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    Graphical depiction of rK39 results. Points above dashed line are ELISA positive. OD = optical density; CL = cutaneous leishmaniasis; VL = visceral leishmaniasis.

  • 1

    World Health Organization, 2002. Urbanization: an increasing risk factor for leishmaniasis. Wkly Epidemiol Rec 44 :365–372.

  • 2

    Myles O, Wortmann GW, Cummings JF, Barthel RV, Patel S, Crum-Cianflone NF, Negin NS, Weina PJ, Ockenhouse CF, Joyce DJ, Magill AJ, Aronson NE, Gasser RA, 2007. Visceral leishmaniasis: clinical observations in 4 US Army soldiers deployed to Afghanistan or Iraq, 2002–2004. Arch Intern Med 167 :1899–1901.

    • Search Google Scholar
    • Export Citation
  • 3

    Burns JA, Shreffler WG, Benson DR, Ghalib HW, Badaro R, Reed SG, 1993. Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis. Proc Natl Acad Sci USA 90 :775–779.

    • Search Google Scholar
    • Export Citation
  • 4

    Chappuis F, Rijal S, Alonso S, Menten J, Boelaert M, 2006. A meta-analysis of the diagnostic performance of the direct agglutination test and rK39 dipstick for visceral leishmaniasis. BMJ 333 :723–727.

    • Search Google Scholar
    • Export Citation
  • 5

    Schallig H, Canto-Cavalheiro M, Silva E, 2002. Evaluation of the direct agglutination test and the rK39 dipstick test for the sero-diagnosis of visceral leishmaniasis. Mem Inst Oswaldo Cruz 97 :1015–1018.

    • Search Google Scholar
    • Export Citation
  • 6

    Braz R, Nascimento E, Martins D, Wilson ME, Pearson RD, Reed SG, Jeronimo SM, 2002. The sensitivity and specificity of Leishmania chagasi recombinant K39 antigen in the diagnosis of American visceral leishmaniasis and in differentiating active from subclinical infection. Am J Trop Med Hyg 67 :344–348.

    • Search Google Scholar
    • Export Citation
  • 7

    Ozensoy S, Ozbel Y, Turgay N, Alkan MZ, Gul K, Gilman-Sachs A, Chang KP, Reed SG, Ozcel MA, 1998. Serodiagnosis and epidemiology of visceral leishmaniasis in Turkey. Am J Trop Med Hyg 59 :363–369.

    • Search Google Scholar
    • Export Citation
  • 8

    Wortmann G, Hochberg L, Houng HH, Sweeney C, Zapor M, Aronson N, Weina P, Ockenhouse CF, 2005. Rapid identification of Leishmania complexes by a real-time PCR assay. Am J Trop Med Hyg 73 :999–1004.

    • Search Google Scholar
    • Export Citation
  • 9

    Kreutzer RD, Semko ME, Hendricks LD, Wright N, 1983. Identification of Leishmania spp. by multiple isozyme analysis. Am J Trop Med Hyg 32 :703–715.

    • Search Google Scholar
    • Export Citation
  • 10

    Zijlstra E, Nur Y, Desjeux P, Khalil EA, El-Hassan AM, Groen J, 2001. Diagnosing visceral leishmaniasis with the recombinant K39 strip test: experience from the Sudan. Trop Med Int Health 6 :108–113.

    • Search Google Scholar
    • Export Citation
  • 11

    Singh S, Kumari V, Singh N, 2002. Predicting kala-azar disease manifestations in asymptomatic patients with latent Leishmania donovani infection by detection of antibody against recombinant K39 antigen. Clin Diagn Lab Immunol 9 :568–572.

    • Search Google Scholar
    • Export Citation
  • 12

    Sundar S, Pai K, Sahu M, Kumar V, Murray HW, 2002. Immunochromatographic strip-test detection of anti-K39 antibody in Indian visceral leishmaniasis. Ann Trop Med Parasitol 96 :19–23.

    • Search Google Scholar
    • Export Citation
  • 13

    Menzel S, Bienzle U, 1978. Antibody response in patients with cutaneous leishmaniasis of the Old World. Ann Trop Med Parasitol 29 :194–197.

