Cir379 321.325
Evaluation of a Newly Developed Lateral FlowImmunoassay for the Diagnosis of Cryptococcosis
Mark D. Lindsley,1 Nanthawan Mekha,2 Henry C. Baggett,3 Yupha Surinthong,2 Rinrapas Autthateinchai,2Pongpun Sawatwong,3 Julie R. Harris,1 Benjamin J. Park,1 Tom Chiller,1 S. Arunmozhi Balajee,1 andNatteewan Poonwan2
1Mycotic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia; 2National Institute of Health, Department of Medical Sciences,Ministry of Public Health, Nonthaburi, Thailand; and 3International Emerging Infections Program, Thailand Ministry of Public Health–Centers forDisease Control and Prevention Collaboration, Nonthaburi, Thailand
Cryptococcosis is a common opportunistic infection of human immunodeficiency virus (HIV)–
infected individuals mostly occurring in resource-limited countries. This study compares the performance ofa recently developed lateral flow immunoassay (LFA) to blood culture and enzyme immunoassay (EIA) for thediagnosis of cryptococcosis.
Archived sera from 704 HIV-infected patients hospitalized for acute respiratory illness in Thailand
were tested for cryptococcal antigenemia using EIA. All EIA-positive and a subset of EIA-negative sera were tested byLFA, with results recorded after 5 and 15 minutes incubation. Urine from patients with LFA- and EIA-positive sera
was tested by LFA. Antigen results from patients with positive cryptococcal blood cultures were compared.
Of 704 sera, 92 (13%) were positive by EIA; among the 91 EIA-positive sera tested by LFA, 82 (90%)
and 87 (96%) were LFA positive when read after 5 and 15 minutes, respectively. Kappa agreement of EIA and LFA
for sera was 0.923 after 5 minutes and 0.959 after 15 minutes, respectively. Two of 373 EIA-negative sera were LFApositive at both time points. Of 74 urine specimens from EIA-positive patients, 52 (70.3%) were LFA positive. EIAwas positive in 16 of 17 sera from blood culture–positive patients (94% sensitivity), and all sera were positive by LFA(100% sensitivity).
A high level of agreement was shown between LFA and EIA testing of serum. The LFA is a rapid,
easy-to-perform assay that does not require refrigeration, demonstrating its potential usefulness as a point-of-careassay for diagnosis of cryptococcosis in resource-limited countries.
Cryptococcosis, caused by Cryptococcus spp., is one of the
90% of deaths from cryptococcal meningitis occur in
most common opportunistic infections among human
sub-Saharan Africa and Southeast Asia [2]. Primary re-
immunodeficiency virus (HIV)–infected individuals [1].
spiratory illness due to Cryptococcus, while uncommon
Globally, an estimated 1 million new cases of crypto-
in the United States and Europe, is more common in
coccal meningitis occur per year, with more than 600 000
other regions, such as Southeast Asia [2].
deaths. An estimated 88% of global cases and more than
Currently, cryptococcal diagnostics include micros-
copy and/or culture-based methods, or detection ofcryptococcal antigen (CrAg) in body fluids using eitherlatex agglutination (LA) or enzyme immunoassay (EIA)
Received 4 March 2011; accepted 29 April 2011.
Correspondence: S. Arunmozhi Balajee, MD, PhD, Mycotic Diseases Branch,
methods. Although culturing of the organism is con-
Centers for Disease Control and Prevention, Mailstop G11, 1600 Clifton Rd,
sidered the gold standard diagnostic method, it has poor
Atlanta, GA 30333 (
[email protected]).
sensitivity, requires a large quantity of specimen, and
Clinical Infectious Diseases
Published by Oxford University Press on behalf of the Infectious Diseases Society
requires laboratory infrastructure including electricity
of America 2011. This is an Open Access article distributed under the terms of the
(for centrifugation). Antigen tests such as LA or EIA
Creative Commons Attribution Non-Commercial License (http://creativecommons.
org/licenses/by-nc/2.5/), which permits unrestricted non-commercial use, distribution,
performed on cerebral spinal fluid or serum are highly
and reproduction in any medium, provided the original work is properly cited.
