4.3 Results
4.3.8 Trypanosoma vivax blinded serum panel using the indirect ELISA format with
4.3.8 Trypanosoma vivax blinded serum panel using the
The corrected absorbance values at 405 nm obtained were used to calculate the percentage positivity for each of the serum samples comprising the T. vivax blinded serum panel along with the panel of known non-infected serum samples which were tested in the indirect ELISA format. This allowed for the determination of three cut-off values and the subsequent prediction of the infection status of each of the T. vivax blinded panel tested serum samples.
After the panel was unblinded, the sensitivity and specificity of each antigen in the indirect ELISA format was calculated. The blinded panel consisted of 72 samples of which 31 were positive and the remaining 41 were negative.
The decision matrix outlined in Table 4.2 was used to predict the infection status of each of the tested serum samples (Table 4.9). From the data in Table 4.9, the sensitivity, specificity, accuracy, PPV, NPV and 100%-specificity values for each antigen at each serum dilution were calculated (Table 4.10). At some of the cut-off values for each antigen tested in the indirect ELISA format at the different serum dilutions (Table 4.10), higher or lower values for the above-mentioned parameters were obtained and thus the best combination thereof was selected and highlighted.
Table 4.9: Scores from the T. vivax blinded serum panel using the indirect ELISA format at the third cut-offa. ELISA result
Number of serum samples with predicted infection statuses
TvOPB (1:10) TvOPB (1:100) TvCATL (1:10) TvCATL (1:100)
Positive Negative Positive Negative Positive Negative Positive Negative
Positive 0 0 3 7 10 21 18 7
Negative 31 41 25 37 1 40 13 34
a n = 72.
Table 4.10: Calculated specificity and sensitivity values for the T. vivax blinded serum panel using the indirect ELISA formata. Antigen
(serum dilution)
Cut-off (%) Specificity (%) 100-specificity
(%) NPV (%) Sensitivity (%) PPV (%) Accuracy (%)
TvOPB (1:10)
Mean + 1SD 97.69 100.00 0.00 56.94 0.00 ND 56.94
Mean + 2SD 142.32 100.00 0.00 56.94 0.00 ND 56.94
Mean + 3SD 186.94 100.00 0.00 56.94 0.00 ND 56.94
TvOPB (1:100)
Mean + 1SD 43.65 80.49 18.42 56.9 22.58 50 55.56
Mean + 2SD 58.45 82.93 15.79 53.97 9.68 33.33 51.39
Mean + 3SD 73.24 90.24 10.53 56.92 9.68 42.86 55.56
TvCATL (1:10)
Mean + 1SD 14.43 80.49 21.05 67.35 48.39 65.22 66.67
Mean + 2SD 19.66 92.68 7.89 64.41 32.26 76.92 66.67
Mean + 3SD 24.9 97.56 2.63 65.57 32.26 90.91 69.44
TvCATL (1:100)
Mean + 1SD 36.83 17.07 89.47 53.85 80.65 42.37 44.44
Mean + 2SD 51.73 46.34 57.89 73.08 77.42 52.17 59.72
Mean + 3SD 66.63 82.93 18.42 72.34 58.06 72.00 72.22
a cut-off values at 40 min development.
ND: not determined
Using the TvOPB antigen in the indirect ELISA format testing the serum panel at a 1:10 dilution, high cut-off values (97.69 to 186.94%) with large standard deviations were obtained thus discrimination could not be made between infected and non-infected serum samples at any of the cut-offs. This might have been caused by the low absorbance values at 405 nm even before they were corrected for the no-coat control, and as a result when the percentage positivity was calculated, large values were obtained of which many had to be adjusted to fall between the 0 to 100% limits.
Diagnosis of T. vivax infections using the indirect TvOPB ELISA showed that at a 1:100 serum dilution, high specificity (90.24%) with a low sensitivity (9.68%) was obtained at the third cut-off. The accuracy, PPV and NPV values were average (55.56, 42.86 and 56.92% respectively) with a low 100%-specificity value (10.53%) indicating the TvOPB antigen in this ELISA format, using a 1:100 serum dilution, was more able to correctly diagnose the non-infected rather than the infected serum samples due to the low sensitivity and PPV values.
Using the TvCATL antigen in the indirect ELISA format testing the serum panel at a 1:10 serum dilution, high specificity (97.56%), PPV (90.91%) and accuracy (69.44%) values were obtained at the third cut-off with slightly higher NPV and sensitivity values at the second cut-off. A low 100%-specificity value of 2.62% along with the above parameters proves that the TcCATLFL antigen in this ELISA format was able to discriminate between infected and non-infected serum samples, more so for the non-infected samples due to the low sensitivity (32.26%) despite the high PPV value of 90.91%.
The indirect TvCATL ELISA of the serum panel using a 1:100 serum dilution resulted in high specificity (82.93%), NPV (73.34%), PPV (72.00%) and accuracy (72.22%) values at the third cut-off. A low 100%-specificity (18.42%) and an average sensitivity (55.06%) along with the above parameters proves that the TcCATLFL antigen in this ELISA format was able to discriminate between infected and non-infected serum samples, more so for the non-infected serum samples. Using a 1:100 serum dilution along with the TvCATL antigen in an indirect ELISA format yielded higher sensitivity, accuracy and NPV values than when a 1:10 serum dilution was used. Despite the increased 100%-specificity and decreased PPV values, testing the serum at a 1:100 dilution led to better overall results.
The ROC analysis of the T. vivax blinded serum panel in the indirect ELISA format for each of the antigens used is shown in Fig. 4.20 from which the sensitivity and
specificity for each antigen at each serum dilution was determined The percentage positivity values from the TvCATL antigens were used to generate the ROC curves.
The non-corrected absorbance values at 405 nmn were used to create the ROC curves for the TvOPB antigen due to the low non-corrected absorbance values at 405 nm and their resulting high percentage positivity values.
Along with high P values (0.0671 to 0.7740), AUC values close to 0.5 were obtained for the TvCATL antigen using a 1:10 serum dilution and for the TvOPB antigen using a 1:100 serum dilution in the indirect ELISA format. Despite the high Youden index obtained for the TvOPB antigen (0.5167), these values confirm that these tests were unable to discriminate between infected and non-infected serum samples and thus have very little diagnostic potential (Table 4.11). However, low P (<0.0001 to 0.0009148) and high AUC values (0.7345 to 0.8813) along with high Youden indices (0.5745 and 0.6768) were obtained for the TvCATL antigen using a 1:100 serum dilution and for the TvOPB antigen using a 1:10 serum dilution respectively in the indirect ELISA format. This confirms that these tests were able to discriminate between infected and non-infected serum samples and thus have promising diagnostic potential (Table 4.11).
When comparing the cut-off, specificity and sensitivity values calculated in Table 4.10 with those calculated from the ROC analysis (Table 4.11), the values for the TvCATL antigen at both serum dilutions were favourable and all within approximately 12 units of each. Due to the fact that the absorbance values were used instead of the percentage positivity values to create the ROC curves for the TvOPB antigen, the results are only a guideline since using the absorbance values the calculation of the serum sample‟s percentage positivity was not made with respect to the known infected serum control.
When comparing the TvOPB ELISA results in Table 4.10 to those obtained in the ROC analysis (Table 4.12) when the serum was tested at a 1:10 dilution a large difference is obtained. Thus the diagnostic potential of the TvOPB antigen in the indirect ELISA format using a 1:10 serum dilution cannot be confirmed.
Figure 4.20: ROC analysis of the indirect antibody detection ELISA format using the TvOPB and TvCATL antigens for the detection of T. vivax infections in cattle.
The ROC curves for the specified antigens in the indirect ELISA format at a 1:10 and 1:100 serum dilutions are shown in: panel (A) TvOPB antigen using the non-corrected absorbance values at 405 nm and (B) TvCATL antigen using the percentage positivity values.
Table 4.11: ROC-based indices of diagnostic accuracy of the indirect antibody detection ELISA, testing different antigens against the T. vivax blinded serum panel.
Indirect (serum dilution)
TvOPB (1:10)a TvOPB (1:100)a TvCATL (1:10) TvCATL (1:100)
AUC 0.7345 0.6690 0.5240 0.8813
Standard Error 0.07674 0.0924 0.06042 0.03473
95 % CI 0.5841 to 0.8850 0.5177 to 0.8802 0.4055 to 0.6424 0.8132 to 0.9494
P value 0.000915 0.00671 0.7440 <0.0001
a based on the non-corrected absorbance values at 405 nm.
Table 4.12: Optimal sensitivity and specificity of the selected antigens, from the ROC analysis, which were used to test the T. vivax blinded serum panel in the indirect antibody detection ELISA format.
Indirect (serum dilution)
TvOPB (1:10) TvOPB (1:100) TvCATL (1:10) TvCATL (1:100)
Cut-off (%) > 0.1015 > 0.0825 < 4.300 > 49.06
Sensitivity (%) 84.72 91.67 41.67 72.22
95 % CI (%) 74.31 to 92.12 82.74 to 96.88 30.15 to 53.89 60.41 to 82.14
Specificity (%) 72.73 60.00 85 95.45
95 % CI (%) 49.78 to 89.27 36.05 to 80.88 62.11 to 96.79 77.16 to 99.88
Youden index 0.5745 0.5167 0.2667 0.6767
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