    • Search Google Scholar
    • Export Citation

 

 

 

 

Positive rK39 Serologic Assay Results in US Servicemen with Cutaneous Leishmaniasis

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  • 1 Infectious Diseases Division, Walter Reed Army Medical Center, Washington, District of Columbia; Division of Infectious Disease, Uniformed Services University of the Health Sciences, Bethesda, Maryland; Walter Reed Army Institute of Research, Silver Spring, Maryland; Department of Clinical Investigation, Walter Reed Army Medical Center, Washington, District of Columbia

The rK39 test is a serologic assay for the rapid diagnosis of visceral leishmaniasis (VL). Serum from a North American cohort of 59 otherwise asymptomatic soldiers with cutaneous leishmaniasis (CL) was tested with the rK39 dipstick and ELISA assays, and 10.2% and 28.8% had positive results, respectively. CL is associated with a reactive rK39 assay result in some patients without clinical evidence of VL.

INTRODUCTION

Visceral leishmaniasis (VL) is a significant health problem worldwide, with nearly 500,000 new cases occurring annually.1 Rare in the United States, it has been reported primarily in travelers, immigrants from endemic areas, and US military personnel.2 The diagnosis of VL is typically made by performing either bone marrow, liver, or splenic aspirate/biopsy and examining the tissue sample (by culture, microscopy, or poly-merase chain reaction [PCR] assay) for the presence of Leishmania organisms. Recently, a significant advance in the diagnosis of VL has been developed, which allows for the rapid and non-invasive diagnosis of this condition.

The rK39 test (KalazarDetect; InBios, Seattle, WA; and DiaMed-IT LEISH; Cressier sur Morat, Switzerland) was developed from a recombinant antigen containing a 39 amino acid repeat found in Leishmania infantum-chagasi, part of a kinesin-related protein produced mainly by amastigotes.3 The test has been developed in both an ELISA and immunochromatographic strip (dipstick) platform and is designed to detect antibodies against the rK39 antigen, which are produced in response to VL. Advantages of the assay include requiring only phlebotomy rather than invasive tissue sampling, the potential for a rapid turn-around time, and no requirement for an expert interpretation of the result.

The rK39 dipstick has been studied extensively, with a recent meta-analysis of 13 studies reporting an overall sensitivity and specificity of 94% and 91%, respectively, for the diagnosis of VL.4 However, in some reports, the test showed cross-reactivity in patients with cutaneous leishmaniasis (CL), with up to 20% of CL patients having a positive rK39 dipstick result.47 Because these studies were conducted in geographic locales endemic for both CL and VL Leishmania syndromes, it is uncertain whether the detection of anti-rK39 antibodies in patients with active CL represents antibodies produced in response to active tegumentary infection or whether the positive assay represents sub-clinical VL or perhaps prior episodes of CL and/or VL.

With the advent of Operations Iraqi Freedom (Iraq) and Enduring Freedom (Afghanistan), the Walter Reed Army Medical Center (WRAMC) has evaluated and treated > 500 patients with Old World CL. Most of these patients had never previously lived or traveled to Leishmania-endemic regions of the world and thus represented a unique cohort in which to evaluate the prevalence of positive rK39 assays in a heretofore Leishmania-naive population.

MATERIALS AND METHODS

Seventy-three frozen serum samples collected from participants enrolled in an ongoing Leishmania developmental diagnostics protocol were used for this study. Serum samples represented 59 active cases of confirmed (by culture, microscopy, and/or real-time PCR) CL, 3 cases of VL, and 11 control samples. Leishmania species identification was performed for CL and VL patients either by glucose phosphate isomerase (GPI)-based RT-PCR, which has been previously described, or by cellulose acetate electrophoresis (CAE) of cultured pro-mastigotes.8,9 The CL and VL patients were previously healthy American soldiers referred to WRAMC, Washington, DC, for treatment. Controls were otherwise healthy, generally young men, with no exposure history to Leishmania including no travel to Leishmania-endemic regions. Associated demographic, clinical, and microbiologic information was available for all collected sera. All CL patients whose serum was used in this study underwent subsequent treatment with sodium stibogluconate and were assessed with daily clinician visits and frequent laboratory analyses for at least 2 weeks (with no evidence of visceral infection) and then followed for up to 1 year. Both the current project and the diagnostics protocol were approved by the Institutional Review Boards at the investigators’ institutions.

Serum samples were thawed once, and both the Kalazar DetectELISA (InBios) and Kalazar Detect Rapid Test (InBios) were performed on each test in accordance with the manufacturer’s directions. For the ELISA, samples were run in duplicate, and a result was considered positive when the optical density (OD) exceeded 0.19 (mean from the negative controls plus three times the SD).

Statistical analysis was completed using SPSS statistical software, version 12.0.1 (SPSS, Chicago, IL). Demographic variables at baseline were compared using the Mann-Whitney test and Fisher exact tests to determine differences that existed between CL patients with positive and negative rK39 test results. P < 0.05 was considered statistically significant.

RESULTS

Using the rK39 dipstick, six CL patients (10.2%; 95% CI: 4–21%) and all three VL patients tested positive. Using the ELISA assay, the 6 patients with positive dipstick results, the 3 VL patients, and an additional 17 CL patients with negative dipstick results tested positive (28.8%; 95% CI: 18–42%). The 11 control and remaining CL patients (N = 36) had negative rK39 ELISA assays and dipstick results (Figure 1).

The demographic and clinical characteristics of the study patients are listed in Table 1 and separated into categories of positive and negative rk39 test results (dipstick and ELISA). Patients with more lesions (3 to 1, P = 0.001) and larger lesions (1,102 versus 300 mm2, P < 0.0005) were more likely to have positive rK39 assays. Patients who had traveled to the Middle East (Iraq or Afghanistan) were also more likely to have positive results (P = 0.002). There was no difference among the other variables collected.

The majority of infections were caused by L. L. major (N = 46; 87%), and all were acquired in Iraq (N = 42) and Afghanistan (N = 4). There were two cases of L. L. tropica from Afghanistan. The other species acquired in Central America included L. L. mexicana (N = 3), L. V. braziliensis (N = 1), and L. V. panamensis (N = 1). For patients with a positive rK39 dipstick, five were infected with L. L. major and one with L. L. tropica. Of those with additional positive rK39 ELISA, there were 15 L. L. major, 1 L. L. tropica, and 1 that was unable to be identified. There were no patients with cutaneous L. L. infantum-chagasi or L. L. donovani included in this study. The three VL patients were confirmed by culture or PCR to have L. L. infantum-chagasi or L. L. donovani infection.

DISCUSSION

In this first study to examine the test characteristics of the Leishmania rK39 dipstick assay in US patients with CL, 10% of patients tested positive using the dipstick assay and 29% with the ELISA format. This rate is similar to the rates of positive assays reported in previous studies conducted in Leishmania-endemic locales.57 ELISA was more sensitive in our study, which is consistent with previous reports as well.10

Previous studies have suggested that positive rK39 test results in patients with CL could represent incubating or previous VL infection by L. L. infantum-chagasi.10,11 One study reported that in contacts of patients with VL, 69% of those initially asymptomatic with a positive rK39 assay developed VL within 1 year.11 It is unlikely that our patients had subclinical VL infection because there have been very few cases of VL diagnosed in our general patient population and none of the current cohort of CL patients had clinical evidence of a systemic infectious process.2 Although the possibility of subclinical VL infection cannot be totally excluded based on our methods, we believe that our results support the notion that CL itself (infection by L. L. major) rather previous or incubating VL can cause a positive rK39 assay.

Besides leishmaniasis, positive rK39 assay results have been reported in patients with malaria, Chagas disease, tuberculosis, leprosy, and African trypanosomiasis.5,12 None of the patients in this study had evidence of these infections at the time of diagnosis or during the year of follow-up. It is unlikely that cross-reactivity from one of these infections caused their positive rK39 assay.