sensitive and specific diagnostic options that are less
labor- and time-intensive than culture [3, 4]. However,
LFA for Cryptococcal Diagnosis d CID 2011:53 (15 August) d 321
these methods require refrigeration, a cold chain for specimen
Control and Prevention and the Ethical Review Committee of
transport, and technical expertise; therefore, they are often
the Thailand Ministry of Health.
performed only in reference/diagnostic labs far removed from
All testing was performed at the National Institute of Health,
patients, potentially limiting their clinical utility. In addition, the
Nonthaburi, Thailand. The results of the LFA were compared
costs of these tests are not affordable for many clinics. As a re-
with those obtained from 2 commonly used diagnostic tests for
sult, cryptococcosis often goes undiagnosed in resource-limited
detecting Cryptococcus infection, blood culture and EIA. Blood
cultures were obtained from a subset of study participants as
Point-of-care tests (POCTs) show promise for enabling di-
a part of routine clinical care. Cultures were processed by au-
agnosis of infectious diseases in remote care centers in low-
tomated BacTAlert blood culture system (bioMe´rieux), and
resource countries. POCTs are assays that can produce results
pathogen identification of isolates from positive blood cultures
quickly, are simple to perform and interpret by personnel with
was performed using standard microbiology methods (mor-
no or minimal laboratory training, and ideally can be used
phological and physiological tests).
without cold chain or advanced laboratory equipment [5]. To-
Stored sera from all HIV-infected patients enrolled in the
day, reliable and affordable POCTs are available for the de-
study from 2004 to 2009 were tested by EIA, using the Premier
tection of hepatitis B infection [6], HIV infection [7], malaria
Cryptococcal Antigen enzyme immunoassay (Meridian Bio-
[8, 9], syphilis [10], cholera [11], and some neglected tropical
sciences). LFA testing was performed on serum specimens from
diseases [12–14]. Additional advantages of POCTs include re-
all patients with a positive serum EIA test and a random subset
duction of patient anxiety and waiting time and decreases in
of patients with a negative serum EIA test. Additionally, urine
patient loss to follow-up [15]. To serve the communities most in
from patients whose sera were positive by LFA and EIA were
need, the World Health Organization (WHO) recommended
tested by LFA. No LFA testing was performed on urine of pa-
that POCTs should be Affordable, Sensitive, Specific, User-
tients who had serum that tested EIA-negative.
friendly, Rapid, Equipment-free, and Delivered to those who
The EIA was performed according to the manufacturer's
need it (ASSURED) [16, 17].
protocol using 50 lL of specimen. For the EIA, wells with re-
In 2009, a lateral flow immunoassay (LFA) for the detection
actions of optical densities ,0.07 units were considered negative;
of cryptococcal antigen was developed by IMMY (Immuno-
$0.07 to ,0.100 was considered indeterminate; and $0.100 was
Mycologics) as a potential POCT for diagnosis of cryptococcal
considered positive. The LFA is a semiquantitative test system for
infection. The LFA is stable at room temperature, has a rapid
the detection of capsular polysaccharide antigens of Cryptococcus
turnaround time, requires very little technical skill, and can be
species complex (Cryptococcus neoformans and Cryptococcus
performed with minimal laboratory infrastructure. In this study,
gattii). The LFA kit consists of immunochromatic test strips
the performance of the LFA for CrAg detection was evaluated by
impregnated with monoclonal antibodies optimized to detect all
testing archived specimens from HIV-infected patients hospi-
4 cryptococcal serotypes and a diluent. The LFA kit can be stored
talized with acute respiratory illness in Thailand, and comparing
at room temperature for up to a year. To perform the assay,
these results with culture and EIA.
20 lL of patient specimen was mixed with 2 drops of diluentin a 2-mL screw cap microtube (Sarstedt AG). The LFA strip
was placed in the specimen and diluent cocktail and in-cubated at room temperature. Results were read after two
The serum and urine specimens used in this study were collected
incubation times: 5 minutes according to the manufacturer's
as part of a pneumonia etiology study that included HIV-
instructions and a prolonged incubation time of 15 minutes.
infected patients in Thailand, described elsewhere [18]. In brief,
The presence of 2 bands (control band and test band) in the test
patients admitted to 1 of 8 hospitals in Sa Kaeo or 12 hospitals in
zone of the LFA strip was interpreted as a positive result and
Nakhon Phanom provinces in Thailand between 2004 and 2009
a single band in the test zone (control band) was interpreted
were enrolled if they displayed any sign or symptom of active
as a negative result.
