which were caused by P. vivax, 26 by Microscopy 91 205 95.7 97.9
P. falciparum and one was caused by both OptiMAL 83 213 87.0 97.9
species. In the population-based study car- PCR 92 204 98.0 100
ried out in Sucre, there was a prevalence of Plasmodium vivax
2% for P. vivax infection and no cases of P. Microscopy 65 230 97.1 99.1
falciparum or P. malariae, as expected from OptiMAL 64 231 85.4 98.2
PCR 66 229 98.7 100
previous reports. In the population-based
study carried out in Amazonas, the preva- Plasmodium falciparum
Microscopy 27 268 97.0 99.1
lences of 12.2% for P. vivax and 15.4% for OptiMAL 23 272 88.2 98.2
P. falciparum infection were detected. No PCR 26 269 98.7 100
infection caused by P. malariae was de-
Sensitivity and specificity were calculated according to the criteria stated in the
tected by PCR or microscopy. Material and Methods section.
The microscopic diagnosis for malaria
showed a high sensitivity (95.7%) but some-
what lower than for PCR (98.0%), and both
higher than for OptiMAL (87.0%), with these Table 2. Comparison between the microscopic and PCR diagnosis in the 295 blood
samples tested in the present study.
differences being statistically significant
(Table 1). Specificity values were high PCR Total
(≥98%) for all methods either for species-
specific or nonspecific diagnosis. Plasmodium Plasmodium Mixed Negative
Microscopy vivax falciparum infection results
When comparing the microscopic and
PCR methods, there was agreement in the Plasmodium vivax 62 1 0 2 65
diagnosis of 85 positive and 199 negative Plasmodium falciparum 0 23 1 3 27
Mixed infection 0 0 0 0 0
samples (Table 2). Two samples were posi- Negative results 3 1 0 199 203
tive for P. vivax by microscopy and negative Total 65 25 1 204 295
by PCR. Of these, one was a true-positive for
All PCR results were true results except for 1 false-negative result for Plasmodium
microscopy undetected by PCR, and one
vivax and 1 for P. falciparum.
was a false-positive for microscopy. Simi-
larly, three samples were positive for P.
falciparum by microscopy and negative by
PCR. Of these, 1 was a true-positive for Table 3. Comparison between the OptiMAL and PCR diagnosis in the 295 blood
microscopy which went undetected by PCR, samples from this study.

and 2 were false-positives for microscopy. PCR Total

The 2 false-negative results obtained by PCR
were reanalyzed (new DNA extraction and Plasmodium Plasmodium Mixed Negative
OptiMAL vivax falciparum infection results
amplification) and found to be positive. The
false-positive and -negative results obtained Plasmodium vivax 59 3 1 1 64
by microscopy were rescored by a highly Plasmodium falciparum 1 18 0 4 23
Mixed infection 0 0 0 0 0
trained microscopist (Malaria Reference Negative results 5 4 0 199 208
Center) who found either parasites or no Total 65 25 1 204 295
sign of infection, agreeing with both the first
and second PCR analysis, as well as with the All PCR results were true results except for 1 false-negative result for Plasmodium
vivax and 1 for P. falciparum.
OptiMAL test. Additionally, one sample

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540 H. Rodulfo et al.