Univariate analysis showed that patients with more lesions and larger lesions were more likely to have positive rK39 test results. The association between number and size of lesions and a positive rK39 dipstick result makes intuitive sense and suggests that a greater parasitologic burden results in antibody production rising to the limits of detection by the rK39 assay. These results are supported by an earlier study in CL patients that reported increasing antibody titers with increasing lesion size.13 Travel history to the Iraq and Afghanistan was also associated with positive tests; however, it is likely that these results were based on the large size of lesions from patients in Afghanistan. There were too few patients from each travel location to perform further multivariate analysis.

In conclusion, this study showed that, in a cohort of North American patients infected with CL, 10% will have a positive rK39 dipstick test. Patients with larger lesions and more lesions were more likely to have a positive result. A positive rK39 assay result is not always indicative of VL, and positive results should be interpreted with caution in patients who are at risk for or have CL or those without classic VL symptoms. These patients may deserve further monitoring or diagnostic evaluation to better determine the etiology of their positive test. In patients who are suspected of having VL (the population for which the test was developed), the rK39 assay has performed well diagnostically, and our results should not change the current practice of those who use the test in this setting.

Table 1

Demographic and clinical characteristics of 59 CL patients

rK39 negative (N = 36)rK39 positive (N = 23)P
Numbers are percentages unless noted.
*Six subjects without speciation.
Median age in years (range)27 (19–56)26 (20–44)0.91
Sex (% male)97910.55
Race [N (%)]0.089
    African American2 (6)5 (22)
    White31 (86)14 (61)
    Other3 (8)4 (17)
Travel history [N (%)]0.002
    Iraq29 (80)17 (77)
    Afghanistan0 (0)5 (23)
    Central America6 (17)0
    Europe1 (3)0
Duration symptoms [days (range)]90 (42–750)98 (30–270)0.54
Number of lesions (range)1 (1–15)3 (1–9)0.001
Lesion size (length × width) mm2 (range)300 (25–2,160)1102 (132–2,500)< 0.0005
Lesion appearance [N (%)]0.90
    Ulcer23 (64)16 (70)
    Papulonodular3 (8)1 (4)
    Eschar2 (6)2 (9)
    Plaque8 (22)4 (17)
Adenopathy present [N (%)]1 (3)3 (13)0.29
L. major [N (%)]*25/31 (81)20/22 (91)0.45
Figure 1.
Figure 1.

Graphical depiction of rK39 results. Points above dashed line are ELISA positive. OD = optical density; CL = cutaneous leishmaniasis; VL = visceral leishmaniasis.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 79, 6; 10.4269/ajtmh.2008.79.843

*

Address correspondence to Joshua D. Hartzell, US Army Infectious Disease Fellow, Walter Reed Army Medical Center BLD 2, Ward 63, 6900 Georgia Avenue NW, Washington, DC 20307. E-mail: joshua.hartzell@amedd.army.mil

Authors’ addresses: Joshua D. Hartzell, Walter Reed Army Medical Center BLD 2, Ward 63, 6900 Georgia Ave. NW, Washington, DC 20307, Tel: 202-782-1663, Fax: 202-782-3765. Naomi E. Aronson, Room A3060, USUHS, 4301 Jones Bridge Rd., Bethesda, MD 20814, Tel: 301-295-3621, Fax: 301-295-3557. Peter J. Weina, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring, MD 20910, Tel: 301-319-9956, Fax: 301-319-7360. Robin S. Howard, Biometrics Service, Department of Clinical Investigation, Walter Reed Army Medical Center, 6900 Georgia Ave. NW, Bldg. 6, Rm. 4045, Washington, DC 20307-5001, Tel: 202-782-7878. Anjali Yadava, US Military Malaria Vaccine Program, Walter Reed Army Institute of Research, Silver Spring, MD 20910, Tel: 301-319-9577, Fax: 301-319-7358. Glenn W. Wortmann, Walter Reed Army Medical Center, Bldg. 2, Ward 63, 6900 Georgia Ave. NW, Washington, DC 20307, Tel: 202-782-1663, Fax: 202-782-3765.

Acknowledgments: The authors thank Oral Cephas, James Cummings, Kim Moran, Juan Mendez, and Mark Polhemus for assistance with this study.

Financial support: This study was supported by the Infectious Disease Clinical Research Program and Military Infectious Disease Research Program.