infection (temperature .38.2°C or ,35.5°C within 24 hours of
A positive blood culture result for C. neoformans was used as
admission; chills; abnormal total white blood cell count or dif-
the gold standard for confirmed Cryptococcus infection. Sensi-
ferential), and had evidence of lower respiratory illness (ab-
tivity was calculated among the group of patients with a positive
normal breath sounds, tachypnea, cough, sputum production,
blood culture result as: (number of patients with positive serum
or dyspnea). All patients provided informed consent for sample
LFA) / (number of patients with positive blood culture) * 100.
collection and use of samples for research purposes. Serum and
Specificity was not calculated in this group, as blood culture was
urine specimens were collected soon after hospital admission
not considered to be a method with sufficiently low false neg-
and immediately after study consent. This study was approved
ative rates. Agreement between serum or urine LFA results and
by the Institutional Review Board of the Centers for Disease
serum EIA results was calculated using the j statistic.
322 d CID 2011:53 (15 August) d Lindsley et al
LFA using a 5-minute incubation, 52 (70.3%) were positive,and 22 (29.7%) were negative. Among urine specimens from
A total of 704 HIV-infected study participants with available
63 patients with LFA-positive sera, 51 (81.0%) were LFA positive,
specimens were identified and were tested as outlined in Figure 1.
and 12 (19.1%) were LFA negative.
Blood culture was performed on 325 patient specimens, with 18
All 91 EIA-positive sera were also tested by LFA using an
of 325 yielding C. neoformans. Serum was not available from 1 of
extended incubation time. When the results of the serum LFA
the 18 culture-positive patients for analyses in the study; thus the
were read after 15 minutes, 5 additional sera became positive
EIA testing was performed on 17 culture-positive sera. Of the 17
(n 5 87; 95.6%). Of these 5 sera, only 1 came from a patient with
serum specimens tested, 16 were positive by EIA and 17 were
blood culture performed, which was negative for Cryptococcus
positive by LFA with a 5-minute incubation time yielding a sen-
spp. All EIA-negative specimens remained LFA negative after
sitivity of 94% and 100%, respectively. Thirteen of the 18 patients
the extended incubation period, increasing the j statistic to
whose blood culture was positive for Cryptococcus also had
a corresponding urine specimen, 12 of which were positive byLFA (92% sensitivity).
Sera from all 704 patients were tested using EIA; 92
(13.1%) were positive. No sera tested in this study gave in-
Resource-limited countries, especially those in sub-Saharan
determinate results by EIA. Of 91 EIA-positive sera that were
Africa and Southeast Asia, continue to experience a high in-
available for further analysis, 82 (90.1%) were positive by
cidence of cryptococcosis; managing this disease is a persistent
LFA with a 5-minute incubation time. Discordant results
public health challenge [2]. Although several CrAg tests are
between EIA and LFA were most often observed in serum
currently available for cryptoccoccal diagnosis, these tests are
exhibiting lower EIA values (data not shown). A random
not readily accessible in resource-limited settings, resulting in no
subset of 373 serum samples was selected from the 612 EIA-
diagnosis or underdiagnosis of these often fatal infections. In the
negative sera and tested using LFA: 371 of 373 (99.5%) were
present study, a recently developed cryptococcal assay, the LFA,
LFA negative, and 2 (0.5%) were LFA positive. The resultant
was evaluated for test sensitivity and agreement with other
j statistic describing agreement between LFA and EIA was
available diagnostic methods using archived serum and urine
0.923 (95% CI, .877–.967).
specimens from HIV-infected patients in Thailand. Results
Urine specimens were available from 74 patients whose sera
showed that, with serum specimens, the LFA was 100% sen-
were EIA positive. When these urine specimens were tested by
sitive when compared with the gold standard blood culture.
Flow chart of the specimen testing algorithm. A total of 704 sera were tested by cryptococcal antigen enzyme immunoassay (CrAg-EIA);
91 EIA-positive sera were tested by lateral flow immunoassay (LFA); a subset of CrAg-EIA–negative sera (n 5 373) was further tested by LFA. Specimensfrom Cryptococcus blood culture–positive patients (n 5 17) were tested by LFA and EIA. A total of 74 urine samples from serum CrAg-EIA–positivepatients were tested by LFA. HIV, human immunodeficiency virus.
