Serum samples from 26 suspected CA cases were evaluated for LF toxemia, seroconversion to anti-PA IgG and serum toxin neutralization activity (TNA) and compared with culture and direct observation diagnostic tests for anthrax. Acute stage samples were collected from 1 to 14 days after symptom onset and convalescent stage samples from 16 to 28 days. Antimicrobial use was reported in 11 individuals from 2 to 7 days prior to collection of the first available (acute) sample. In the remaining 15 case patients, antimicrobial treatment commenced with the acute sample collection ().
Of the 26 suspected CA cases in this study, there were 6 (pab4, pab16, pab23, pab25, pab27, and pab30) for which a sample was not available for analysis by culture/IHC/M’Fadyean (culture/observation) (). Of the 20 case patients for which a lesion sample was available, 6 (pab1, pab3, pab5, pab12, pab18, and pab21) were confirmed by culture/IHC/M’Fadyean ( and ). Serum from these 6 individuals was also reactive for LF toxemia, anti-PA IgG, and serum TNA. The LF concentrations for these 6 CA cases ranged from 0.041 to 1.26 ng/mL. There was not a consistent trend between the magnitude of toxemia and eschar reactivity by culture/IHC/M’Fadyean. One case patient (pab20) that was indeterminate by culture/IHC/M’Fadyean was nonreactive by any test except the anti-PA IgG ELISA. However, this individual was classified as indeterminate as there was not a 4-fold increase in anti-PA IgG between acute (<LLOQ) and convalescent (4.0 μg/mL) sera. There were 13 case patients (pab2, pab7, pab8, pab9, pab10, pab11, pab13, pab14, pab15, pab19, pab29, pab31,and pab32) for which there was a sample available but that were by negative for culture observation by either culture, IHC or M’Fadyean.
Figure 1. IHC staining of abundant granular and bacilliform antigens in skin biopsy samples with anti–B. anthracis capsule antibody (A) and anti–B. anthracis cell wall antibody (B). Immunoalkaline phosphatase with napthol fast red substrate and (more ...)
M’Fadyean (MFad) stain for capsulated B. anthracis in a cutaneous lesion smear (pab21). The pink-stained poly-d-glutamic acid (PGA) capsule can be seen surrounding the purple stained vegetative bacilli.
Twelve case patients (pab2, pab4, pab7, pab8, pab9, pab10, pab11, pab13, pab14, pab15, pab31, and pab32) were reactive by toxemia and serology only (). Three of these (pab14, pab31, and pab32) were reactive but did not meet the required 4-fold change between acute and convalescent paired sera for anti-PA and 1 (pab8) did not meet the ≥4-fold increase required for TNA reactivity. For pab14 and pab31, anti-PA IgG levels increased from <LLOQ to 10 μg/mL and from 4.0 μg/mL to 12.3 μg/mL between acute and convalescent sera, respectively. Thus, they did not meet the criterion for seroconversion. Pab32 had a high level of anti-PA IgG in the acute serum (679.5 μg/mL) and convalescent serum (487.2 μg/mL) and therefore also did not achieve a 4-fold change in anti-PA required for seroconversion. Serum TNA for these 3 cases increased from <LLOQ to an ED50 titer of 40 and 45 for pab14 and pab31, respectively, and from 58 to 487.2 for pab32. LF levels in pab14, pab31 and pab32 were 0.040, 0.035, and 0.086 ng/mL, respectively. Case pab8, although not reactive by TNA assay, had measureable levels of both LF (0.11 ng/mL) and anti-PA antibody (convalescent, 26.8 μg/mL), indicating that this also was a true infection. The combined data from all 3 serological tests indicate that these were true infections.
Four case patients (pab3, pab15, pab31, and pab32) had detectable levels of anti-PA IgG in their acute stage sera. Acute stage samples for pab3, pab15, and pab31 were obtained at days 8, 3, and 5 after reported onset of symptoms, respectively. The onset of an anti-PA antibody response in these 4 individuals was earlier than the day 12 onset reported previously for bioterrorism associated cutaneous anthrax [8
]. The acute stage sample for pab32 was obtained at day 14 and had a high level of anti-PA IgG (679.5 μg/mL), which declined to 487.2 μg/mL in the convalescent serum (day 25). This demonstrates that the immune response to infection had passed its peak level and that exposure may have been considerably earlier than assessed from the development of the cutaneous lesion. Of note is that this was the only case for which an acute serum sample had a detectable TNA titer and the titer increased ≥4-fold as discussed above. Linear regression analysis indicated a positive correlation (r2
= 0.41) between log10 transformed TNA ED50 titer and anti-PA IgG concentration for all values ≥ LLOQ (). This correlation is lower than that reported previously for inhalation anthrax (r2
= 0.83). The reason for this difference is unclear and may be a consequence of the different disease presentations. Of particular interest to note is that no convalescent stage sample contained detectable LF and all case patients that seroconverted to anti-PA had detectable acute stage toxemia.
Figure 3. Linear regression analysis of log10 TNA ED50 titers with log10 anti–protective antigen (anti-PA) immunoglobulin G (IgG) (μg/mL) for all values greater or equal to the assays’ lower limits of quantification indicated a positive (more ...)
There are few opportunities to compare laboratory diagnostic data for human anthrax. We compared the anti-PA responses for naturally acquired CA in this study with those previously reported for bioterrorism (BT) associated CA [8
]. The BT and naturally acquired CA samples were evaluated using the same validated anti-PA ELISA, reference standards, and QC criteria. The long-term performance stability of this assay has also been described [17
] and provides confidence in the comparison. Comparisons were made using first the Shapiro-Wilk statistic test for normality, followed by the t
test for normally distributed data. Both the bioterrorism-acquired and the naturally acquired maximum anti-PA IgG values were normally distributed in log space. The maximum measured anti-PA IgG level was significantly lower in BT associated CA than in naturally acquired CA (25.0 μg/mL [n = 13] vs 52.6 μg/mL [n = 21] [P
= .046, 2-sided unequal variance] ().
Figure 4. Box Plot comparisons of maximum determined anti–protective antigen (anti-PA) immunoglobulin G (IgG) responses from bioterrorism (BT) associated and naturally occurring cutaneous anthrax cases. Geometric means are 25.0 μg/mL (n = 13) and (more ...)