which was originally scored as positive for was shown to be a P. vivax false-negative
P. vivax by microscopy was rescored as result by PCR. Similarly, four samples found
positive for P. falciparum (species misdiag- to be positive for P. falciparum by OptiMAL,
nosis), agreeing with the PCR and OptiMAL failed to be diagnosed by PCR. Three of
result. Finally, 3 samples infected with P. these were found to be negative when re-
vivax and one with P. falciparum were diag- tested by PCR and OptiMAL, and one was
nosed as negative by microscopy. Rescoring the true-positive not detected by PCR dis-
of these samples agreed with the PCR and cussed above. Additionally, three samples
OptiMAL results. which originally tested positive for P. vivax
When comparing the OptiMAL and PCR and one that tested positive for P. falciparum
methods (Table 3), we found agreement in were retested by OptiMAL and PCR and
the diagnosis of 77 positive and 199 nega- found to be positive for P. falciparum and P.
tive samples. One sample positive for P. vivax, respectively (species misdiagnosis),
vivax by OptiMAL and negative by PCR agreeing with the original PCR and micro-
scopic results. Finally, 5 samples infected
with P. vivax and 4 with P. falciparum were
Table 4. Comparisons between the OptiMAL and microscopic diagnosis in the 295 diagnosed as negative by OptiMAL. Retest-
blood samples tested in the present study. ing of these samples agreed with the original
Microscopy Total
PCR and microscopic results. Mixed infec-
tion with P. vivax/P. falciparum was de-
Plasmodium Plasmodium Mixed Negative tected as a P. vivax infection by OptiMAL
OptiMAL vivax falciparum infection results
and as a P. falciparum infection by micros-
Plasmodium vivax 60 2 0 2 64 copy. The results for the 28 (9.5%) samples
Plasmodium falciparum 1 20 0 2 23 with disagreement in the diagnosis by any
Mixed infection 0 0 0 0 0 method were concordant after retesting.
Negative results 4 5 0 199 208
Total 65 27 0 203 295
When comparing the OptiMAL and mi-
croscopic methods (Table 4), there was agree-
ment in the diagnosis of 80 positive and 199
negative samples. Two samples were found
to be positive for P. vivax by OptiMAL and
Table 5. Sensitivity of each diagnostic method according to parasitemia. negative by microscopy. Similarly, one sam-
Parasitemia True- False- Total Diagnostic
ple found to be positive for P. falciparum by
positive negative agreement OptiMAL was diagnosed as P. vivax by
microscopy; all three samples were species
Microscopy
misdiagnoses by OptiMAL. Additionally, of
>5000 parasites/µL 68 0 68 100
500-5000 parasites/µL 17 2 19 89.5 4 samples that were negative by microscopy
50-500 parasites/µL 3 4 7 42.9 2 tested positive for P. vivax and 2 for P.
OptiMAL falciparum, while of the 9 negative samples
>5000 parasites/µL 63 5 68 92.7 that tested negative by OptiMAL 4 tested
500-5000 parasites/µL 14 5 19 73.7 positive for P. vivax and 5 for P. falciparum.
50-500 parasites/µL 3 4 7 42.9
Most of the samples (72.2%) contained
PCR more than 5000 parasites/µL blood, while
>5000 parasites/µL 67 1 68 98.5
500-5000 parasites/µL 18 1 19 94.7
20.2% were between 500-5000 parasites/µL
50-500 parasites/µL 7 0 7 100 and 7.4% showed 50-500 parasites/µL and
none was found showing fewer than 50 para-
These values were calculated according to the criteria stated in the Material and
sites/µL (Table 5). The diagnostic agree-
Methods section.
ment of the microscopic examination and

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Diagnosis of malaria in endemic areas of Venezuela 541

OptiMAL test decreased with lower parasi- In addition, the lower sensitivity found
temia, but the PCR test showed the highest for P. falciparum antigens detected by the
diagnostic agreement when samples had 50- antibodies used on the OptiMAL strips, in
500 parasites/µL. The mixed infection this study, agrees with previous reports (17,
showed a high number of P. vivax parasites 18,20,24) with 6-57% differences in sensi-
(higher than 5000 parasites/µL) but a low tivity but with specificities similar to those
number of P. falciparum parasites (less than found in our study. Moody (4), in a large
500 parasites/µL). study with 636 patients with malaria symp-
toms from Sub-Saharan Africa, showed sen-
Discussion sitivities for the OptiMAL test of 96, 95.3,
57 and 47% for P. vivax, P. falciparum, P.
Even though conventional microscopy is ovale, and P. malariae, respectively, and
the reference method and the one most used specificities of 100% for P. falciparum and
for the diagnosis of Plasmodium spp, its 94% for the other species, when compared
sensitivity and specificity are limited to the with the microscopic diagnosis. They sug-
number of tests that can be analyzed per gested that the lower sensitivity of the tests
microscopist and his/her training, especially for P. falciparum infections were due to the
for low-parasite densities, when more time fact that only gametocytes at low densities
is needed for an accurate diagnosis (2,14). were present.
These limitations could explain the false Miller et al. (27), evaluating the treat-
results obtained in the microscopic diagno- ment of malaria by OptiMAL and conven-
sis of the endemic populations of Sucre and tional microscopy in 12 patients from Thai-
Amazonas. land infected with P. falciparum without
Zaman et al. (15) suggested that the great- clinical complications, found that the color
est disadvantage of the microscopic diagno- intensity in the reactive strips for the
sis is the possibility of misdiagnosis of Plas- OptiMAL test decreased with the levels of
modium species, particularly for low parasi- parasitemia, also showing that sensitivity
temia, mixed infections and when only ring was 88% when the density of gametes was
forms are seen. Postigo et al. (16) reported ≥100/µL and 35% for densities of <100/µL.
that false-negative results by microscopy for This agrees with our results regarding para-
P. vivax are probably due to very low parasi- sitemia and the sensitivity of OptiMAL.
temia which is very difficult to detect by However, even at high parasitemia (>5000
routine microscopic methods. This agrees parasites/µL), 5 false-negative results were
with the present study, where most of the reported for this test, which showed lower
false-negative results were obtained for para- sensitivities than the other two methods at
sitemia lower than 500 parasites/µL. any parasitemia level.
The significantly lower sensitivity of the The OptiMAL test shows sensitivity simi-
OptiMAL test agrees with many reports from lar to those reported for other dipsticks, such
Afghanistan, Turkey, Kuwait, Honduras, and as ParaSight and ICT Malaria Pf/Pv, which
Peru (17-21) in which the test showed sensi- ranged from 86-100 and 58-96%, respec-
tivities ranging from 79.3 to 94% but speci- tively (3,20,28-33). Iqbal et al. (20) com-
ficities ranging from 97 to 100%. Other stud- pared the OptiMAL and ICT Malaria Pf/Pv
ies from Thailand, USA, Honduras and Co- tests, showing that OptiMAL had a higher
lombia (22-25) have shown sensitivities and sensitivity for the diagnosis of P. vivax (79%)
specificities close to 100%, while one study and P. falciparum (87%), than ICT Malaria
from Canada (26) showed a sensitivity of Pf/Pv (58 and 81% respectively), but with
29.1 but a specificity of 95.6%. both tests showing specificities higher than