LFA for Cryptococcal Diagnosis d CID 2011:53 (15 August) d 323
Additionally, the LFA had a high level of agreement with the
submitted to the United States Food and Drug Administration
cryptococcal EIA. When urine was evaluated, the LFA was
for approval (personal communication, Sean Bauman, IMMY).
found to be very sensitive (92%) when compared with blood
For POCTs to have maximum value in remote settings, the use
culture, and moderately sensitive (70.7%) when compared with
of minimally invasive, easily obtained, processing-free specimens is
optimal. This study was performed with sera and urine as test
One of the limitations of this study was that the specimens
specimens in a controlled laboratory setting (reference laboratory);
were collected from patients hospitalized with acute respiratory
however, in remote areas with insufficient technical expertise
illness for whom complete clinical details were not available,
or specimen processing capabilities, sera may not be the ideal
including whether they had meningitis. Patients with more in-
specimen type. The lower sensitivity of the LFA for urine
vasive infections (ie, meningitis) may have a higher burden of
compared with serum could be due to reduced excretion of
circulating organisms and therefore antigen, and this may im-
CrAg into the urine, compared with the blood. HIV infection
pact the performance of tests that measure CrAg. Accordingly,
[20] and treatment of HIV with the antiretroviral drugs te-
discrepant results between serum EIA and LFA were more
nofovir and indinavir [21, 22] have been previously demon-
often observed in serum with lower EIA optical density values,
strated to reduce the glomerular filtration rate and,
possibly reflecting the presence of low levels of circulating
potentially, reduce the amount of CrAg excreted in the urine.
Urine has been used for antigen detection in other fungal
This study was performed in a reference laboratory and
[23–25] and nonfungal diseases [26, 27]. The kinetics of ex-
therefore the performance of the LFA in a field or hospital set-
cretion of CrAg antigen in urine is unclear, and additional
ting is unclear at this time. However, the LFA was simple to
studies testing CrAg in urine need to be performed, including
perform and did not require any additional laboratory equip-
studies where urine is collected under controlled conditions.
ment. Incubation could be performed at room temperature and
Another minimally invasive specimen type is whole blood
the assay itself could be accomplished in 3 easy steps since this
from a finger stick. Point-of-care assays for whole blood have
method does not require any pretreatment of specimen (to re-
been developed for viral and parasitic diseases [8, 9, 12, 13], all of
move rheumatoid factor). Finally, the LFA yielded results that
which have been useful in resource-limited countries. Further
were unambiguous. Thus, the LFA has many characteristics that
studies evaluating the LFA using finger-stick blood would en-
may make it a valuable POCT in resource-limited areas. In ad-
hance the accessibility of this assay. Additionally the test's cost,
dition, this study demonstrates that the CrAg LFA satisfies most
ranging from $1.25 to $2.50 per test (depending on the country
of the WHO ASSURED criteria [16, 17]: specifically, the assay is
and volume of purchase) is based on the World Bank's list of
sensitive, user-friendly (small specimen volume, simple to use),
economies, thereby ensuring affordability to the countries most
rapid (10–15 minutes to perform), and equipment-free (including
no requirement for refrigeration). The rapid turnaround time
In summary, this study demonstrates that the LFA is a sensi-
will allow diagnoses to be potentially provided during patient
tive test for Cryptococcus spp compared with the gold standard
visits, allowing treatment to begin immediately if warranted.
culture, and has a high level of agreement with EIA. Given the
Recently, Jarvis et al [19] strongly recommended the integration
ease of use, temperature stability, minimal requirements for
of CrAg screening into national antiretroviral treatment pro-
laboratory infrastructure, and potential low cost of this test, the
grams in sub-Saharan Africa to reduce the human and economic
LFA shows great promise as a POCT for diagnosis of crypto-
costs due to the disease [19]. Although not tested in this study,
coccosis. The availability of this assay as a POCT for use in
the LFA may also have utility as a screening tool for early
remote locations could have a meaningful impact on crypto-
diagnosis of cryptococcosis.
coccal diagnosis.
Extending the incubation time for sera from 5 to 15 minutes
improved the sensitivity of the LFA when compared with EIA,
thereby increasing agreement between the EIA and the LFA.
