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Keywords:

  • ELISA;
  • rk-28;
  • serodiagnosis;
  • visceral Leishmaniasis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Funding
  9. Transparency Declaration
  10. References

Clin Microbiol Infect 2012; 18: 81–85

Abstract

Antibody detection is a safely applied method at the wide scale in diagnosis of visceral Leishmaniasis (VL). In order to further advance serodiagnosis, the rK28 antigen has been recently introduced as a candidate for diagnosis of VL. We evaluated the sensitivity and specificity of the rK28 antigen in a micro-ELISA format in comparison to the rk39 antigen. The test was conducted on 252 parasitologically confirmed VL cases, 103 endemic healthy controls, 95 non-endemic healthy controls, 88 other infectious disease and 53 follow-up cases. Of 252 parasitologically confirmed VL cases, 251 cases were reported positive by rK28 antigen, yielding 99.6% sensitivity (95% CI, 0.97–0.99), which was similar to the sensitivity of rK39 ELISA (99.6%) (95% CI, 0.97–0.99). Specificity of the rK28 antigen in non-endemic and endemic healthy controls was 100% (95% CI 0.96–1) and 94.17% (95% CI, 0.88–0.97), respectively. In 88 different diseases, specificity was 95.45% (95% CI, 0.84–0.96). With the rK39 antigen, specificity of non-endemic and endemic controls and different diseases was 100% (95% CI 0.96–1), 92.23% (95% CI 0.85–0.96) and 96.59% (95% CI 0.90–0.98), respectively. Our results show that rK39 and rK28 antigens have similar sensitivity and specificity and rK28 can also be used as a serodiagnostic tool in the endemic population of Bihar.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Funding
  9. Transparency Declaration
  10. References

Visceral Leishmaniasis (VL) is commonly known as kala-azar in endemic areas of the Indian subcontinent, where definitive diagnosis and treatment still need attention. The demonstration of amastigotes in splenic and/or bone marrow aspirates remains the reference standard for accurate and definitive diagnosis of VL. Though this procedure is most specific its sensitivity widely depends on smear preparation, proper staining and appropriate working of the microscope [1]. Spleen aspirates are risky, and therefore require technical expertise. Serodiagnosis based on IFA, Western blot, DAT and ELISA are poorly adapted in the field conditions of an endemic region. Immunochromatographic tests (ICT) are ready to use and require low maintenance in field settings. In the current scenario, the rK39 ICT assay is used as a reference standard for the diagnosis of VL but its inability to discriminate between clinical and subclinical infection in an endemic population drew our attention towards more specific and sensitive novel antigens. Many other L. donovani-specific antigens have been characterized, demonstrating variable specificity and sensitivity [2,3]. rK9, rK26, rKRP42 and other antigens have been largely evaluated on symptomatic VL of endemic populations but the diagnostic accuracy of the rK39 antigen is the strongest worldwide [4]. The sensitivity of the rK26 antigen developed from Leishmania chagasi is only 20–40% in India [5,6]. The K9 antigen, which possesses 11 copies of a 14-amino-acid repeat in the open reading frame of K26 [5], yields only 78% sensitivity [7]. Important drawbacks of rK39 in any format are its low sensitivity in Africa [8–10], which to some extent has improved with the rapid tests manufactured by DiaMed Cressier sur Morat (Switzerland), and a large proportion (up to 32%) of asymptomatic healthy individuals from an endemic area testing positive [11]. Thus to resolve these drawbacks, a new-generation fusion antigen rK28 has been designed, which can perform better [12]. K28 is a recombinant synthetic gene of the kinesin region, consisting of multiple tandem repeat sequences of the L. donovani haspb1 and k39 kinesin genes to the complete open reading frame of haspb2, increasing antigen epitope density [12]. Though rK28 RDT has shown improved performance in Sudan [12], its evaluation in the Indian subcontinent is yet to be carried out. Thus the aim of this study is to evaluate the diagnostic accuracy of the novel rK28 antigen in the endemic population of Bihar.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Funding
  9. Transparency Declaration
  10. References

This prospective study was conducted at the Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, and at its field site at Kala-azar Medical Research Centre, Muzaffarpur, Bihar. The study was approved by the ethical committee of the Institute of Medical Sciences, BHU and KAMRC, Muzaffarpur. Written informed consent was obtained from all subjects.

In this study, 591 subjects were enrolled during January 2010 to August 2010, including 252 parasitologically confirmed patients with VL, 103 endemic healthy controls (EHC) selected from the VL endemic region of Muzaffarpur, and 95 non-endemic healthy controls from Varanasi. All these individuals had no past history of Kala-azar. In a panel of 88 other diseases, there were patients with malaria (n = 15), tuberculosis (n = 10), amoebic liver abscess (n = 17), dengue fever (n = 15), leprosy (n = 15) and typhoid (n = 16). These subjects were recruited and treated at SS Hospital, Banaras Hindu University, Varanasi. Patients who were below the age of 2 years, had a past history of kala-azar, or had positive HIV serology or positive pregnancy tests, were excluded. The median age of patients was 25 years. Among the recruited patients, 48% subjects were male and 52% were female.

Serum sample collection

Serum was collected from patients with VL before the onset of treatment. Serum was separated from 1 mL of blood collected from different groups of controls and confirmed VL patients in cryovials and stored at −20°C. The rK39 antigen was used as a comparator in this study.

ELISA

rK28 and rK39 antigens were received as a kind gift from S. G. Reed, Seattle, USA. ELISA was carried out as described earlier [13]. Briefly, flat-bottom 96-well microtitre plates were coated with 25 ng/well (100μL) of rK28 and rK39 antigen in coating buffer and incubated overnight at 4°C. The plates were then blocked with blocking buffer (1% BSA in 0.05 m phosphate buffer) for 2 h at room temperature. Plates were loaded with serum sample diluted 1:400 and incubated at room temperature for 1 h. The plates were washed five times with PBS containing 0.1% Tween-20 (pH 7.4) and incubated with peroxidase-conjugated goat anti-human IgG (1: 32 000 dilution in serum dilution buffer) at 37°C for 1 h. Then the plates were incubated with TMB substrate (GeNei) for 5 min at room temperature in the dark. Finally, the reaction was stopped with 0.1 N H2SO4.

The optical density was measured at 450 nm. Each sample was assayed in duplicate. Serum pools of pretreated VL patients were used as a positive control and pooled non-endemic controls were used as a negative control in each plate.

Statistical analysis

Determination of sample size for the evaluation of the new antigen rK28 in an endemic population was carried out by McNemar’s test. The cut-off values for anti-rK39 antibodies were set as the mean plus three standard deviations of the healthy non-endemic controls. The serological data thus obtained were analysed with EPIInfo (version-6) and SPSS 16 softwares. The generated predicted values were used to construct a receiver-operator characteristic (ROC) curve that plots the true-positive rate against the false-positive rate for different potential cut-off points [14]. Data from the repeatability experiment were used to estimate the kappa value for the ELISA method for both the antigens. The kappa coefficient of agreement quantifies reproducibility, correcting for agreement expected by chance. A kappa value of 1 represents perfect agreement.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Funding
  9. Transparency Declaration
  10. References

The pool of sera from VL patients was serially diluted from 100 to 4 × 105 times to find out the baseline of reactivity of anti-rK39 antibodies and anti-rK28 antibody responses with their respective antigens. Optical density at 4 × 105 dilution lies just above the cut-off range of non-endemic healthy controls. 1:400 dilution of serum from a parasitologically confirmed VL patient reacted strongly with rK28 antigens at a concentration as low as 25 ng/well.

To determine the serodiagnostic performance of the rK28 antigen, sensitivity and specificity of rK28 ELISA was compared with sensitivity and specificity of rK39 ELISA. In symptomatic VL cases, the mean titre of antibodies against rK28 and rK39 is 31 times higher than the mean titre of non-endemic controls (Fig. 1).

image

Figure 1.  Anti-rK39 and anti-rK28 antibody levels in serum determined by ELISA in parasitologically confirmed cases (VL) and three groups of controls: non-endemic healthy (NEHC), endemic healthy (EHC) and different diseases (DD). Results are expressed as optical density at 450 nm. Dotted line represents cut-off values between cases and control groups. Each dot represents an individual subject, bars display median value.

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Of 252 VL patients, 251 were positive by the rK28 antigen, yielding sensitivity of 99.6%, which was exactly similar to the sensitivity of rK39 ELISA (99.6%) (Table 1). There was excellent agreement between the assays (kappa 0.97, p <0.001) for serodiagnosis. The data for specificity are given in Table 2. The specificity for rk28 was very high in all groups, being 100% in non-endemic controls, and 94.17% and 95.45% for endemic controls and different diseases (Table 2). The data were comparable between rK28 and rK39.

Table 1.   Comparative performance of rK39 and rK28 antigens in visceral Leishmaniasis patients
Subjects, VL (n = 252)rK39 antigenrK28 antigenKappa
Positive (%)95% CIPositive (%)95% CI
 251 (99.6%)97.3–99.9251 (99.6%)97.3–99.90.97
Table 2.   Comparative specificity of rK39 and rK28 antigens in different control groups
SubjectsrK39 antigenrK28 antigenKappa
Negative (%)95% CINegative (%)95% CI
Endemic healthy (n = 103)95 (92.2)0.85–0.9697 (94.2)0.87–0.970.86
Non-endemic healthy (n = 95)95 (100)0.96–1.095 (100)0.96–1.01
Different diseases (n = 88)85 (96.6)0.90–0.9884 (95.5)0.84–0.960.86

The area under the curve of the ROC plot by both the antigens was 0.995, which shows the excellent performance and diagnostic accuracy of rK28 and rK39 antigens.

For 53 patients with VL, sera at 6 month follow-up were also available. There were 3.6- and 2.98-fold declines in the titre of antibodies against rK28 and rK39 antigens, respectively (Fig. 2). However, no significant decline in titres was observed at day 30 (p >0.05) in both antigens. Five out of eight relapse cases showed the same value of antibody titre persisting till 6 months of follow-up. In the remaining 45 cases, only four had no significant decrease in antibody titre.

image

Figure 2.  Trend of anti-rK39 antibody (a) and anti-rK28 antibody (b) titre in pretreated (day 0) and post-treated (day 180) subjects (p ≤ 0.001).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Funding
  9. Transparency Declaration
  10. References

In this study, the sensitivity of the rK28 antigen was excellent as out of 252 only one was negative in symptomatic VL cases. The only rK28 antigen-negative patient was positive with rK39 though the OD was low for this patient. VL sera with borderline/low ELISA reactivity against the rK39 antigen could be detected with similar accuracy with the rK28 antigen. This can be clearly demonstrated by the fact that there was no significant difference between mean optical densities of ELISA against both the antigens in the case of VL. Hence sensitivity of rK28 was similar (99.6%) to the rK39 antigen. The area under the ROC curve also shows excellent diagnostic accuracy for the rK28 antigen, which was similar to the rK39 antigen. Specificity of the rK28 antigens was slightly higher (approximately 2%) than for the rK39 antigens for healthy subjects from the endemic population. In a panel of patients with different diseases, all 15 malaria patients were negative and one tuberculosis patient was positive with rK39. The situation was the opposite for the rK28 antigen (i.e. one was positive in the malaria group and all were negative in the tuberculosis group). No subjects were positive in the group of patients suffering from dengue fever and leprosy, while one individual was positive in the typhoid group with both the antigens. In a group with amoebic liver abscesses, one subject was positive with rk39 and two were positive by the rK28 antigen. Hence, there was only 4.5% cross-reactivity with rK28, which was quite similar to the rK39 antigen. Overall specificity in the control group against both antigens was quite similar (>94%) and agreement between both assays was also excellent (Kappa 0.86).

In 53 follow-up cases, there was a similar decrease in antibody level at follow-up with both the antigens, though the titres remained high with both antigens. Thus we can conclude that the synthetic new-generation rK28 antigen shows excellent sensitivity and specificity, similar to the rK39 antigen in ELISA format, and is an additional tool in the armamentarium for VL diagnosis.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Funding
  9. Transparency Declaration
  10. References

We thank Professor R.N. Mishra (Department of Community Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India) for help with statistical analysis.

Funding

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Funding
  9. Transparency Declaration
  10. References

Funded by the National Institute of Allergy and Infectious disease (NIAID), DMID funding mechanism: Tropical Medicine Research Center Grant number: P50AI074321.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Funding
  9. Transparency Declaration
  10. References
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