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542 H. Rodulfo et al.

97%. On the other hand, Grobusch et al. tion-based studies that will allow the estab-
(34), comparing the ParaSight, ICT Malaria lishment of better relationships among the
Pf/Pv and OptiMAL tests, found similarly epidemiological factors that can affect the
high sensitivities for the first two (95.1 and endemicity of malaria. In addition, the use of
95.7%, respectively) and significantly lower PCR-based techniques is most valuable for
sensitivity for OptiMAL (76.2%), although the evaluation of drug treatments as well as
all tests showed again specificities higher for diagnosing the emergence of drug resis-
than 97%. tance in specific areas. Thus, we recommend
Coleman et al. (35) evaluated the effi- the incorporation of the PCR technique into
cacy of the ICT Malaria Pf/Pv in a large the Reference Centers of government insti-
study of 559 asymptomatic patients from an tutions responsible for malaria control, the
endemic area of Thailand using conventional evaluation of diagnostic tests, the verifica-
microscopy as the reference method, and tion of the quality of the microscopic diag-
found that the sensitivity for P. falciparum nosis at each diagnostic center, as well as the
dropped from 100 to 23.3% when the parasi- monitoring of the emergence of resistant
temia was below 500 trophozoites/µL. For strains of Plasmodium parasites.
P. vivax infections, the sensitivity dropped
from 66.7% to 0 at parasitemia below 500 Acknowledgments
trophozoites/µL. In our case, diagnostic
agreement for OptiMAL, was similar for The authors wish to acknowledge to
both species but also dropped drastically in Gerencia de Saneamiento Ambiental y Ma-
samples with <500 parasites/µL. lariología, Region XI, Carupano, Venezuela
Our results allow us to recommend the who donated the OptiMAL kits, as well as
use of both microscopy and PCR for the Isaurea Quijada (IIBCA, Universidad de
characterization of any diagnostic test, since Oriente, Cumana, Venezuela) and Melcenia
by using both techniques we can obtain 100% Moreno (Instituto de Biomedicina, Ministerio
certainty of the results. It is especially im- de Ciencia, Tecnologia e Innovacion, Cara-
portant to use PCR for the species-specific cas, Venezuela) who helped at various stages
diagnosis to evaluate the treatment of ma- in the research. We also wish to thank Dr.
laria, since resistance to the drugs used is Frances Osborn for revising and correcting
species-specific, so that the treatment of one the English text. Finally, we wish to thank
type of malaria will probably not cure the Elier Diaz (IIBCA, Universidad de Oriente,
other (14). Cumana, Venezuela), who has been of great
We recommend the use of PCR for the assistance in this study, working beyond the
accurate diagnosis of infections in popula- call of duty in order to complete the mission.

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