Disclaimer: The views expressed are those of the authors and should not be construed to represent the positions of Walter Reed Army Medical Center, the Department of the Army, or the Department of Defense.

REFERENCES

  • 1

    World Health Organization, 2002. Urbanization: an increasing risk factor for leishmaniasis. Wkly Epidemiol Rec 44 :365–372.

  • 2

    Myles O, Wortmann GW, Cummings JF, Barthel RV, Patel S, Crum-Cianflone NF, Negin NS, Weina PJ, Ockenhouse CF, Joyce DJ, Magill AJ, Aronson NE, Gasser RA, 2007. Visceral leishmaniasis: clinical observations in 4 US Army soldiers deployed to Afghanistan or Iraq, 2002–2004. Arch Intern Med 167 :1899–1901.

    • Search Google Scholar
    • Export Citation
  • 3

    Burns JA, Shreffler WG, Benson DR, Ghalib HW, Badaro R, Reed SG, 1993. Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis. Proc Natl Acad Sci USA 90 :775–779.

    • Search Google Scholar
    • Export Citation
  • 4

    Chappuis F, Rijal S, Alonso S, Menten J, Boelaert M, 2006. A meta-analysis of the diagnostic performance of the direct agglutination test and rK39 dipstick for visceral leishmaniasis. BMJ 333 :723–727.

    • Search Google Scholar
    • Export Citation
  • 5

    Schallig H, Canto-Cavalheiro M, Silva E, 2002. Evaluation of the direct agglutination test and the rK39 dipstick test for the sero-diagnosis of visceral leishmaniasis. Mem Inst Oswaldo Cruz 97 :1015–1018.

    • Search Google Scholar
    • Export Citation
  • 6

    Braz R, Nascimento E, Martins D, Wilson ME, Pearson RD, Reed SG, Jeronimo SM, 2002. The sensitivity and specificity of Leishmania chagasi recombinant K39 antigen in the diagnosis of American visceral leishmaniasis and in differentiating active from subclinical infection. Am J Trop Med Hyg 67 :344–348.

    • Search Google Scholar
    • Export Citation
  • 7

    Ozensoy S, Ozbel Y, Turgay N, Alkan MZ, Gul K, Gilman-Sachs A, Chang KP, Reed SG, Ozcel MA, 1998. Serodiagnosis and epidemiology of visceral leishmaniasis in Turkey. Am J Trop Med Hyg 59 :363–369.

    • Search Google Scholar
    • Export Citation
  • 8

    Wortmann G, Hochberg L, Houng HH, Sweeney C, Zapor M, Aronson N, Weina P, Ockenhouse CF, 2005. Rapid identification of Leishmania complexes by a real-time PCR assay. Am J Trop Med Hyg 73 :999–1004.

    • Search Google Scholar
    • Export Citation
  • 9

    Kreutzer RD, Semko ME, Hendricks LD, Wright N, 1983. Identification of Leishmania spp. by multiple isozyme analysis. Am J Trop Med Hyg 32 :703–715.

    • Search Google Scholar
    • Export Citation
  • 10

    Zijlstra E, Nur Y, Desjeux P, Khalil EA, El-Hassan AM, Groen J, 2001. Diagnosing visceral leishmaniasis with the recombinant K39 strip test: experience from the Sudan. Trop Med Int Health 6 :108–113.

    • Search Google Scholar
    • Export Citation
  • 11

    Singh S, Kumari V, Singh N, 2002. Predicting kala-azar disease manifestations in asymptomatic patients with latent Leishmania donovani infection by detection of antibody against recombinant K39 antigen. Clin Diagn Lab Immunol 9 :568–572.

    • Search Google Scholar
    • Export Citation
  • 12

    Sundar S, Pai K, Sahu M, Kumar V, Murray HW, 2002. Immunochromatographic strip-test detection of anti-K39 antibody in Indian visceral leishmaniasis. Ann Trop Med Parasitol 96 :19–23.

    • Search Google Scholar
    • Export Citation
  • 13

    Menzel S, Bienzle U, 1978. Antibody response in patients with cutaneous leishmaniasis of the Old World. Ann Trop Med Parasitol 29 :194–197.

    • Search Google Scholar
    • Export Citation
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