Since the time of this study, the manufacturer of the LFA has
The authors express their gratitude to Apiwat Lapamnouysup for the
technical assistance in this study, and acknowledge Immuno-Mycologics,
modified the testing protocol, now recommending testing twice
Inc. (Norman, Oklahoma), for the donation of the lateral flow immuno-
the volume of patient specimen, reducing the recommended
assay kits for this study.
specimen:diluent ratio from 1:5 to 1:2, increasing the amount
The findings and conclusions in this article are those of the author(s)
and do not necessarily represent the views of the CDC. The use of product
of conjugate in the chromatographic strip, and increasing the
names in this manuscript does not imply their endorsement by the US
recommended incubation time of the LFA to 10 minutes. Cur-
Department of Health and Human Services.
rently the LFA has CE marking (a mandatory conformance
Potential conflicts of interest. All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
marking for the European Economic Area) for use in the Euro-
Conflicts of Interest. Conflicts that the editors consider relevant to the content
pean Union and in countries that use CE approval and has been
of the manuscript have been disclosed in the Acknowledgments section.
324 d CID 2011:53 (15 August) d Lindsley et al
14. Peeling RW, Mabey D. Point-of-care tests for diagnosing infections in
the developing world. Clin Microbiol Infect 2010; 16:1062–9.
1. Ong EL. Common AIDS-associated opportunistic infections. Clin Med
15. Loubiere S, Moatti JP. Economic evaluation of point-of-care diagnostic
2008; 8:539–43.
technologies for infectious diseases. Clin Microbiol Infect 2010; 16:
2. Park BJ, Wannemuehler KA, Marston BJ, Govender N, Pappas PG,
Chiller TM. Estimation of the current global burden of cryptococcal
16. Mabey D, Peeling RW, Ustianowski A, Perkins MD. Diagnostics for
meningitis among persons living with HIV/AIDS. AIDS 2009; 23:525–30.
the developing world. Nat Rev Microbiol 2004; 2:231–40.
3. Gade W, Hinnefeld SW, Babcock LS, et al. Comparison of the PRE-
17. WHO. Mapping the landscape of diagnostics for sexually transmitted
MIER cryptococcal antigen enzyme immunoassay and the latex ag-
infections. Key findings and recommendations.: UNICEF/UNDP/
glutination assay for detection of cryptococcal antigens. J Clin
World Bank/WHO, 2004.
Microbiol 1991; 29:1616–9.
18. Olsen SJ, Thamthitiwat S, Chantra S, et al. Incidence of respiratory
4. Sekhon AS, Garg AK, Kaufman L, et al. Evaluation of a commercial
pathogens in persons hospitalized with pneumonia in two provinces
enzyme immunoassay for the detection of cryptococcal antigen. My-
in Thailand. Epidemiol Infect 2010; 138:1811–22.
coses 1993; 36:31–4.
19. Jarvis JN, Wainwright H, Harrison TS, Rebe K, Meintjes G. Pulmonary
5. Anderson D, Crowe S, Garcia M. Point-of-care testing. Curr HIV/AIDS
cryptococcosis misdiagnosed as smear-negative pulmonary tubercu-
Rep 2011; 8:31–7.
losis with fatal consequences. Int J Infect Dis 2010; 14(Suppl 3):
6. Lin YH, Wang Y, Loua A, et al. Evaluation of a new hepatitis B virus
surface antigen rapid test with improved sensitivity. J Clin Microbiol
20. Krawczyk CS, Holmberg SD, Moorman AC, Gardner LI, McGwin G Jr.,
2008; 46:3319–24.
Group HIVOS. Factors associated with chronic renal failure in HIV-
7. Pavie J, Rachline A, Loze B, et al. Sensitivity of five rapid HIV tests on
infected ambulatory patients. AIDS 2004; 18:2171–8.
oral fluid or finger-stick whole blood: a real-time comparison in
21. Campbell LJ, Ibrahim F, Fisher M, Holt SG, Hendry BM, Post FA.
a healthcare setting. PLoS One 2010; 5:e11581.
