Veterinary Parasitology 205 (2014) 338–342
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short Communication
Evaluation of a novel dried blood spot collection device (HemaSpotTM ) to test blood samples collected from dogs for antibodies to Leishmania infantum Alexa C. Rosypal a,∗ , Leanne D. Pick a , Jaime O. Esquivel Hernandez a , David S. Lindsay b a Department of Natural Sciences and Mathematics, College of Science, Technology, Engineering and Mathematics, Johnson C. Smith University, Charlotte, NC 28216, USA b Department of Biomedical Science and Pathology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, 1410 Prices Fork Road, Blacksburg, VA 24061, USA
a r t i c l e
i n f o
Article history: Received 3 June 2014 Received in revised form 22 July 2014 Accepted 29 July 2014 Keywords: Leishmania infantum Dog Dried blood spot Antibody
a b s t r a c t Collection of blood samples from veterinary and wildlife patients is often challenging because the samples have to be collected on farm or in the wild under various environmental conditions. This poses many technical problems associated with venipuncture materials, their safe use and disposal, transportation and processing of collected samples. Dried blood spot (DBS) sample collection techniques offer a simple and practical alternative to traditional blood collection methods to obtain blood samples from animals for parasite antibody evaluation. The DBS collection devices are compact, simple to use, and are particularly useful for large number of samples. Additionally, DBS samples take up less space and they are easier to transport than traditional venipuncture-collected blood samples. Visceral leishmaniasis (VL) is a potentially fatal parasitic disease of dogs and humans and it is frequently diagnosed by antibody tests. Immunochromatographic tests (ICT) for antibodies to Leishmania infantum are commercially available for dogs and they produce qualitative results in minutes. Measurement of canine antibodies to L. infantum with the ICT using traditional venipuncture has been validated previously, but the use of DBS samples has not been evaluated using this method. The purpose of the present study was to determine the ability of DBS samples to detect antibodies to L. infantum in dogs using a commercial ICT assay. One hundred plasma samples from dogs experimentally infected with the LIVT-1 strain of L. infantum were collected by venipuncture and frozen. Individual samples were thawed, and then 80 l plasma (2 drops) was aliquotted onto the 8-spoked disk pad on individual DBS sample collection devices (HemaSpotTM , Spot-On Sciences, Austin, TX), dried, and stored in the dark at room temperature. After one month and six months, respectively, 2 spokes of the 8 spokes of the disk pad of each DBS sample were removed and eluted in 200 l PBS. The eluate was used to test for antibodies in the ICT and compared to ICT results using thawed plasma (same initial source). Sensitivity and specificity of the ICT using DBS were determined by using ICT results from traditional blood collection samples for comparison. After 1 month, DBS samples showed 100% sensitivity and specificity when
∗ Corresponding author. Tel.: +1 704 378 1206; fax: +1 704 378 1213. E-mail address: [email protected] (A.C. Rosypal). http://dx.doi.org/10.1016/j.vetpar.2014.07.031 0304-4017/© 2014 Elsevier B.V. All rights reserved.
A.C. Rosypal et al. / Veterinary Parasitology 205 (2014) 338–342
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compared to ICT results on thawed plasma samples collected by traditional venipuncture. After six months storage at room temperature, DBS samples demonstrated 79% sensitivity and 100% specificity compared to traditional blood collection. Results from this study indicate that dried blood spot collection may be a useful tool for screening dogs for antibodies to L. infantum with the ICT assay. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Visceral leishmaniasis (VL) is a sand fly vector-borne disease of dogs and humans caused by infection with parasites in the Leishmania donovani complex, which includes Leishmania infantum. In dogs, L. infantum infections may result in fatal disease and infected domestic dogs are the most important reservoir host for human infections. Leishmaniasis caused by L. infantum is endemic in many parts of the world including parts of Africa and Asia, the Middle East, the Mediterranean region, North and South America. In the United States, L. infantum is established in the foxhound breed (Rosypal et al., 2003a) and maintained by maternal transmission in foxhounds in North America (Rosypal et al., 2005c). Serological detection of specific anti-Leishmania antibodies is the most useful and most routinely performed