Abstract
Background
We have previously demonstrated that eosinophil-associated processes underlie some of the differences in clinical presentation among patients with Loa loa infection prior to therapy and that some posttreatment adverse events appear to be dependent on eosinophil activation.
Methods
We first conducted a retrospective review of 204 patients (70 microfilaria [MF] positive/134 negative) with Loa loa both before and following definitive therapy. We then measured filarial-specific antibodies, eosinophil- and Th2-associated cytokines, and eosinophil granule proteins in their banked serum prior to and at 1 year following definitive treatment. We also evaluated the influence of pretreatment corticosteroids and/or apheresis in altering the efficacy of treatment.
Results
Patients without circulating microfilariae (MF negative) not only had a higher likelihood of peripheral eosinophilia and increased antifilarial antibody levels but also had significantly increased concentrations of granulocyte-macrophage colony–stimulating factor, interleukin (IL) 5, and IL-4 compared with MF-positive patients. However, these differences had all resolved by 1 year after treatment, when all parameters approached the levels seen in uninfected individuals. Neither pretreatment with corticosteroids nor apheresis reduced the efficacy of the diethylcarbamazine used to treat these subjects.
Conclusions
Our results highlight that, by 1 year following treatment, infection-associated immunologic abnormalities had resolved in nearly all patients treated for loiasis, and pretreatment corticosteroids had no influence on the resolution of the immunologic perturbations nor on the efficacy of diethylcarbamazine as a curative agent in loiasis.
Clinical Trials Registration
Keywords: Loa loa, eosinophil, posttreatment, immunology
This study identified several baseline immunologic derangements in subjects with Loa loa, which had resolved in most patients by 1 year following treatment. Pretreatment corticosteroids had no influence on the resolution of the immunologic perturbations or the efficacy of diethylcarbamazine.
Up to 14 million people live in high-risk areas for Loa loa [1]. The most common presenting symptoms of loiasis include transient localized angioedema (Calabar swelling) or the presence of an eyeworm, although the infection is frequently subclinical. Rarely, Loa loa infection can lead to serious long-term complications [2–12].
Loiasis also has clinical import because a high Loa loa microfilaria (MF) count (>30 000 MF/mL [13, 14]) is the primary risk factor for severe posttreatment reactions to the antiparasitic medications ivermectin (IVM) and diethylcarbamazine (DEC) [15, 16]. As IVM is used in mass drug administration programs for onchocerciasis and (in combination with either DEC or albendazole) for lymphatic filariasis, many elimination programs have been halted (or slowed) in areas of co-endemicity with Loa loa [17].
Loa loa infection causes 2 very distinct clinical syndromes: (1) an asymptomatic condition associated with microfilaremia, seen most commonly in individuals from areas endemic for Loa loa, or (2) amicrofilaremic infections that are typically clinically symptomatic and seen in those who acquired the infection as temporary residents in endemic areas (TRs) [18–20]. These distinct syndromes are thought to be caused by differing immune responses to the parasite [19].
Diethylcarbamazine, the drug of choice for loiasis in Europe and North America, has a rapid microfilaricidal effect and is also macrofilaricidal. However, in up to 60% of cases, the adult worm can survive initial treatment with DEC [21–25]. Factors associated with an increased likelihood of treatment failure are not completely understood. Treatment of Loa loa infection initiates a cascade of inflammatory responses thought to be driven by parasite antigen release. A prospective study comparing the effects of single-dose DEC or IVM in Loa loa MF-positive patients over the first 14 days following drug administration showed that both medications induced a rise in the absolute eosinophil counts, serum levels of interleukin (IL) 5 and eosinophil granule proteins, and eosinophil surface expression of CD69 [26]. However, the long-term posttreatment immunology of loiasis has not been studied.
Posttreatment reactions to DEC and IVM are believed to be primarily mediated by eosinophils. Therefore, steroids have been suggested as a possible method to prevent such reactions. Given the importance of the MF count as a risk factor for posttreatment symptoms, apheresis has also been proposed as a potential intervention to prevent serious adverse events by physically lowering the MF count prior to treatment [27].
