- Email: [email protected]
Diagnosis of Mediterranean visceral leishmaniasis by detection of Leishmania-related antigen in urine and oral fluid samples
Accepted Manuscript Title: Diagnosis of Mediterranean visceral leishmaniasis by detection of Leishmania-related antigen in urine and oral fluid samples Author: Meriem Ben-Abid Yousr Gala¨ı Zakia Habboul Rim Ben-Abdelaziz Ines Ben-Sghaier Karim Aoun A¨ıda Bouratbine PII: DOI: Reference:
S0001-706X(16)30608-8 http://dx.doi.org/doi:10.1016/j.actatropica.2016.12.026 ACTROP 4159
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
16-8-2016 16-12-2016 21-12-2016
Please cite this article as: Ben-Abid, Meriem, Gala¨ı, Yousr, Habboul, Zakia, Ben-Abdelaziz, Rim, Ben-Sghaier, Ines, Aoun, Karim, Bouratbine, A¨ıda, Diagnosis of Mediterranean visceral leishmaniasis by detection of Leishmania-related antigen in urine and oral fluid samples.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2016.12.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Diagnosis of Mediterranean visceral leishmaniasis by detection of Leishmania-related antigen in urine and oral fluid samples
Meriem Ben-Abid a, Yousr Galaï a, Zakia Habboul b, Rim Ben-Abdelaziz c, Ines Ben-Sghaier a, Karim Aoun a, Aïda Bouratbine a# a
Department of clinical Parasitology, Laboratoire de recherche LR 11-IPT-06 « Parasitologie
Médicale, Biotechnologies et Biomolécules », Institut Pasteur de Tunis, Université Tunis ElManar, Tunis, Tunisia ; b Pediatric Department of the Regional Hospital of Kairouan, Tunisia ; c
Pediatric Department, Hospital la Rabta, Tunis, Tunisia
# Address correspondence to Aïda Bouratbine, Laboratoire de Parasitologie, Institut Pasteur de Tunis, 13 Place Pasteur, BP 74, 1002 Tunis Belvédère, Tunisia. E-mail: [email protected].
Graphical Abstract Summary Sensitivity and specificity of KAtex® kit in the diagnosis of visceral leishmaniasis caused by Leishmania infantum varied according to biological sample used (urine or oral-fluid).
Highlights
Sensitivity and specificity of KAtex® kit in the diagnosis of visceral leishmaniasis caused by Leishmania infantum were 51.4% and 98.3% respectively.
Sensitivity and specificity of KAtex® kit in the diagnosis of visceral leishmaniasis using oral-fluid were 80% and 88.3% respectively.
Lack of specificity for detection of related Leishmania antigens in oral fluid is probably due to cross-reaction with soluble bacteria related-antigen that may be present in saliva
Abstract Implementation of simple diagnostic tests using non-invasive collection of biological specimens is of great importance in the diagnosis of pediatric visceral leishmaniasis caused by Leishmania infantum. Latex agglutination kit (KAtex®) is widely used in the diagnosis mainly in L. donovani endemic areas. However its utilization in L. infantum endemic regions remains limited and its use on noninvasive biological specimen apart urine was not reported. In this study, KAtex® kit was used to detect Leishmania-related antigen in urine and oral fluid of 35 L. infantum visceral leishmaniasis cases and 62 controls including non-infectious disease and infectious disease controls (34 and 28 respectively). Sensitivity and specificity of urine based KAtex® were 51.4% and 98.3% respectively, whereas, sensitivity and specificity of oral-fluid based KAtex® were 80% and 88.3% respectively. Although, sensitivity of oral-fluid KAtex® was high, its specificity varied significantly according to the presence or the absence of an infectious disease (71.4% versus 97%, p=0.01).
Keywords: Mediterranean visceral leishmaniasis; Leishmania infantum; oral fluid; urine; latex agglutination assay; KAtex®
Diagnosis of Mediterranean visceral leishmaniasis by detection of Leishmania-related antigen in urine and oral fluid samples
Introduction Visceral leishmaniasis (VL) is endemic in the Mediterranean basin, where Leishmania infantum is the causative species. In Tunisia, North Africa, VL is primarily a pediatric disease that is life threatening if not early and accurately diagnosed (Galai et al., 2011). Its accurate diagnosis requires the availability of reliable laboratory methods, as microscopic identification of Leishmania parasites in bone marrow aspirates, DNA detection by PCR assays on blood samples and detection of anti-Leishmania antibodies in sera (Galai et al., 2011). Latex agglutination kit (KAtex®) manufactured by Kalon Biological Ltd, UK, is an easy to use test currently available to identify Leishmania-related antigen. It applies latex beads coated with anti-Leishmania polyclonal antibodies and detects presence of a heat-stable disease-specific parasite antigen in urine of VL patients with active infection (Sarkari, et al., 2002). This commercially available immunoassay is widely used in VL diagnosis, mainly in L. donovani endemic areas such Sudan, India and Nepal (Boelaert et al., 2008; Boelaert et al., 2014; Diro et al., 2007; Rijal et al., 2004; Sundar et al., 2005). However, its utilization in L. infantum endemic regions remains limited (Boelaert et al., 2014) and its use on another noninvasive biological specimen type such as saliva was not reported. In fact, oral fluid-based diagnostic tests were already validated for antigen detection in several parasitic systemic diseases, namely amoebiasis and malaria (Abd-Alla et al., 2000; Wilson et al., 2008). Our objective was to test KAtex® on urine and to evaluate the relevance of its use on oral fluid specimen in the diagnosis of Mediterranean VL.
Material and methods The study included 35 VL patients (mean age = 19 + 17 months) hospitalized at the Pediatric Department of the Regional Hospital of Kairouan (Tunisia) and 62 controls. Visceral leishmaniasis was suspected upon clinical signs and confirmed by the microscopic observation of Leishmania amastigotes in Giemsa stained bone marrow smears. Controls subjects were matched according to the age of VL patients and included non-infectious disease (NID) controls (n=34) and infectious disease (ID) controls (n=28). All controls with NID had no fever and were hospitalized for investigation of chronic diseases such as diabetes, metabolic diseases, asthma .... Whereas, controls with ID presented with fever and were hospitalized for medical management of acute bronchiolitis (n=20), meningitis (n=5) and pneumonia (n=3). Urine collection bags were used to collect urine. Oral fluid was collected by an ORACOLTM device (Malvern Medical Developments, Worcester, United Kingdom) as described previously (Galai et al., 2011). Biological samples were stored at -20°C until use. The study was reviewed and approved by the Pasteur Institute of Tunis ethics committee. Detection of Leishmania-related antigens by KAtex® kit (Kalon Biological, UK) was performed according to the manufacturer's instructions on both specimens. Briefly, urine and oral fluid specimens were placed 5 min in a boiling water bath and after cooling, were mixed with the latex reagent on a ceramic slide. The degree of agglutination was recorded as described by the manufacturer. Positive results were considered for a reading threshold > ++. Statistical analysis was done using the MedCalc Statistical software (version 83 11.4.4.0). A test was considered significant if p value was less than 0.05. Results 18 out of 35 VL patients had a urine-based KAtex® positive test, which represents a sensitivity of 51.4%. However, 28 out of 35 VL patients had a oral-fluid based KAtex® positive test, which represents a sensitivity of 80%. While positive percent agreement (PPA)
between saliva and urine (as reference standard) was 83.3%. Specificity of urine based assay was 98.3%. Nevertheless, specificity of oral-fluid based assay was 88.3% and varied significantly according to ID or NID controls (71.4% versus 97%, p=0.01) (Table).
Table Sensitivity and Specificity of urine-based and oral fluid-based KAtex® Kit KAtex® Kit Urine
Sensitivity 51.4%
NID* ID** Oral fluid 80% NID* ID** *Non Infectious Diseases; **Infectious Diseases
Specificity 98.3% 98.3% 97% 71.4%
98.3% 88.3%
Discussion The results obtained with KAtex® indicated that the test works well regardless of the biological origin of samples (oral fluid versus urine). The low sensitivity of urine-based KAtex® in L. infantum cases was already reported in Spain (Cruz et al., 2006). Low sensitivity of urine-based KAtex® in our study may also be attributed to the effect of storage of urine at −20 ◦C for long period, possibly leading to degradation of antigens, as reported by other authors (Diro et al., 2007; Rijal et al., 2004). Sensitivity of oral fluid-based KAtex® was higher than in urine. The positive percent agreement (PPA) between saliva and urine suggests that the immunoassay probably detects in oral fluid the similar low molecular heat stable antigenic fraction than in urine. Circulating Leishmania antigens evidenced in serum samples from immune-competent individuals with Mediterranean VL are probably detected in oral fluid specimens when reaching a high level (Ferrua et al., 2009). Disparity in specificity of oral-fluid based assay according to ID or NID controls (71.4% versus 97%, p=0.01) is probably due to cross-reaction with soluble bacteria related-antigen that may be present in saliva (Streckfus and Bigler, 2002). We previously demonstrated the usefulness of oral-fluid
collection in the detection of both Leishmania antibodies and Leishmania DNA (Galai et al., 2011). This biological fluid seems to lack specificity for detection of related Leishmania antigens.
Funding information This study was supported by the Ministry of Higher Education and Scientific Research, Tunisia in the setting of the Research Laboratory Medical Parasitology, Biotechnologies and Biomolecules (PMBB), Institut Pasteur of Tunis, Tunisia (LR11-IPT06) and the U.S. Agency for International Development (USAID), Middle East Regional Cooperation Program (MERC), grant agreement N ° TA-MOU-08-M27-072, proposal entitled “Surveillance and Control of Visceral Leishmaniasis in the Middle East and North Africa”
References Abd-Alla MD, Jackson TF, Reddy S, Ravdin JI. 2000. Diagnosis of invasive amebiasis by enzyme-linked immunosorbent assay of saliva to detect amebic lectin antigen and anti-lectin immunoglobulin G antibodies. J Clin Microbiol 38: 2344-7. Boelaert M, El-Safi S, Hailu A, Mukhtar M, Rijal S ,Sundar S, Wasunna M, Aseffa A, Mbui J, Mentena J, Desjeux P, Peeling RW. 2008. Diagnostic tests for kala-azar: a multi-centre study ofthe freeze-dried DAT, rK39 strip test and KAtex inEast Africa and the Indian subcontinent. Trans R Soc Trop Med Hyg 102: 32-40. Boelaert M, Verdonck K, Menten J, Sunyoto T, van Griensven J, Chappuis F, Rijal S. 2014. Rapid tests for the diagnosis of visceral leishmaniasis in patients with suspected disease (Review). The Cochrane Library, Issue 6
Cruz I, Chicharro C, Nieto J, Bailo B,Canavate C, Figueras M, Alvar J. 2006. Comparison of New Diagnostic Tools for Management of Pediatric Mediterranean Visceral Leishmaniasis. J Clin Microbiol 44: 2343–2347. Diro E, Techane Y, Tefera T, Assefa Y, Kebede T, Genetu A, Kebede Y, TesfayeA, Ergicho B, Gebre-Yohannes A, Mengistu G, Engers H, Aseffa A, Desjeux P, Boelaert M, Hailu A. 2007. Field evaluation of FD-DAT, rK39 dipstick and KATEX (urine latex agglutination) for diagnosis of visceral leishmaniasis in northwest Ethiopia. Trans R Soc Trop Med Hyg 101, 908-914. Ferrua B, Rol N, Michel G, Marty P. 2009. Antigenemia in Patients with Mediterranean Visceral Leishmaniasis. J Clin Microbiol 47 :3760–3762 Galaï Y, Chabchoub N, Ben-Abid M, Ben-Abda I, Ben-Alaya-Bouafif N, Amri F, Aoun K, Bouratbine A.
2011. Diagnosis of mediterranean visceral leishmaniasis by detection of
leishmania antibodies and leishmania DNA in oral fluid samples collected using an Oracol device. J Clin Microbiol 49:3150-3153 Rijal S, Boelaert M, Regmi S, Karki BM, Jacquet D, Singh R, Chance ML, Chappuis F, Hommel M, Desjeux P, Van der Stuyft P, Le Ray D, Koirala S. 2004. Evaluation of a urinary antigen-based latex agglutination test in the diagnosis of kala-azar in eastern Nepal. Trop Med Int Health 9 :724-729. Sarkari B, Chance M, Hommel M. 2002. Antigenuria in visceral leishmaniasis: detection and partial characterization of a carbohydrate antigen. Acta Trop 82 :339-348. Streckfus CF, Bigler LR. 2002. Saliva as a diagnostic fluid. Oral Dis. Review 8(2) : 69-76. Sundar S, Agrawal S, Pai K, Chance M, Hommel M. 2005. Detection of leishmania antigen in the urine of patients with visceral leishmaniasis by a latex agglutination test. Am J Trop Med Hyg 73 :269-271.
Wilson NO, Adjei AA, Anderson W, Baidoo S, Stiles JK. 2008. Detection of Plasmodium falciparum histidine-rich protein II in saliva of malaria patients. Am J Trop Med Hyg 78:7335.
S0001-706X(16)30608-8 http://dx.doi.org/doi:10.1016/j.actatropica.2016.12.026 ACTROP 4159
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
16-8-2016 16-12-2016 21-12-2016
Please cite this article as: Ben-Abid, Meriem, Gala¨ı, Yousr, Habboul, Zakia, Ben-Abdelaziz, Rim, Ben-Sghaier, Ines, Aoun, Karim, Bouratbine, A¨ıda, Diagnosis of Mediterranean visceral leishmaniasis by detection of Leishmania-related antigen in urine and oral fluid samples.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2016.12.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Diagnosis of Mediterranean visceral leishmaniasis by detection of Leishmania-related antigen in urine and oral fluid samples
Meriem Ben-Abid a, Yousr Galaï a, Zakia Habboul b, Rim Ben-Abdelaziz c, Ines Ben-Sghaier a, Karim Aoun a, Aïda Bouratbine a# a
Department of clinical Parasitology, Laboratoire de recherche LR 11-IPT-06 « Parasitologie
Médicale, Biotechnologies et Biomolécules », Institut Pasteur de Tunis, Université Tunis ElManar, Tunis, Tunisia ; b Pediatric Department of the Regional Hospital of Kairouan, Tunisia ; c
Pediatric Department, Hospital la Rabta, Tunis, Tunisia
# Address correspondence to Aïda Bouratbine, Laboratoire de Parasitologie, Institut Pasteur de Tunis, 13 Place Pasteur, BP 74, 1002 Tunis Belvédère, Tunisia. E-mail: [email protected].
Graphical Abstract Summary Sensitivity and specificity of KAtex® kit in the diagnosis of visceral leishmaniasis caused by Leishmania infantum varied according to biological sample used (urine or oral-fluid).
Highlights
Sensitivity and specificity of KAtex® kit in the diagnosis of visceral leishmaniasis caused by Leishmania infantum were 51.4% and 98.3% respectively.
Sensitivity and specificity of KAtex® kit in the diagnosis of visceral leishmaniasis using oral-fluid were 80% and 88.3% respectively.
Lack of specificity for detection of related Leishmania antigens in oral fluid is probably due to cross-reaction with soluble bacteria related-antigen that may be present in saliva
Abstract Implementation of simple diagnostic tests using non-invasive collection of biological specimens is of great importance in the diagnosis of pediatric visceral leishmaniasis caused by Leishmania infantum. Latex agglutination kit (KAtex®) is widely used in the diagnosis mainly in L. donovani endemic areas. However its utilization in L. infantum endemic regions remains limited and its use on noninvasive biological specimen apart urine was not reported. In this study, KAtex® kit was used to detect Leishmania-related antigen in urine and oral fluid of 35 L. infantum visceral leishmaniasis cases and 62 controls including non-infectious disease and infectious disease controls (34 and 28 respectively). Sensitivity and specificity of urine based KAtex® were 51.4% and 98.3% respectively, whereas, sensitivity and specificity of oral-fluid based KAtex® were 80% and 88.3% respectively. Although, sensitivity of oral-fluid KAtex® was high, its specificity varied significantly according to the presence or the absence of an infectious disease (71.4% versus 97%, p=0.01).
Keywords: Mediterranean visceral leishmaniasis; Leishmania infantum; oral fluid; urine; latex agglutination assay; KAtex®
Diagnosis of Mediterranean visceral leishmaniasis by detection of Leishmania-related antigen in urine and oral fluid samples
Introduction Visceral leishmaniasis (VL) is endemic in the Mediterranean basin, where Leishmania infantum is the causative species. In Tunisia, North Africa, VL is primarily a pediatric disease that is life threatening if not early and accurately diagnosed (Galai et al., 2011). Its accurate diagnosis requires the availability of reliable laboratory methods, as microscopic identification of Leishmania parasites in bone marrow aspirates, DNA detection by PCR assays on blood samples and detection of anti-Leishmania antibodies in sera (Galai et al., 2011). Latex agglutination kit (KAtex®) manufactured by Kalon Biological Ltd, UK, is an easy to use test currently available to identify Leishmania-related antigen. It applies latex beads coated with anti-Leishmania polyclonal antibodies and detects presence of a heat-stable disease-specific parasite antigen in urine of VL patients with active infection (Sarkari, et al., 2002). This commercially available immunoassay is widely used in VL diagnosis, mainly in L. donovani endemic areas such Sudan, India and Nepal (Boelaert et al., 2008; Boelaert et al., 2014; Diro et al., 2007; Rijal et al., 2004; Sundar et al., 2005). However, its utilization in L. infantum endemic regions remains limited (Boelaert et al., 2014) and its use on another noninvasive biological specimen type such as saliva was not reported. In fact, oral fluid-based diagnostic tests were already validated for antigen detection in several parasitic systemic diseases, namely amoebiasis and malaria (Abd-Alla et al., 2000; Wilson et al., 2008). Our objective was to test KAtex® on urine and to evaluate the relevance of its use on oral fluid specimen in the diagnosis of Mediterranean VL.
Material and methods The study included 35 VL patients (mean age = 19 + 17 months) hospitalized at the Pediatric Department of the Regional Hospital of Kairouan (Tunisia) and 62 controls. Visceral leishmaniasis was suspected upon clinical signs and confirmed by the microscopic observation of Leishmania amastigotes in Giemsa stained bone marrow smears. Controls subjects were matched according to the age of VL patients and included non-infectious disease (NID) controls (n=34) and infectious disease (ID) controls (n=28). All controls with NID had no fever and were hospitalized for investigation of chronic diseases such as diabetes, metabolic diseases, asthma .... Whereas, controls with ID presented with fever and were hospitalized for medical management of acute bronchiolitis (n=20), meningitis (n=5) and pneumonia (n=3). Urine collection bags were used to collect urine. Oral fluid was collected by an ORACOLTM device (Malvern Medical Developments, Worcester, United Kingdom) as described previously (Galai et al., 2011). Biological samples were stored at -20°C until use. The study was reviewed and approved by the Pasteur Institute of Tunis ethics committee. Detection of Leishmania-related antigens by KAtex® kit (Kalon Biological, UK) was performed according to the manufacturer's instructions on both specimens. Briefly, urine and oral fluid specimens were placed 5 min in a boiling water bath and after cooling, were mixed with the latex reagent on a ceramic slide. The degree of agglutination was recorded as described by the manufacturer. Positive results were considered for a reading threshold > ++. Statistical analysis was done using the MedCalc Statistical software (version 83 11.4.4.0). A test was considered significant if p value was less than 0.05. Results 18 out of 35 VL patients had a urine-based KAtex® positive test, which represents a sensitivity of 51.4%. However, 28 out of 35 VL patients had a oral-fluid based KAtex® positive test, which represents a sensitivity of 80%. While positive percent agreement (PPA)
between saliva and urine (as reference standard) was 83.3%. Specificity of urine based assay was 98.3%. Nevertheless, specificity of oral-fluid based assay was 88.3% and varied significantly according to ID or NID controls (71.4% versus 97%, p=0.01) (Table).
Table Sensitivity and Specificity of urine-based and oral fluid-based KAtex® Kit KAtex® Kit Urine
Sensitivity 51.4%
NID* ID** Oral fluid 80% NID* ID** *Non Infectious Diseases; **Infectious Diseases
Specificity 98.3% 98.3% 97% 71.4%
98.3% 88.3%
Discussion The results obtained with KAtex® indicated that the test works well regardless of the biological origin of samples (oral fluid versus urine). The low sensitivity of urine-based KAtex® in L. infantum cases was already reported in Spain (Cruz et al., 2006). Low sensitivity of urine-based KAtex® in our study may also be attributed to the effect of storage of urine at −20 ◦C for long period, possibly leading to degradation of antigens, as reported by other authors (Diro et al., 2007; Rijal et al., 2004). Sensitivity of oral fluid-based KAtex® was higher than in urine. The positive percent agreement (PPA) between saliva and urine suggests that the immunoassay probably detects in oral fluid the similar low molecular heat stable antigenic fraction than in urine. Circulating Leishmania antigens evidenced in serum samples from immune-competent individuals with Mediterranean VL are probably detected in oral fluid specimens when reaching a high level (Ferrua et al., 2009). Disparity in specificity of oral-fluid based assay according to ID or NID controls (71.4% versus 97%, p=0.01) is probably due to cross-reaction with soluble bacteria related-antigen that may be present in saliva (Streckfus and Bigler, 2002). We previously demonstrated the usefulness of oral-fluid
collection in the detection of both Leishmania antibodies and Leishmania DNA (Galai et al., 2011). This biological fluid seems to lack specificity for detection of related Leishmania antigens.
Funding information This study was supported by the Ministry of Higher Education and Scientific Research, Tunisia in the setting of the Research Laboratory Medical Parasitology, Biotechnologies and Biomolecules (PMBB), Institut Pasteur of Tunis, Tunisia (LR11-IPT06) and the U.S. Agency for International Development (USAID), Middle East Regional Cooperation Program (MERC), grant agreement N ° TA-MOU-08-M27-072, proposal entitled “Surveillance and Control of Visceral Leishmaniasis in the Middle East and North Africa”
References Abd-Alla MD, Jackson TF, Reddy S, Ravdin JI. 2000. Diagnosis of invasive amebiasis by enzyme-linked immunosorbent assay of saliva to detect amebic lectin antigen and anti-lectin immunoglobulin G antibodies. J Clin Microbiol 38: 2344-7. Boelaert M, El-Safi S, Hailu A, Mukhtar M, Rijal S ,Sundar S, Wasunna M, Aseffa A, Mbui J, Mentena J, Desjeux P, Peeling RW. 2008. Diagnostic tests for kala-azar: a multi-centre study ofthe freeze-dried DAT, rK39 strip test and KAtex inEast Africa and the Indian subcontinent. Trans R Soc Trop Med Hyg 102: 32-40. Boelaert M, Verdonck K, Menten J, Sunyoto T, van Griensven J, Chappuis F, Rijal S. 2014. Rapid tests for the diagnosis of visceral leishmaniasis in patients with suspected disease (Review). The Cochrane Library, Issue 6
Cruz I, Chicharro C, Nieto J, Bailo B,Canavate C, Figueras M, Alvar J. 2006. Comparison of New Diagnostic Tools for Management of Pediatric Mediterranean Visceral Leishmaniasis. J Clin Microbiol 44: 2343–2347. Diro E, Techane Y, Tefera T, Assefa Y, Kebede T, Genetu A, Kebede Y, TesfayeA, Ergicho B, Gebre-Yohannes A, Mengistu G, Engers H, Aseffa A, Desjeux P, Boelaert M, Hailu A. 2007. Field evaluation of FD-DAT, rK39 dipstick and KATEX (urine latex agglutination) for diagnosis of visceral leishmaniasis in northwest Ethiopia. Trans R Soc Trop Med Hyg 101, 908-914. Ferrua B, Rol N, Michel G, Marty P. 2009. Antigenemia in Patients with Mediterranean Visceral Leishmaniasis. J Clin Microbiol 47 :3760–3762 Galaï Y, Chabchoub N, Ben-Abid M, Ben-Abda I, Ben-Alaya-Bouafif N, Amri F, Aoun K, Bouratbine A.
2011. Diagnosis of mediterranean visceral leishmaniasis by detection of
leishmania antibodies and leishmania DNA in oral fluid samples collected using an Oracol device. J Clin Microbiol 49:3150-3153 Rijal S, Boelaert M, Regmi S, Karki BM, Jacquet D, Singh R, Chance ML, Chappuis F, Hommel M, Desjeux P, Van der Stuyft P, Le Ray D, Koirala S. 2004. Evaluation of a urinary antigen-based latex agglutination test in the diagnosis of kala-azar in eastern Nepal. Trop Med Int Health 9 :724-729. Sarkari B, Chance M, Hommel M. 2002. Antigenuria in visceral leishmaniasis: detection and partial characterization of a carbohydrate antigen. Acta Trop 82 :339-348. Streckfus CF, Bigler LR. 2002. Saliva as a diagnostic fluid. Oral Dis. Review 8(2) : 69-76. Sundar S, Agrawal S, Pai K, Chance M, Hommel M. 2005. Detection of leishmania antigen in the urine of patients with visceral leishmaniasis by a latex agglutination test. Am J Trop Med Hyg 73 :269-271.
Wilson NO, Adjei AA, Anderson W, Baidoo S, Stiles JK. 2008. Detection of Plasmodium falciparum histidine-rich protein II in saliva of malaria patients. Am J Trop Med Hyg 78:7335.