diagnostic test for canine leishmaniasis (CanL) (Kalayou et al., 2011; Solano-Gallego et al., 2009; Gradoni and Gramiccia, 2008). Several serological tests are available for CanL including, IFAT, ELISA, DAT and immunochromatographic tests (ICT) (Solano-Gallego et al., 2009; Rosypal et al., 2003a). Immunochromatographic devices are particularly useful because they are simpler to perform compared to other traditional antibody tests and they produce a qualitative result in minutes (Kalayou et al., 2011; Rosypal et al., 2005a; Solano-Gallego et al., 2009). In a study of experimentally infected dogs using a United States’ isolate of L. infantum, a commercial ICT identified more infected dogs compared to IFAT (Rosypal et al., 2005a). Traditional blood collection is the current “gold standard” for collecting samples to measure biomarkers such as antibodies (McDade, 2014). Traditional collection of serum or plasma involves venipuncture which must be performed by trained personnel, centrifugation to separate the liquid fraction from blood cells and storage by refrigeration or freezing. For research purposes, dried blood spots (DBS) are increasingly recognized as a preferable alternative to traditional venipuncture for sample collection (McDade, 2014; Ostler et al., 2014). The use of DBS could relieve major barriers to blood collection, storage and transportation. Blood collection by DBS can be obtained from a finger or ear-prick or toe nail snip which can eliminate the need for specifically trained technicians, additional supplies (needles, collection and storage tubes, syringes), and the need for post-collection processing required for traditional blood collection (Fajardo et al., 2014; Ostler et al., 2014). The small amount of blood needed (80 l) is usually readily available and less stressful on the patient. In contrast, DBS samples are simply allowed to air dry after collection. Additionally, in contrast to traditional plasma or serum samples which
must be refrigerated or stored frozen to prevent degradation, DBS samples may be stored at room temperature (Fajardo et al., 2014; Ostler et al., 2014). Also, DBS samples are smaller and do not require refrigeration which reduces shipping costs (Fajardo et al., 2014; Ostler et al., 2014) although storage at −20 C may help increase sample survival time (McDade et al., 2007). Thus, DBS samples are less costly than traditional venipuncture samples which make them particularly useful for large scale studies (Ostler et al., 2014; McDade, 2014). The majority of serological tests are developed for use with plasma or serum obtained by traditional blood collection and, therefore, the performance of these tests using DBS samples needs to be evaluated. HemaSpotTM (SpotOn Sciences, Austin, TX) is a novel DBS collection device designed for storage and transport at ambient temperature. The purpose of the present study was to evaluate the ability of DBS samples collected and stored on HemaSpotTM devices to detect antibodies to L. infantum in dogs using a commercial ICT assay when compared to ICT results using thawed plasma from the same initial source. 2. Materials and methods 2.1. Source of dog plasma Dogs were intravenously infected with promastigotes of the LIVT-1 strain of Leishmania infantum (Rosypal et al., 2005a) under a protocol approved by the Institutional Animal Care and use Committee of Virginia Tech. The LIVT-1 strain was originally isolated from a naturally infected foxhound from Virginia (Rosypal et al., 2003b). Eight beagles were injected with either 2 × 108 promastigotes (N = 4) or 1 × 107 promastigotes (N = 4) suspended in 1 ml Hanks balanced salt solution (HBSS). For controls, 2 beagles were injected with 1 ml HBSS. Dogs were followed for a period of 62–112 weeks after infection. Over the course of the study, dogs were bled monthly by traditional blood collection using jugular venipuncture into heparinized tubes (Rosypal et al., 2005b). Plasma was separated from whole blood by centrifugation and stored at −20 ◦ C. 2.2. HemaSpotTM DBS collection devices HemaSpotTM (Spot-On Sciences, Austin, TX) is a novel collection device designed for finger prick sampling of human patients and safe storage and transport at ambient temperature. HemaSpotTM is designed as an individual cartridge with an 8-spoked disk pad of absorbent paper and desiccant covered by an application disk. The application surface contains a small hole for blood to enter into the device (Fig. 1A). After the sample is loaded, the
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Fig. 1. HemaSpotTM collection device. (A) open device with 8-spoked disk pad loaded with plasma; (B) closed device with barcode for sample identification; (C) manufacturer-provided tool for opening closed devices.
cartridge is closed and the desiccant quickly dries the sample (Fig. 1B). Once dried, the samples are stable at room temperature for both storage and transport. The label on each HemaSpotTM device has a unique barcode that can be used to store sample identification records which can be read by a smart phone application. The cartridge latch is tamper-resistant and is opened with an included tool (Fig. 1C). Once opened, the individual spokes of the 8spoked disk pad can be removed for analysis while the remaining sample can be stored at ambient temperature for later testing. 2.3. Preparation of DBS samples Prior to preparing the DBS samples, frozen plasma samples were thawed at room temperature. Thawed samples were vortexed and 80 l of plasma, which approximated the volume of 2 drops of blood as recommended by the manufacturer, was aliquotted onto individual HemaSpotTM devices. Once loaded with sample, the cartridge was closed, allowed to dry rapidly with the desiccant layer, and stored at room temperature in the dark until testing. The DBS samples were tested by the ICT for canine leishmaniasis one month and 6 months post inoculation (PI) of samples. Prior to examination 2 of the 8 spokes were removed and placed in 200 l of PBS. Samples were incubated at room temperature for 30 min and the spokes removed. The ICT was conducted on 20 l of the eluated sample. 2.4. ICT assays for canine leishmaniasis Canine plasma or eluate from HemaSpotTM devices was tested by using the recombinant K39 antigen which utilizes
an immunodominant protein specific to members of the L. donovani complex, which includes L. infantum. The ICT used in this study is commercially available for domestic dogs (Kalazar DetectTM Canine Rapid Test, InBios International Ltd., Seattle, Washington). The test procedure has been described previously (Rosypal et al., 2005a). Briefly, for each ICT, 20 l of sample was loaded on the test strip, placed in the well of a 96-well plate and 3 drops of buffer was added to the well. The ICT results were read after 10 min. According to test instructions, a positive test was indicated by 2 red or pink bands and a negative result demonstrated only one red or pink band in the control region. Positive and negative control plasma samples were obtained from dogs proven to be infected and uninfected, respectively, by both serology and culture.
2.5. Sensitivity and specificity determination Sensitivity and specificity were calculated using standard formulas. Sensitivity is defined as the number of canine samples with L. infantum antibodies divided by the number of canine samples with L. infantum antibodies plus the number of canine samples with false positive antibodies multiplied by 100. Specificity is defined as the number of canine samples without L. infantum antibodies divided by the number of canine samples without L. infantum antibodies plus the number of canine samples with false negative antibodies multiplied by 100. The ICT results from DBS samples were compared to ICT results performed on thawed plasma samples (same initial source) as the “gold standard” for comparison. The ICT on thawed plasma samples were conducted at the time of DBS sample preparation.
A.C. Rosypal et al. / Veterinary Parasitology 205 (2014) 338–342
3. Results The results from this study indicate that the novel DBS collection device is effective as a sampling tool to detect antibodies to L. infantum in experimentally infected dogs. The “gold standard” for comparison in this study was represented by ICT performed on thawed plasma from experimentally infected dogs. The ICT results from thawed plasma samples detected 33 positive and 67 negative dogs for antibodies to L. infantum. After one month PI, the ICT results on DBS samples showed 100% agreement with the thawed samples. Thus, after one month PI, the sensitivity and specificity of the ICT using DBS samples were both 100%. The ICT performed on DBS samples 6 months PI identified 24 positive and 76 negative plasma samples for canine antibodies to L. infantum. The ICT conducted 6 months PI identified 9 falsely negative samples and it did not detect any false positives. The sensitivity of the ICT was 79% and the specificity was 100% on DBS samples tested after 6 months of storage. 4. Discussion Leishmaniasis is a potentially fatal disease in both dogs and humans. Domestic dogs are considered the primary reservoir host for human infections with L. infantum (Rosypal et al., 2003a; Gramiccia and Gradoni, 2005). Serological detection of specific anti-leishmanial antibodies is one of the most useful methods to diagnose CanL (SolanoGallego et al., 2009). The most commonly used serological tests for antileishmanial antibodies in dogs include the IFAT, ELISA, and ICT devices (reviewed by Solano-Gallego et al., 2009). Most laboratory procedures such as serology, however, require either plasma or serum. The use of DBS samples in the same assays, therefore, requires validation for accuracy and consistency (McDade et al., 2007). While venipuncture has been used traditionally for blood collection, it has several distinct disadvantages compared to dried blood spot sampling. Compared to venipuncture, DBS samples are smaller in volume and simple to collect by a minimally invasive prick with a lancet which does not require medically trained phlebotomists or additional supplies (needles, tubes, tourniquets) (McDade et al., 2007; McDade, 2014). Additionally, once collected, DBS samples do not need to be centrifuged and separated in contrast to traditional blood collection. DBS samples are compact and they can be stacked, stored and shipped at room temperature (McDade et al., 2007; McDade, 2014). Thus, DBS sampling offers a low-cost alternative to traditional venipuncture, which makes it particularly well-suited for use in large scale research studies (McDade et al., 2007; McDade, 2014; Ostler et al., 2014). The HemaSpotTM blood collection device used in this study will be useful as a tool for DBS samples to test dogs with the ICT for antibodies to L. infantum. The ICT is a common test for human and canine leishmaniasis because it is easy to perform, it can be utilized in field conditions and it gives results in minutes (Srivastava et al., 2011). After one month of storage at ambient
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temperature, we found that the sensitivity and specificity of the ICT using HemaSpotTM samples were both 100%. After 6 months of the HemaSpotTM sample storage, the ICT sensitivity was 79% and specificity was 100%. After 6 months of storage at room temperature, the ICT using HemaSpotTM samples did not detect any false positives. Our study describes the use of DBS samples collected on HemaSpotTM devices in the ICT for canine antibodies to L. infantum. This test is highly sensitive and specific for antibodies to L. infantum when using HemaSpotTM samples after one month of storage at room temperature. Although the specificity was reduced to 79% after 6 months of storage, it is possible that if the HemaSpotTM samples were stored under cold conditions rather than room temperature, the specificity may improve after longer storage times. The HemaSpotTM device described in this study represents an easy to use and cost-effective alternative for blood collection to test dogs for antibodies to L. infantum. In the future, it should be evaluated as a source of blood for other serological tests for canine leishmaniasis. Acknowledgements This study was supported in part by the CATO Par Excellence Teaching Award from Johnson C. Smith University to ACR and special funds from the Department of Pathobiology and Biomedical Sciences, Virginia Tech to DSL. References Fajardo, E., Metcalf, C.A., Chaillet, P., Aleixo, L., Pannus, P., Panunzi, I., Trivino, L., Ellman, T., Likaka, A., Mwenda, R., 2014. Prospective evaluation of diagnostic accuracy of dried blood spots from finger prick samples for determination of HIV-1 load with the NucliSENS Easy-Q HIV-1 Version 2.0 assay in Malawi. J. Clin. Microbiol. 52, 1343–1351. Gradoni, L., Gramiccia, M., 2008. Leishmaniosis, OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Mammals, Birds and Bees), 6th ed. Office International des Epizooties, Paris, France, pp. 240–250. Gramiccia, M., Gradoni, L., 2005. The current status of zoonotic leishmaniases and approaches to disease control. Int. J. Parasitol. 35, 223–226. Kalayou, S., Tadelle, H., Bsrat, A., Abebe, N., Haileselassie, M., Schallig, H.D.F.H., 2011. Serological evidence of Leishmania donovani infection in apparently healthy dogs using direct agglutination test (DAT) and rk39 dipstick tests in Kafta Humera, north-west Ethiopia. Transbound. Emerg. Dis. 58, 255–326. McDade, T.W., Williams, S.A., Snodgrass, J.J., 2007. What a drop can do: dried blood spots as a minimally invasive method for integrating biomarkers into population-based research. Demography 44, 899–925. McDade, T.W., 2014. Development and validation of assay protocols for use with dried blood spot samples. Am. J. Hum. Biol. 26, 1–9. Ostler, M.W., Porter, J.H., Buxton, O.M., 2014. Dried blood spot collection of health biomarkers to maximize participation in population studies. J. Vis. Exp. 83, e50973. Rosypal, A.C., Zajac, A.M., Lindsay, D.S., 2003a. Canine visceral leishmaniasis and its emergence in the United States. Vet. Clin. N. Am. Small Anim. Pract. 33, 921–937. Rosypal, A.C., Troy, G.C., Zajac, A.M., Duncan Jr., R.B., Waki, K., Chang, K.P., Lindsay, D.S., 2003b. Emergence of zoonotic canine leishmaniasis in the United States: isolation and immunohistochemical detection of Leishmania infantum from foxhounds from Virginia. J. Eukaryot. Microbiol. 50 (Suppl.), 691–693. Rosypal, A.C., Troy, G.C., Duncan, R.B., Zajac, A.M., Lindsay, D.S., 2005a. Utility of diagnostic tests used in diagnosis of infection in dogs experimentally inoculated with a North American isolate of Leishmania infantum infantum. J. Vet. Intern. Med. 19, 802–809.
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Rosypal, A.C., Gogal Jr., R.M., Zajac, A.M., Troy, G.C., Lindsay, D.S., 2005b. Flow cytometric analysis of cellular immune responses in dogs experimentally infected with a North American isolate of Leishmania infantum. Vet. Parasitol. 131, 45–51. Rosypal, A.C., Troy, G.C., Zajac, A.M., Frank, G., Lindsay, D.S., 2005c. Transplacental transmission of a North American isolate of Leishmania infantum in an experimentally infected beagle. J. Parasitol. 91, 970–972.
Solano-Gallego, L., Koutinas, A., Miro, G., Cardosa, L., Pennisi, M.G., Ferrer, L., Bourdeau, P., Oliva, G., Baneth, G., 2009. Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. Vet. Parasitol. 165, 1–18. Srivastava, P., Dayama, A., Mehrotra, S., Sundar, S., 2011. Diagnosis of visceral leishmaniasis. Trans. R. Soc. Med. Hyg. 105, 1–6.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short Communication
Evaluation of a novel dried blood spot collection device (HemaSpotTM ) to test blood samples collected from dogs for antibodies to Leishmania infantum Alexa C. Rosypal a,∗ , Leanne D. Pick a , Jaime O. Esquivel Hernandez a , David S. Lindsay b a Department of Natural Sciences and Mathematics, College of Science, Technology, Engineering and Mathematics, Johnson C. Smith University, Charlotte, NC 28216, USA b Department of Biomedical Science and Pathology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, 1410 Prices Fork Road, Blacksburg, VA 24061, USA
a r t i c l e
i n f o
Article history: Received 3 June 2014 Received in revised form 22 July 2014 Accepted 29 July 2014 Keywords: Leishmania infantum Dog Dried blood spot Antibody
a b s t r a c t Collection of blood samples from veterinary and wildlife patients is often challenging because the samples have to be collected on farm or in the wild under various environmental conditions. This poses many technical problems associated with venipuncture materials, their safe use and disposal, transportation and processing of collected samples. Dried blood spot (DBS) sample collection techniques offer a simple and practical alternative to traditional blood collection methods to obtain blood samples from animals for parasite antibody evaluation. The DBS collection devices are compact, simple to use, and are particularly useful for large number of samples. Additionally, DBS samples take up less space and they are easier to transport than traditional venipuncture-collected blood samples. Visceral leishmaniasis (VL) is a potentially fatal parasitic disease of dogs and humans and it is frequently diagnosed by antibody tests. Immunochromatographic tests (ICT) for antibodies to Leishmania infantum are commercially available for dogs and they produce qualitative results in minutes. Measurement of canine antibodies to L. infantum with the ICT using traditional venipuncture has been validated previously, but the use of DBS samples has not been evaluated using this method. The purpose of the present study was to determine the ability of DBS samples to detect antibodies to L. infantum in dogs using a commercial ICT assay. One hundred plasma samples from dogs experimentally infected with the LIVT-1 strain of L. infantum were collected by venipuncture and frozen. Individual samples were thawed, and then 80 l plasma (2 drops) was aliquotted onto the 8-spoked disk pad on individual DBS sample collection devices (HemaSpotTM , Spot-On Sciences, Austin, TX), dried, and stored in the dark at room temperature. After one month and six months, respectively, 2 spokes of the 8 spokes of the disk pad of each DBS sample were removed and eluted in 200 l PBS. The eluate was used to test for antibodies in the ICT and compared to ICT results using thawed plasma (same initial source). Sensitivity and specificity of the ICT using DBS were determined by using ICT results from traditional blood collection samples for comparison. After 1 month, DBS samples showed 100% sensitivity and specificity when
∗ Corresponding author. Tel.: +1 704 378 1206; fax: +1 704 378 1213. E-mail address: [email protected] (A.C. Rosypal). http://dx.doi.org/10.1016/j.vetpar.2014.07.031 0304-4017/© 2014 Elsevier B.V. All rights reserved.
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compared to ICT results on thawed plasma samples collected by traditional venipuncture. After six months storage at room temperature, DBS samples demonstrated 79% sensitivity and 100% specificity compared to traditional blood collection. Results from this study indicate that dried blood spot collection may be a useful tool for screening dogs for antibodies to L. infantum with the ICT assay. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Visceral leishmaniasis (VL) is a sand fly vector-borne disease of dogs and humans caused by infection with parasites in the Leishmania donovani complex, which includes Leishmania infantum. In dogs, L. infantum infections may result in fatal disease and infected domestic dogs are the most important reservoir host for human infections. Leishmaniasis caused by L. infantum is endemic in many parts of the world including parts of Africa and Asia, the Middle East, the Mediterranean region, North and South America. In the United States, L. infantum is established in the foxhound breed (Rosypal et al., 2003a) and maintained by maternal transmission in foxhounds in North America (Rosypal et al., 2005c). Serological detection of specific anti-Leishmania antibodies is the most useful and most routinely performed diagnostic test for canine leishmaniasis (CanL) (Kalayou et al., 2011; Solano-Gallego et al., 2009; Gradoni and Gramiccia, 2008). Several serological tests are available for CanL including, IFAT, ELISA, DAT and immunochromatographic tests (ICT) (Solano-Gallego et al., 2009; Rosypal et al., 2003a). Immunochromatographic devices are particularly useful because they are simpler to perform compared to other traditional antibody tests and they produce a qualitative result in minutes (Kalayou et al., 2011; Rosypal et al., 2005a; Solano-Gallego et al., 2009). In a study of experimentally infected dogs using a United States’ isolate of L. infantum, a commercial ICT identified more infected dogs compared to IFAT (Rosypal et al., 2005a). Traditional blood collection is the current “gold standard” for collecting samples to measure biomarkers such as antibodies (McDade, 2014). Traditional collection of serum or plasma involves venipuncture which must be performed by trained personnel, centrifugation to separate the liquid fraction from blood cells and storage by refrigeration or freezing. For research purposes, dried blood spots (DBS) are increasingly recognized as a preferable alternative to traditional venipuncture for sample collection (McDade, 2014; Ostler et al., 2014). The use of DBS could relieve major barriers to blood collection, storage and transportation. Blood collection by DBS can be obtained from a finger or ear-prick or toe nail snip which can eliminate the need for specifically trained technicians, additional supplies (needles, collection and storage tubes, syringes), and the need for post-collection processing required for traditional blood collection (Fajardo et al., 2014; Ostler et al., 2014). The small amount of blood needed (80 l) is usually readily available and less stressful on the patient. In contrast, DBS samples are simply allowed to air dry after collection. Additionally, in contrast to traditional plasma or serum samples which
must be refrigerated or stored frozen to prevent degradation, DBS samples may be stored at room temperature (Fajardo et al., 2014; Ostler et al., 2014). Also, DBS samples are smaller and do not require refrigeration which reduces shipping costs (Fajardo et al., 2014; Ostler et al., 2014) although storage at −20 C may help increase sample survival time (McDade et al., 2007). Thus, DBS samples are less costly than traditional venipuncture samples which make them particularly useful for large scale studies (Ostler et al., 2014; McDade, 2014). The majority of serological tests are developed for use with plasma or serum obtained by traditional blood collection and, therefore, the performance of these tests using DBS samples needs to be evaluated. HemaSpotTM (SpotOn Sciences, Austin, TX) is a novel DBS collection device designed for storage and transport at ambient temperature. The purpose of the present study was to evaluate the ability of DBS samples collected and stored on HemaSpotTM devices to detect antibodies to L. infantum in dogs using a commercial ICT assay when compared to ICT results using thawed plasma from the same initial source. 2. Materials and methods 2.1. Source of dog plasma Dogs were intravenously infected with promastigotes of the LIVT-1 strain of Leishmania infantum (Rosypal et al., 2005a) under a protocol approved by the Institutional Animal Care and use Committee of Virginia Tech. The LIVT-1 strain was originally isolated from a naturally infected foxhound from Virginia (Rosypal et al., 2003b). Eight beagles were injected with either 2 × 108 promastigotes (N = 4) or 1 × 107 promastigotes (N = 4) suspended in 1 ml Hanks balanced salt solution (HBSS). For controls, 2 beagles were injected with 1 ml HBSS. Dogs were followed for a period of 62–112 weeks after infection. Over the course of the study, dogs were bled monthly by traditional blood collection using jugular venipuncture into heparinized tubes (Rosypal et al., 2005b). Plasma was separated from whole blood by centrifugation and stored at −20 ◦ C. 2.2. HemaSpotTM DBS collection devices HemaSpotTM (Spot-On Sciences, Austin, TX) is a novel collection device designed for finger prick sampling of human patients and safe storage and transport at ambient temperature. HemaSpotTM is designed as an individual cartridge with an 8-spoked disk pad of absorbent paper and desiccant covered by an application disk. The application surface contains a small hole for blood to enter into the device (Fig. 1A). After the sample is loaded, the
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Fig. 1. HemaSpotTM collection device. (A) open device with 8-spoked disk pad loaded with plasma; (B) closed device with barcode for sample identification; (C) manufacturer-provided tool for opening closed devices.
cartridge is closed and the desiccant quickly dries the sample (Fig. 1B). Once dried, the samples are stable at room temperature for both storage and transport. The label on each HemaSpotTM device has a unique barcode that can be used to store sample identification records which can be read by a smart phone application. The cartridge latch is tamper-resistant and is opened with an included tool (Fig. 1C). Once opened, the individual spokes of the 8spoked disk pad can be removed for analysis while the remaining sample can be stored at ambient temperature for later testing. 2.3. Preparation of DBS samples Prior to preparing the DBS samples, frozen plasma samples were thawed at room temperature. Thawed samples were vortexed and 80 l of plasma, which approximated the volume of 2 drops of blood as recommended by the manufacturer, was aliquotted onto individual HemaSpotTM devices. Once loaded with sample, the cartridge was closed, allowed to dry rapidly with the desiccant layer, and stored at room temperature in the dark until testing. The DBS samples were tested by the ICT for canine leishmaniasis one month and 6 months post inoculation (PI) of samples. Prior to examination 2 of the 8 spokes were removed and placed in 200 l of PBS. Samples were incubated at room temperature for 30 min and the spokes removed. The ICT was conducted on 20 l of the eluated sample. 2.4. ICT assays for canine leishmaniasis Canine plasma or eluate from HemaSpotTM devices was tested by using the recombinant K39 antigen which utilizes
an immunodominant protein specific to members of the L. donovani complex, which includes L. infantum. The ICT used in this study is commercially available for domestic dogs (Kalazar DetectTM Canine Rapid Test, InBios International Ltd., Seattle, Washington). The test procedure has been described previously (Rosypal et al., 2005a). Briefly, for each ICT, 20 l of sample was loaded on the test strip, placed in the well of a 96-well plate and 3 drops of buffer was added to the well. The ICT results were read after 10 min. According to test instructions, a positive test was indicated by 2 red or pink bands and a negative result demonstrated only one red or pink band in the control region. Positive and negative control plasma samples were obtained from dogs proven to be infected and uninfected, respectively, by both serology and culture.
2.5. Sensitivity and specificity determination Sensitivity and specificity were calculated using standard formulas. Sensitivity is defined as the number of canine samples with L. infantum antibodies divided by the number of canine samples with L. infantum antibodies plus the number of canine samples with false positive antibodies multiplied by 100. Specificity is defined as the number of canine samples without L. infantum antibodies divided by the number of canine samples without L. infantum antibodies plus the number of canine samples with false negative antibodies multiplied by 100. The ICT results from DBS samples were compared to ICT results performed on thawed plasma samples (same initial source) as the “gold standard” for comparison. The ICT on thawed plasma samples were conducted at the time of DBS sample preparation.
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3. Results The results from this study indicate that the novel DBS collection device is effective as a sampling tool to detect antibodies to L. infantum in experimentally infected dogs. The “gold standard” for comparison in this study was represented by ICT performed on thawed plasma from experimentally infected dogs. The ICT results from thawed plasma samples detected 33 positive and 67 negative dogs for antibodies to L. infantum. After one month PI, the ICT results on DBS samples showed 100% agreement with the thawed samples. Thus, after one month PI, the sensitivity and specificity of the ICT using DBS samples were both 100%. The ICT performed on DBS samples 6 months PI identified 24 positive and 76 negative plasma samples for canine antibodies to L. infantum. The ICT conducted 6 months PI identified 9 falsely negative samples and it did not detect any false positives. The sensitivity of the ICT was 79% and the specificity was 100% on DBS samples tested after 6 months of storage. 4. Discussion Leishmaniasis is a potentially fatal disease in both dogs and humans. Domestic dogs are considered the primary reservoir host for human infections with L. infantum (Rosypal et al., 2003a; Gramiccia and Gradoni, 2005). Serological detection of specific anti-leishmanial antibodies is one of the most useful methods to diagnose CanL (SolanoGallego et al., 2009). The most commonly used serological tests for antileishmanial antibodies in dogs include the IFAT, ELISA, and ICT devices (reviewed by Solano-Gallego et al., 2009). Most laboratory procedures such as serology, however, require either plasma or serum. The use of DBS samples in the same assays, therefore, requires validation for accuracy and consistency (McDade et al., 2007). While venipuncture has been used traditionally for blood collection, it has several distinct disadvantages compared to dried blood spot sampling. Compared to venipuncture, DBS samples are smaller in volume and simple to collect by a minimally invasive prick with a lancet which does not require medically trained phlebotomists or additional supplies (needles, tubes, tourniquets) (McDade et al., 2007; McDade, 2014). Additionally, once collected, DBS samples do not need to be centrifuged and separated in contrast to traditional blood collection. DBS samples are compact and they can be stacked, stored and shipped at room temperature (McDade et al., 2007; McDade, 2014). Thus, DBS sampling offers a low-cost alternative to traditional venipuncture, which makes it particularly well-suited for use in large scale research studies (McDade et al., 2007; McDade, 2014; Ostler et al., 2014). The HemaSpotTM blood collection device used in this study will be useful as a tool for DBS samples to test dogs with the ICT for antibodies to L. infantum. The ICT is a common test for human and canine leishmaniasis because it is easy to perform, it can be utilized in field conditions and it gives results in minutes (Srivastava et al., 2011). After one month of storage at ambient
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temperature, we found that the sensitivity and specificity of the ICT using HemaSpotTM samples were both 100%. After 6 months of the HemaSpotTM sample storage, the ICT sensitivity was 79% and specificity was 100%. After 6 months of storage at room temperature, the ICT using HemaSpotTM samples did not detect any false positives. Our study describes the use of DBS samples collected on HemaSpotTM devices in the ICT for canine antibodies to L. infantum. This test is highly sensitive and specific for antibodies to L. infantum when using HemaSpotTM samples after one month of storage at room temperature. Although the specificity was reduced to 79% after 6 months of storage, it is possible that if the HemaSpotTM samples were stored under cold conditions rather than room temperature, the specificity may improve after longer storage times. The HemaSpotTM device described in this study represents an easy to use and cost-effective alternative for blood collection to test dogs for antibodies to L. infantum. In the future, it should be evaluated as a source of blood for other serological tests for canine leishmaniasis. Acknowledgements This study was supported in part by the CATO Par Excellence Teaching Award from Johnson C. Smith University to ACR and special funds from the Department of Pathobiology and Biomedical Sciences, Virginia Tech to DSL. References Fajardo, E., Metcalf, C.A., Chaillet, P., Aleixo, L., Pannus, P., Panunzi, I., Trivino, L., Ellman, T., Likaka, A., Mwenda, R., 2014. Prospective evaluation of diagnostic accuracy of dried blood spots from finger prick samples for determination of HIV-1 load with the NucliSENS Easy-Q HIV-1 Version 2.0 assay in Malawi. J. Clin. Microbiol. 52, 1343–1351. Gradoni, L., Gramiccia, M., 2008. Leishmaniosis, OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Mammals, Birds and Bees), 6th ed. Office International des Epizooties, Paris, France, pp. 240–250. Gramiccia, M., Gradoni, L., 2005. The current status of zoonotic leishmaniases and approaches to disease control. Int. J. Parasitol. 35, 223–226. Kalayou, S., Tadelle, H., Bsrat, A., Abebe, N., Haileselassie, M., Schallig, H.D.F.H., 2011. Serological evidence of Leishmania donovani infection in apparently healthy dogs using direct agglutination test (DAT) and rk39 dipstick tests in Kafta Humera, north-west Ethiopia. Transbound. Emerg. Dis. 58, 255–326. McDade, T.W., Williams, S.A., Snodgrass, J.J., 2007. What a drop can do: dried blood spots as a minimally invasive method for integrating biomarkers into population-based research. Demography 44, 899–925. McDade, T.W., 2014. Development and validation of assay protocols for use with dried blood spot samples. Am. J. Hum. Biol. 26, 1–9. Ostler, M.W., Porter, J.H., Buxton, O.M., 2014. Dried blood spot collection of health biomarkers to maximize participation in population studies. J. Vis. Exp. 83, e50973. Rosypal, A.C., Zajac, A.M., Lindsay, D.S., 2003a. Canine visceral leishmaniasis and its emergence in the United States. Vet. Clin. N. Am. Small Anim. Pract. 33, 921–937. Rosypal, A.C., Troy, G.C., Zajac, A.M., Duncan Jr., R.B., Waki, K., Chang, K.P., Lindsay, D.S., 2003b. Emergence of zoonotic canine leishmaniasis in the United States: isolation and immunohistochemical detection of Leishmania infantum from foxhounds from Virginia. J. Eukaryot. Microbiol. 50 (Suppl.), 691–693. Rosypal, A.C., Troy, G.C., Duncan, R.B., Zajac, A.M., Lindsay, D.S., 2005a. Utility of diagnostic tests used in diagnosis of infection in dogs experimentally inoculated with a North American isolate of Leishmania infantum infantum. J. Vet. Intern. Med. 19, 802–809.
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