The goals of this study were as follows: (1) to assess the impact of steroids on DEC efficacy and (2) to evaluate the long-term immunologic consequences following treatment for loiasis among MF-positive and MF-negative individuals.
METHODS
Patient Selection and Demographics
The study population included all patients treated by the Clinical Parasitology Section of the Laboratory of Parasitic Diseases at the National Institutes of Health between 1976 and 2019 with a definitive diagnosis of loiasis. Amicrofilaremic loiasis was defined as the presence of either an eyeworm or Calabar swelling in individuals with a relevant exposure history and positive testing for antifilarial antibodies, microfilarial DNA by polymerase chain reaction, or an adult worm on biopsy. Clinical cure was defined as being amicrofilaremic for at least 1 year following the final dose of treatment. Individuals who had been amicrofilaremic at baseline were also required to be asymptomatic (ie, absence of Calabar swellings and eyeworm) during this time period to be considered cured.
The baseline clinical and immunologic features of a subset (n = 186) of the patients included in this evaluation (n = 204) have been previously described [18, 20, 28]. All patients were treated for loiasis with a standard 21-day course of DEC [29]. The decision to treat with either steroids or apheresis prior to DEC therapy was made at the discretion of the treating physician and, per standard of care, was primarily determined by the patient’s pretreatment MF counts. All steroid courses were fewer than 6 days in duration and dosing was empirical. Informed written consent was obtained from all patients, and the studies were approved by the Institutional Review Board of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. Between 1988 and the present, the study was conducted under the registered protocol NCT00001230.
Clinical Evaluation
All of the patients evaluated in this study underwent a history and physical examination along with the collection of blood, stool, and urine samples during multiple time points before and after therapy as described previously [19].
Laboratory Evaluations
Serum samples corresponding to each of the patients’ visits (before and multiple time points following definitive therapy) were collected and stored at -80°C. These stored samples were utilized for measurement of analytes as described below.
Eosinophil Granule Proteins
Eosinophil granule protein levels (eosinophil cationic protein [ECP], eosinophil-derived neurotoxin [EDN], eosinophil peroxidase [EPO], and major basic protein [MBP]) were measured in serum using a multiplexed Luminex immunoassay (Luminex Corporation, Austin, TX), as described previously [30].
Cytokine Measurement
Eosinophil-associated serum cytokine levels were measured using the standard protocol for xMAP human cytokine magnetic bead kits (EMD-Millipore Corporation).
Immunoassays
Filaria-specific (Brugia malayi adult antigen–immunoglobulin G [BMA-IgG] and BMA-IgG4) and Loa loa–specific antibodies (Ll-SXP-1) were measured by enzyme-linked immunosorbent assay as described previously [31–34].
Statistical analysis
Unless stated otherwise, geometric means (GMs) were used for measurement of central tendency. Statistical analyses were performed using Fisher’s exact test to compare categorical variables and Mann-Whitney U test for continuous variables. Repeated measures within the same groups were compared using the Wilcoxon matched-pairs signed rank test.
RESULTS
Demographic Information and Clinical Presentation
Patient demographic information for the subjects included in this study is summarized in Table 1. Of the 204 individuals included, 134 (65.7%) were amicrofilaremic and 70 (34.3%) had detectable MF in their blood. There was no difference between the 2 groups in gender distribution, but subjects in the MF-negative group were slightly younger (median age, 27 years) than the MF-positive group (median age, 29 years) (P = .03). Most of the MF-negative group (120/134, 89.6%) were infected as TRs, whereas less than half of the MF-positive group were TRs (32/70, 45.7%, P < .0001).
Table 1.
Baseline Demographics and Laboratory Results of Microfilaria-Positive and Microfilaria-Negative Patients With Loiasis
| MF Negative (n = 134) | MF Positive (n = 70) | Total (N = 204) | P | |
|---|---|---|---|---|
| Median age, years (range) | 27 (4–66) | 29 (6–62) | 28 (4–66) | .03 |
| Gender, n (%) | ||||
| Male | 73 (54.5) | 44 (62.9) | 117 (57.4) | .3 |
| Female | 61 (45.5) | 26 (37.1) | 87 (42.6) | |
| Patient demographics, n (%) | ||||
| Temporary residents | 120 (89.6) | 32 (45.7) | 152 (74.5) | <.0001 |
| Endemic individuals | 14 (10.4) | 38 (54.3) | 52 (25.5) | |
| Subjects with history of eyeworm, n (%) | 25 (21.9a) | 36 (53.7b) | 61 (33.7) | <.0001 |
| MF count, GM MF/mL (range) | N/A | 343.6 (2–1717 500) | N/A | N/A |
| Absolute eosinophil count, GM cells/μL (range) | 1453 (1–1 010 910) | 748.2 (1–8541) | 1158 (1–1 010 910) | <.001 |
| IL-4, GM pg/mL (range) | 1.7 (0.1–792.5) | 0.2 (0.1–31.4) | 0.8 (0.1–792.5) | <.001 |
| Eotaxin-1, GM pg/mL (range) | 45.9 (1–431.9) | 20 (0.1–135.1) | 34.7 (0.1–431.9) | .3 |
| GM-CSF, GM pg/mL (range) | 1.8 (0.1–1854) | 0.4 (0.1–178) | 1.1 (0.1–1 854) | .006 |
| IL-5, GM pg/mL (range) | 1.3 (0.1–483.3) | 0.5 (0.01–12.1) | 0.9 (0.01–483.3) | .01 |
| IL-6, GM pg/mL (range) | 0.5 (0.1–206) | 0.1c (0.1–0.1) | 0.3 (0.1–206) | .007 |
| IL-3, GM pg/mL (range) | 0.3 (0.04–6.7) | 0.2 (0.05–1) | 0.2 (0.04–6.7) | .1 |
| Baseline BMA-specific IgG, GM μg/mL (range) | 646.9 (10.7–4141 370) | 282.9 (3.1–2424 510) | 475.2 (3.1–4141 370) | .003 |
| Baseline BMA-specific IgG4, GM ng/mL (range) | 19.5 (0–3232 924) | 670.3 (0–6565 950) | 74.3 (0–6565 950) | <.001 |
| Baseline IgG anti–Ll-SXP-1 RDT,d number positive (% positive) | 127 (94.8) | 68 (97.1) | 195 (95.6) | .7 |
Abbreviations: BMA, Brugia malayi adult antigen; GM, geometric mean; GM-CSF, granulocyte-macrophage colony-stimulating factor; IgG, immunoglobulin G; IL, interleukin; MF, microfilaria; RDT, rapid diagnostic test.
aOf 114 subjects in the MF-negative group for whom this information was available.
bOf 67 subjects in the MF-positive group for whom this information was available.
cAll pretreatment IL-6 levels for each individual in the MF-negative group were 0.1 pg/mL.
dAccession number AF174420.
Baseline Laboratory Data
As shown in Table 1 and as reported previously in a subset of these patients [19], the GM baseline absolute eosinophil count (AEC) was higher in the MF-negative group (1453 cells/μL compared with 748.2 cells/μL in the MF-positive group) (P < .001). For many of the measured cytokines (including IL-4, granulocyte-macrophage colony–stimulating factor [GM-CSF], IL-5, and IL-6), GM levels at baseline were higher among MF-negative subjects (Table 1). Although not statistically significant, baseline GM levels of all eosinophil granule proteins except for ECP were also higher in the MF-negative group (Supplementary Table 1). Both baseline BMA-IgG and BMA-IgG4 differed between groups (646.9 μg/mL for BMA-IgG and 19.5 ng/mL for BMA-IgG4 in the MF-negative group vs 282.9 μg/mL for BMA-IgG and 670.3 ng/mL for BMA-IgG4 among MF-positive individuals; both P values < .01). Nearly all (95.6%) of the subjects in both groups tested positive for loiasis using the Loa loa IgG anti–Ll-SXP-1 rapid diagnostic test (Table 1).
Posttreatment Symptoms
As shown in Table 2, posttreatment symptoms, such as pruritus and subcutaneous papules, were relatively common in both groups. Whereas subjects in the MF-positive group were more likely to have had hematuria and arthralgias following treatment (both P values ≤ .02), they did not have a greater number of symptoms following treatment than those who were amicrofilaremic (GM number of symptoms, 0.4 vs 0.2; P = .2).
Table 2.
Posttreatment Symptoms Following Initial Treatment With Diethylcarbamazine in Microfilaria-Positive and Microfilaria-Negative Patients
| Number of Subjects Reporting Symptoms (%) | ||||
|---|---|---|---|---|
| MF Negative (n = 134) | MF Positive (n = 70) | Total (N = 204) | OR (95% CI), P Valuea | |
| Pruritus | 48 (35.8) | 27 (38.6) | 75 (36.8) | 1.1, (.6–2), .8 |
| Subcutaneous papules | 52 (38.8) | 20 (28.6) | 72 (35.3) | 0.6 (.3–1.2) .2 |
| Hematuria | 12 (9) | 15 (21.4) | 27 (13.2) | 2.8 (1.3–6.5), .02 |
| Arthralgias/myalgias | 22 (16.4) | 33 (47.1) | 55 (27) | 4.5 (2.4–8.6), <.0001 |
| Rash | 24 (17.9) | 9 (12.9) | 33 (16.2) | 0.7 (.3–1.5), .4 |
| Fever | 5 (3.7) | 8 (11.4) | 13 (6.4) | 3.3 (1–9.3), .07 |
Abbreviations: CI, confidence interval; MF, microfilaria; OR, odds ratio.
aBy Fisher’s exact test.
Among subjects who were MF positive, 34 of 64 subjects for whom the information was definitively available (53.1%) were treated with steroids prior to DEC therapy; only 2 MF-negative individuals (of 98; 2%) received steroids. The GM MF count among MF-positive subjects pretreated with steroids (681 MF/mL) was higher compared with those who were not pretreated (181 MF/mL) (P = .03). Therapeutic apheresis, performed only on MF-positive individuals, was utilized for 26 of 70 (37.1%) MF-positive subjects. Among subjects who underwent apheresis, the GM MF levels were markedly increased compared with those who were not apheresed (mean MF count for apheresed subjects, 1706 MF/mL compared with 64 MF/mL among those not apheresed; P < .001). These results were expected since high levels of MF were the determining factor for therapeutic apheresis and were also used as an indication for pretreatment with steroids. Among the small number of subjects who underwent apheresis but did not receive steroids, a mean reduction of 67% was seen in 24-hour postapheresis MF counts compared with preapheresis values (Supplementary Figure 1).
Long-term Immunologic Changes Posttreatment
Changes in Absolute Eosinophil Counts Following Therapy
Whereas the mean AEC was 2-fold higher among MF-negative subjects compared with MF-positive subjects at baseline, by 1 year posttreatment, most patients in both groups had eosinophil counts within the normal range. Further, at this time point, the GM AEC count was similar between the 2 groups (184.6 cells/μL among MF-negative compared with 201.3 cells/μL among MF-positive individuals; P = .7) (Supplementary Table 1 and Figure 1A).
Figure 1.
Eosinophil counts and eosinophil granule protein levels, pretreatment and at 1 year after treatment. A, The AECs in the MF-negative (left, denoted by circles) and MF-positive (right, triangles) groups prior to treatment and at the 1-year follow-up time point. B, The concentration of each of the 4 eosinophil granule proteins prior to treatment and at the 1-year posttreatment time point for the MF-negative (left, circles) and MF-positive (right, triangles) groups. P values for the difference in concentrations at 1 year compared with baseline values, as determined by the Wilcoxon matched-pairs signed rank test, are shown above the horizontal bracket on each graph. P values listed as “ns” were not statistically significant (>.05). Abbreviations: AEC, absolute eosinophil count; ECP, eosinophil cationic protein; EDN, eosinophil-derived neurotoxin; EPO, eosinophil peroxidase; MBP, major basic protein; MF, microfilaria; Rx, treatment; Yr, year.