Spectrum of chronic kidney disease in HIV-infected patients. HIV Med
8. Ghanchi NK, Beg MA, Hussain R. Estimation of parasite load using
2009; 10:329–36.
rapid diagnostic test ICT Now Malaria P.f/P.v in Plasmodium falcipa-
22. Horberg M, Tang B, Towner W, et al. Impact of tenofovir on renal
rum malaria. Scand J Infect Dis 2009; 41:597–601.
function in HIV-infected, antiretroviral-naive patients. J Acquir Im-
9. Quintana M, Piper R, Boling HL, et al. Malaria diagnosis by dipstick
mune Defic Syndr 2010; 53:62–9.
assay in a Honduran population with coendemic Plasmodium falci-
23. Durkin M, Witt J, Lemonte A, Wheat B, Connolly P. Antigen assay with
parum and Plasmodium vivax. Am J Trop Med Hyg 1998; 59:868–71.
the potential to aid in diagnosis of blastomycosis. J Clin Microbiol
10. Peeling RW, Ye H. Diagnostic tools for preventing and managing
2004; 42:4873–5.
maternal and congenital syphilis: an overview. Bull World Health
24. Swartzentruber S, Rhodes L, Kurkjian K, et al. Diagnosis of acute
Organ 2004; 82:439–46.
pulmonary histoplasmosis by antigen detection. Clin Infect Dis 2009;
11. Mukherjee P, Ghosh S, Ramamurthy T, et al. Evaluation of a rapid
immunochromatographic dipstick kit for diagnosis of cholera em-
25. Wheat LJ. Current diagnosis of histoplasmosis. Trends Microbiol
phasizes its outbreak utility. Jpn J Infect Dis 2010; 63:234–8.
2003; 11:488–94.
12. Rocha A, Braga C, Belem M, et al. Comparison of tests for the detection
26. Dirven K, Ieven M, Peeters MF, van der Zee A, De Schrijver K,
of circulating filarial antigen (Og4C3-ELISA and AD12-ICT) and ul-
Goossens H. Comparison of three Legionella urinary antigen assays
trasound in diagnosis of lymphatic filariasis in individuals with mi-
during an outbreak of legionellosis in Belgium. J Med Microbiol
crofilariae. Mem Inst Oswaldo Cruz 2009; 104:621–5.
2005; 54:1213–6.
13. Weil GJ, Lammie PJ, Weiss N. The ICT Filariasis Test: a rapid-format
27. Neuman MI, Harper MB. Evaluation of a rapid urine antigen assay for
antigen test for diagnosis of bancroftian filariasis. Parasitol Today 1997;
the detection of invasive pneumococcal disease in children. Pediatrics
LFA for Cryptococcal Diagnosis d CID 2011:53 (15 August) d 325
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Mechanism of TranscriptionalBursting in Bacteria Shasha Chongyi Hao Ge,and X. Sunney 1Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA2Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA3Biodynamic Optical Imaging Center (BIOPIC), Peking University, Beijing 100871, China4Beijing International Center for Mathematical Research (BICMR), Peking University, Beijing 100871, China5Co-first author*Correspondence:
Estatutos Sociales Capítulo Primero De la denominación, domicilio, idiomas oficiales, duración y objeto Artículo Primero - La Asociación Civil se denominará RED DE AGRICULTURA SOSTENIBLE, denominación que deberá ir siempre seguida de las palabras, "Asociación Civil" o de su abreviatura "A.C.". Artículo Segundo – El domicilio de la Asociación será la Ciudad de México, Distrito Federal, y no se entenderá cambiado por el establecimiento de oficinas o dependencias, o por la realización de actividades en cualquier otro lugar de la República de México o en el extranjero. Artículo Tercero – Los idiomas oficiales de la Asociación serán el español y el inglés. Los principales documentos y materiales publicados por la Asociación estarán disponibles en ambos idiomas. Todos los documentos preparados o a ser presentados ante la Asociación, tales como estándares y solicitud de trámites estarán disponibles tanto en español como en inglés por parte de la Asociación. Estos estatutos han sido redactados tanto en inglés como en español, y ambas versiones son igualmente vinculantes y constituyen un solo documento; en el entendido, no obstante, que en caso de diferencias entre una y otra versión, la que prevalecerá será la versión en español. Artículo Cuarto – La duración de la Asociación Civil será de tiempo indefinido. Artículo Quinto – La Asociación Civil tiene por objeto llevar a cabo las siguientes actividades: