Measles IgM detection by POCT on OF from suspected cases
OF samples were collected from all 103 suspected measles cases and serum from 96 of these subjects. After testing OF using the Microimmune measles IgM capture EIA, sufficient OF specimens were available for testing with POCT from 99/103 suspected cases. For 92/99 cases, serum samples were also available for testing with the Siemens measles IgM EIA.
The results for these 92 cases are shown in Table 1. There was agreement between the three readers in interpreting the results of POCT on all OF samples and the majority of serum samples. For four serum samples two of three readers agreed on the POCT results and these were interpreted as described in the Methods section. The POCT detected measles IgM in 47/57 cases that were positive in the serum EIA and 29/31 cases that were serum IgM negative were also negative by POCT. OF from 2/4 cases giving serum EIA equivocal results were positive for IgM by POCT. The sensitivity and specificity of the POCT on OF for measles IgM detection compared to the EIA on serum were 83·9% and 93·5%, respectively.
Table 1. Comparison of the detection of measles specific IgM in serum by EIA and in OF by POCT
OF samples from the 47 cases that were positive in both the POCT and serum EIA were also positive in Microimmune measles IgM EIA, confirming measles in these cases. Measles H and N genes were detected by PCR in OF in 45/47 cases. The first of the two measles IgM-positive cases which was negative by PCR was from a 13-year-old whose date of onset was not known. The second case was a 5-month-old whose specimens were collected 2 days post-onset of rash symptoms, and their OF was very strongly positive in the measles IgM EIA (OD450nm 2·265, test/cut-off >25·0). Further, the measles N gene was amplified and genotyped as B3 from POCT strips that had been used for IgM testing of the OF but it was not amplified directly from the OF.
OF from 8/10 serum IgM-positive, POCT-negative cases were positive in the Microimmune measles IgM capture EIA and one of the two remaining cases gave an equivocal result. H and N genes were amplified by PCR in all 10 of these cases. In addition, H and N genes were amplified from OF in the four serum IgM EIA equivocal cases, shown in Table 1, and from a further serum IgM EIA equivocal case for whom insufficient volume of OF was available for testing by POCT.
Two of the 31 serum IgM EIA-negative cases gave a positive result in the OF POCT. The OF from each case was PCR negative. One of these cases was a 17-month-old child who had received measles vaccine 266 days prior to sample collection, which indicates a false positive result in the POCT. The other case was an 8-year-old from whom the OF sample was taken 7 days post-onset of rash; the measles IgM EIA result on this sample was also positive. Although this case may be a true positive, with viral nucleic acid declining to undetectable levels at day 7, it was interpreted as a false positive according to the criteria used here for laboratory-confirmed measles case.
Of the 29 cases that were measles IgM negative by both serum EIA and POCT on OF, H and N genes were amplified in OF from four cases, two of which were also positive by Microimmune measles IgM capture EIA. The OF and serum samples from two of these four cases were taken 1 day after onset of symptoms, and for the other two cases 2 and 3 days after onset.
Applying the criteria for a laboratory-confirmed case defined in the Methods section, 68/92 suspected cases shown in Table 1 were confirmed as measles cases. In addition to the 92 cases described in Table 1, there were seven suspected cases from whom only OF samples were available, and these were investigated by EIA and PCR. For a further four cases where matched serum and OF samples were available for investigation by EIA, three of these had sufficient OF available for PCR. Results from these 11 additional cases brought the total number of laboratory-confirmed measles cases to 75 from the 103 suspected cases investigated in this outbreak. The sensitivity, specificity, positive and negative predictive values of the Siemens' measles-specific IgM EIA on serum, OF IgM POCT, and H gene detection from POCT strips for the 103 suspected cases investigated are shown in Table 2. The results show that the OF IgM POCT has good sensitivity compared to the gold standard Siemens measles IgM EIA, detecting 75% of the measles cases. Furthermore, after testing OF for measles-specific IgM using the POCT, amplification of the H gene from the test strips detected all but two of the 72 measles cases tested.
Table 2. Performance characteristics of the Siemens measles IgM EIA, measles IgM POCT on OF and H gene PCR on nucleic acid extracted from OF tested POCT strips
Timing of specimen collection in relation to onset of symptoms and the method used to detect measles in cases is shown in Table 3. As indicated in the Methods section, serum samples were not available from all suspected cases for testing by EIA and sufficient OF was available for testing 99 suspected cases by POCT. Timing of specimen collection for all 103 cases is shown in the first column. For each test method, the number of positive cases detected and the total number of laboratory-confirmed cases tested is shown in the corresponding columns of Table 3.
Table 3. Comparison of the laboratory method used to confirm measles cases on differing days post-onset of symptoms
Of the nine suspected cases from whom specimens were taken on the day of rash onset, six were identified as measles cases by all the serological and PCR methods used indicating that detectable measles-specific IgM antibody and viral nucleic acid were present in acute cases.
For the 27 suspected cases from whom specimens were collected 1 day post-onset of symptoms, 20 measles cases were laboratory confirmed using the criteria outlined above. However, serological methods using serum IgM EIA and serum IgM POCT each detected only 12/19 (57·8%) and 11/19 (57·8%) confirmed cases, respectively. Of the seven measles cases not detected by the serum IgM EIA, four gave repeatedly equivocal results and three were negative. The eight measles cases not detected by the serum IgM POCT included four cases giving an EIA equivocal result and two cases giving a positive EIA result. The OF IgM POCT detected measles in 10/19 cases (52·6%) from specimens collected 1 day after onset of symptoms. Of the nine measles cases not identified by OF IgM POCT, four were equivocal, two were low positive (OD450nm of 0·43 and 0·59, test/cut-off <3·0) and two negative in the serum IgM EIA and for one case there was no serum sample available for testing.
The POCT for measles IgM on OF identified only 4/7 measles cases (57·1%) when the specimens were collected 3 days post-onset of symptoms. One of three cases not identified by the POCT was also negative in serum IgM EIA; the other two were positive in the serum IgM EIA, and EIA on the OF from these cases indicated very high levels of measles-specific IgM, giving a test to cut-off signal >25. There was insufficient OF remaining from these two cases to retest these specimens after dilution to investigate if the high levels of measles-specific IgM in the specimen had sequestered the entire rNP added for the POCT reaction, leaving no epitopes available for binding to the gold-conjugated monoclonal anti-NP antibody, as was the case for one such specimen in a previous study .
With the exception of OF specimens collected from cases on days 1 and 3 after symptom onset, >75% of the measles cases were identified using the OF IgM POCT at all other time points.
The OF IgM EIA detected 15/20 confirmed measles cases where specimens were taken 1 day after onset (Table 3, column 4). Two of these 15 cases were equivocal and one negative by serum IgM EIA. For specimens collected 2 days after onset of rash, the OF IgM EIA detected all eight confirmed cases, two of which were missed by the serum IgM EIA. Of the 10 confirmed measles cases where specimens were taken 4 days after rash symptoms the OF IgM EIA detected eight cases with one other case giving an equivocal result. The sensitivity of measles detection using OF IgM EIA and serum IgM EIA were similar for specimens collected at all other time points after rash presentation.
Measles H gene PCR performed directly from OF samples detected all but two of the 75 confirmed cases (Table 3, column 5). The two cases that were missed by PCR were serologically positive for measles IgM as outlined above. For one of these two cases OF was collected 2 days post-onset of rash and for the other case the timing was not known. All the measles cases detected by H gene PCR directly from OF specimens could also be detected after H gene PCR of nucleic acids extracted from POCT strips that had been used for detecting IgM in the OF (Table 3, column 6).
PCR on OF from the 28 non-measles suspected cases did not yield H and N gene amplicons. Measles N gene nested PCR on OF detected 72/75 measles cases. This included the two cases missed by the H gene PCR on OF and one additional case from whom the sample was taken 2 days post-onset of symptoms.
The N gene PCR was more sensitive when testing extracts of POCT strips previously used for OF IgM detection (71/72, 98·6%) than when testing OF directly (71/74, 95·9%). It detected two cases that had been PCR negative directly on OF specimens. For the first of the two cases, the date of OF collection in relation to onset of symptoms was not known. The serum IgM EIA and OF IgM EIA results on this case were positive and the H gene PCR result was negative. Although the N gene was amplified from the used POCT strips, the nucleotide sequence was not consistent with a wild-type measles strain. The N gene sequence obtained after amplification from the strip was identical to the measles strain used to produce the recombinant NP antigen employed in the POCT device. This case was serologically confirmed as measles, with no detectable viral RNA in the OF specimens. The second case was a 5-month-old baby from whom OF was collected 2 days post-onset of symptoms. Measles IgM was detected in both serum and OF by EIA and POCT. The measles H gene was not detected by real-time PCR. The N gene was amplified from the POCT strip, after testing OF for IgM from this case, and sequenced as wild-type measles of genotype B3.
N gene sequence identical to the strain used to produce the recombinant NP was also detected after N gene PCR from POCT strips of five of the 28 non-measles cases indicating that, in the absence of wild-type measles, trace levels of residual N gene present in the recombinant NP antigen amplified sporadically, as observed previously .