Changes in Eosinophil Granule Protein Levels
Serum levels of eosinophil granule proteins are believed to be a reflection of eosinophil activation in the tissues. At baseline, for all granule proteins except for ECP, there was a trend towards higher values among the MF-negative compared with the MF-positive group, although this difference was not statistically significant (all P values > .05) (Supplementary Table 1). MBP values decreased significantly in both groups in the year following treatment, whereas ECP values did not change following treatment in either group (Figure 1B). Levels of EDN and EPO decreased following treatment in all but a few subjects, but this change was only statistically significant in the MF-negative group (EDN, 1098 ng/mL pretreatment and 360.3 ng/mL following treatment; P < .0001; EPO among MF-negative individuals, 18.9 ng/mL pretreatment and 4.2 ng/mL following treatment; P < .001) (Supplementary Table 1 and Figure 1B). In contrast to the MF-negative group, no significant changes were seen in concentrations of either EDN or EPO following treatment in the MF-positive group (both P values ≥ .1).
Cytokine Levels
At baseline, the MF-negative group had significantly increased concentrations of regulators of eosinophil development (GM-CSF) and/or activation (IL-5) as well as increases in T-helper 2 (Th2)–associated cytokines (IL-4) compared with the MF-positive group (Table 1). Baseline IL-4 values were increased almost 10-fold among MF-negative (mean IL-4, 1.7 pg/mL) compared with MF-positive (mean IL-4, 0.2 pg/mL) individuals (P < .001). GM-CSF and IL-5 levels were more moderately increased among MF-negative individuals at baseline (GM-CSF, 1.8 pg/mL; IL-5, 1.3 pg/mL) compared with those who were MF positive (GM-CSF, 0.4 pg/mL; IL-5, 0.5 pg/mL) (both P values ≤ .01).
Most cytokine levels (with the exception of eotaxin-1) decreased in the year following treatment. Eotaxin-1 levels increased significantly in both groups during this time period (from 45.9 pg/mL to 68.6 pg/mL among MF-negative and from 20 pg/mL to 62.9 pg/mL among MF-positive individuals; both P values ≤ .01) (Supplementary Table 1 and Figure 2). For the remainder of the cytokines, proportionally larger decreases were seen in the MF-negative (who had higher baseline values) compared with MF-positive groups, with the result that cytokine levels 1 year after treatment were similar in both groups. This decrease in cytokine levels for the MF-negative group following treatment was statistically significant for IL-5 and IL-3 (mean baseline IL-5 among MF-negative individuals, 1.3 pg/mL compared with 0.4 pg/mL at 1-year follow-up; P < .05; GM IL-3, 0.3 pg/mL at baseline and 0.1 pg/mL at follow-up; P < .01). In contrast, the changes in the MF-positive group during this time period were not significant for either cytokine (both P values > .05).
Figure 2.
Cytokine levels, pretreatment and 1 year following treatment. The concentration of each cytokine measured in the MF-negative (left, circles) and MF-positive (right, triangles) groups prior to and 1 year following treatment. P values for the difference in concentrations at 1 year compared with baseline values, as determined by the Wilcoxon matched-pairs signed rank test, are shown above the horizontal bracket on each graph. P values listed as “ns” were not statistically significant (>.05). Abbreviations: GM-CSF, granulocyte-macrophage colony–stimulating factor; IL, interleukin; MF, microfilaria; Rx, treatment; Yr, year.
Immunoassays
Filaria-specific (BMA) IgG and IgG4 levels showed differing patterns of change in the year following treatment (Supplementary Table 1). In both groups, the BMA-IgG levels decreased significantly from baseline to follow-up (both P values ≤ .01). For both time points, BMA-IgG levels were higher among MF-negative (646.9 μg/mL at baseline and 138.6 μg/mL at follow-up) compared with MF-positive (282.9 μg/mL at baseline and 96.5 μg/mL at follow-up) subjects. This difference between groups, however, was only significant at baseline (P value between groups = .003 at baseline; P = .2 at follow-up).
In contrast to BMA-IgG, GM levels of BMA-IgG4 only decreased significantly in the MF-positive group following treatment (from 670.3 ng/mL at baseline to 296.5 ng/mL at follow-up; P = .01). No significant change was seen in IgG4 levels in the MF-negative group during this same time frame (baseline GM IgG4, 19.5 ng/mL; follow-up, 12.2 ng/mL; P = .2). Therefore, at both time points, BMA-Ig G4 values were significantly higher in the MF-positive group compared with the MF-negative group (both P values < .001).
For a small subgroup of subjects, quantitative levels of IgG anti–Ll-SXP-1 were measured. Levels decreased for all subjects at the 1-year follow-up time point compared with baseline (Figure 3A).
Figure 3.
Response to treatment. A, The Loa loa–specific antibody responses specific for the Ll-SXP-1 (accession number AF174420) gene product at baseline and 1 year following treatment. B, The percentage of subjects in the MF-negative (blue line) and MF-positive (red line) groups who were cured following each round of treatment. Abbreviations: IgG, immunoglobulin G; MF, microfilaria; Rx, treatment.
Treatment Efficacy
As shown in Figure 3B and Table 3, fewer MF-positive individuals (34/69 subjects for whom comprehensive treatment data were available; 49.3%) experienced definitive cure following the first treatment course with DEC compared with the MF-negative group (79/132 subjects; 59.8%), although this difference was not statistically significant (P > .05). For each subsequent round of treatment, there were no differences in the percentage of subjects cured between the 2 groups (Figure 3B and Supplementary Figure 2).
Table 3.
Efficacy of Diethylcarbamazine Treatment in Microfilaria-Positive and Microfilaria-Negative Patients
| MF Negative | MF Positive | |||||
|---|---|---|---|---|---|---|
| Number of DEC Courses | Number of Patientsa | Cured, n (%) | Cumulative Efficacy, % | Number of Patientsb | Cured, n (%) | Cumulative Efficacy, % |
| 1 | 132 | 79 (60) | 59.8 | 69 | 34 (49) | 49.3 |
| 2 | 53 | 31 (58) | 83.3 | 35 | 23 (66) | 82.6 |
| 3 | 22 | 10 (45) | 90.9 | 12 | 5 (42) | 89.9 |
| ≥4 | 12 | 9c (75) | 97.7 | 7 | 7 (100) | 100 |
Abbreviations: DEC, diethylcarbamazine; MF, microfilaria.
aOut of 132 patients, 1 patient in the MF-negative group was not treated, and for 1 patient in this group the treatment history was unknown.
bFor 1 patient in the MF-positive group treatment information was not available.
cThe remaining 3 patients were cured after a 3-week course of albendazole.
As shown in Supplementary Table 2, those who acquired their infections as TRs were more likely to require more than 1 treatment course to resolve their infection compared with endemic individuals (ENDs). Subjects were also more likely to require retreatment if they reported a history of Calabar swelling (P < .01), and the mean MF count among those who required retreatment (139.6 MF/mL) was lower than for those who did not (901.6 MF/mL) (P = .02). Of note, both of these factors (Calabar swelling and a lower mean MF count) were characteristic of the TR group, and therefore these findings may simply reflect the increased proportion of TRs among those who were retreated. In a subgroup analysis, the TRs who were MF positive required the largest number of treatment courses (mean number, 2.14) to achieve clinical cure as compared with MF-negative TRs (1.5 treatment courses) or either END group (data not shown; P values < .05 for all comparisons). Other than an increased likelihood to have acquired the infection as a TR, especially if MF positive, there were no differences in baseline demographics, clinical presentation, immune-associated laboratory values, or pretreatment with steroids between subjects who required retreatment and those who did not.
Only 2 individuals in the MF-negative group were pretreated with steroids, precluding analysis of the impact of steroids within this group. There was no difference in the proportion of patients pretreated with steroids among those who did or did not require retreatment in the MF-positive group (16/34 who were cured with 1 round of treatment [47.1%] received steroids vs 18/36 subjects [50%] who required retreatment; P = .8).
DISCUSSION
Previous studies have demonstrated that the immediate posttreatment reactions in Loa loa are associated with immune-mediated responses to the parasite [18–20, 26]. These posttreatment immune changes are believed to be responsible for the serious adverse events to IVM and DEC that have had a negative impact on control programs for onchocerciasis and lymphatic filariasis in Loa-endemic areas. These adverse events have previously been shown to be more common among patients with microfilaremia who have high circulating MF counts [13, 14]. However, much is unknown about the posttreatment immune responses in loiasis, including (1) if the baseline immune differences seen among MF-positive and MF-negative individuals persist over long-term follow-up and (2) if steroids or apheresis can prevent adverse events following treatment.
The impact of microfilariae on the immune responses to infection has been well studied, with MF-positive individuals demonstrating an overall dampened immune response to the parasite [35–37]. Whether this decreased immune response is associated with differing posttreatment symptoms or decreased success rates of therapy compared with those without MF had not been previously studied. In our patients, MF-positive individuals had the same number of posttreatment symptoms as those who were amicrofilaremic. However, subjects who were MF positive were significantly more likely than those who were amicrofilaremic to have had atypical symptoms following treatment, such as hematuria and arthralgias.
Studies have shown that approximately 50% of patients with loiasis are cured following treatment with DEC [25]. Our results suggest that the presence of microfilaremia may have an impact on the likelihood of treatment success, as MF-positive patients were more likely to require retreatment. Interestingly, those who acquired the infection as a TR were also more likely to require retreatment. This finding was surprising as 90% of TRs were MF negative. On subgroup analysis, it appeared that the highest-risk subjects for retreatment may have been the minority (32/152; 21.1%) of TR patients who were also MF positive. However, these results should serve primarily as hypothesis-generating, since the study was not powered to evaluate these subgroups. Overall, success rates following a second round of treatment were similar between the 2 groups, suggesting that, although MF positivity may have an impact on the early posttreatment response, it fails to predict definitive cure.
Interestingly, pretreatment with steroids did not appear to decrease the ability to achieve cure nor to achieve immune homeostasis following treatment. This is important given that corticosteroids have been shown to alter the kinetics of microfilarial killing in a limited number of studies [38, 39]. Our study cohort did not include enough patients with high MF counts to assess the impact of steroids on the development of serious adverse events in such patients. However, in a small case series of such patients, pretreatment with steroids did not appear to decrease the potential for serious adverse events [13].
At baseline, there were multiple differences in immune function between the MF-negative and MF-positive groups, as seen by significant differences in eosinophil counts, eosinophil granule protein concentrations, filarial-specific antibody levels, and Th2 (IL-4) and eosinophil-associated (IL-5 and GM-CSF) cytokine concentrations. However, by the 1-year posttreatment follow-up time point, values had normalized and were similar in both groups. Nearly all cytokine levels were diminished following therapy, with the exception of eotaxin-1 (CCL11), which increased in both groups following treatment. This is not entirely surprising, as previous studies have shown that serum levels of eotaxin-1 and other proinflammatory cytokines are downregulated in helminth infections and exhibit increased levels following treatment [40]. These results indicate that the long-term response to the parasite following treatment may be related to the absence of parasite-driven inflammation.
Overall, our results suggest that, though MF counts are important in determining immediate posttreatment reactions and the initial success rate following the first round of DEC treatment, by 1 year following treatment immune-based biomarkers had returned to normal in both groups and most likely are not influenced long term by corticosteroid use or by physical means (apheresis) of transiently reducing MF numbers.
Supplementary Data
Supplementary materials are available at Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.
Notes
Acknowledgments. The authors thank the clinical staff at the National Institutes of Health who provided care for the patients included in this study.
Financial support. This work was supported by the Division of Intramural Research of the National Institute for Allergy and Infectious Diseases, National Institutes of Health.
Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.
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