The study protocol and consent forms were approved by the ethics committee at Hôpital Ste. Croix and the institutional review board at Centers for Disease Control and Prevention (CDC). Patients were eligible to participate if they were enrolled in the lymphedema treatment clinic at the hospital, had been examined by the clinic physician to rule out other causes of lower limb swelling, gave informed consent to collection of all biopsy specimens, had no medical contraindications to biopsy, had no ADLA episodes during the previous 2 weeks, and lived within a 10-km radius of the hospital.

Patients were tested for filarial infection by using an immunochromatographic card test (ICT), which detects antigen of adult W. bancrofti in the blood (30). Lymphedema stage was assessed by using an adaptation of a three-stage system recommended by WHO (31). Stage 1 lymphedema is characterized by swelling that is reversible on elevation at night. Stage 2 lymphedema is not reversible upon elevation and has no papillomatous changes. Stage 3 lymphedema, sometimes called elephantiasis, is characterized by papillomatous lesions and pronounced dermatosclerosis.

Patients were instructed in lymphedema self-care (27), with emphasis on thorough daily washing of the limb, basic skin care to treat and prevent entry lesions, range-of-motion limb exercises, and elevation of the leg during the day when possible and at night while sleeping. Participants were provided with basic supplies (e.g., soap, towels, wash basin) as needed. To monitor lymphedema self-care, patients returned to the clinic or were visited at home every 4–6 weeks and were asked about compliance with the regimen since the previous visit. Patients were encouraged to seek antimicrobial drugs and symptomatic treatment at the hospital during ADLA attacks. Thus, most ADLA attacks were observed by clinic staff; a few attacks were recorded on the basis of patient history and the presence of residual clinical signs (e.g., peeling of the skin, swelling) at the next follow-up visit.

A total of 91 patients agreed to undergo skin-punch biopsy of their lymphedematous leg or legs, and 26 of these patients also agreed to a biopsy of their non-lymphedematous leg (control). Twenty-seven patients agreed to have follow-up biopsies of their lymphedematous limb ≈12 months later. Biopsy specimens were taken from the anterolateral surface of the leg from a site that was representative in appearance of the rest of the leg. Irregular protrusions and skin lesions were avoided. After the skin was cleaned with betadine and anesthetized with 1% lidocaine, a 4-mm skin-punch biopsy specimen was obtained, and suture or 3M Steri-Strips were used to close the skin at the biopsy site. A short course of oral amoxicillin (250 mg three times a day for 5 days) was given to help prevent bacterial infection.

Skin-punch biopsy specimens were fixed in formalin in Léogâne, Haiti, and sent to CDC in Atlanta, Georgia, where they were embedded in paraffin. Five-micrometer sections were cut and stained with hematoxylin and eosin. All biopsy sections were read by the same pathologist (JG), who was blinded with respect to patient identification, lymphedema stage, and whether the specimen was from an initial or follow-up biopsy. Each biopsy specimen was evaluated for the presence or absence of the histopathologic characteristics presented in Table 1.

Statistical analysis was performed using EpiInfo 6.0. The chi-square and Fischer exact tests were used to compare differences in the proportions of specimens with histopathologic features.

Of the 91 patients enrolled in this study, 73 (80%) were female. Women and men did not differ significantly with regard to age, lymphedema stage, or histologic features (data not shown). Median age was 39 years (range 16–75 years). One patient had bilateral lymphedema; the others had unilateral disease. Two patients had filarial antigen detected in the blood by ICT. Both were treated with diethylcarbamazine, the drug of choice for W. bancrofti infection. The median length of time between enrollment in the lymphedema management program and the first biopsy was 21 days (range 0–866 days).

No clinical signs of infection or inflammation were evident at the biopsy site. Microscopic examination of tissue sections stained with hematoxylin and eosin revealed no evidence of bacterial infection and no W. bancrofti adult worms or microfilariae.

Follow-up biopsy specimens were collected from the lymphedematous limb of 27 patients a median of 365 days (range 317–656 days) after their first biopsy. Of these 27 patients, 20 (74%) were women; the median age was 38 years (range 16–61 years). They did not differ significantly by sex, age, or lymphedema stage from the 64 participants who only had one skin biopsy.

Compliance with self-care practices during the interval between biopsies was high. At 96%, 94%, 87%, and 98% of monthly follow-up visits, respectively, patients reported that, since the previous visit, they had washed the leg daily, practiced range-of-motion exercises, elevated the leg during the daytime, and raised the foot of the bed at night. No changes in lymphedema stage were observed.

The second biopsy specimen was obtained a median of 14 cm (range 6–17 cm) above the sole of the foot, and a median of 1 cm (range <1–5 cm) from the first biopsy site. No postoperative infections developed. Of the 27 biopsy specimens that were collected for follow-up, 2 were from legs with stage 1 lymphedema, 18 from stage 2 lymphedema, and 7 from stage 3 lymphedema.

Of the 27 patients, 21 (78%) had reported one or more ADLA attacks during the 12-month period before entering the program. In contrast, only eight (30%) reported one or more attacks during the interval between biopsies (1, 4, and 3 patients with stage 1, 2, and 3 lymphedema, respectively). The mean reported incidence of attacks during the year before entering the program was 1.7 (range, 0–8) per person-year, compared to 0.5 (range, 0–3) observed between biopsies (p = 0.0009).

The prevalence and intensity of histopathologic abnormalities tended to be greater in initial skin specimens (Figure 1A, B, and C) than in follow-up specimens (Figure 1D, E, and F) (Table 3). Hyperkeratosis and hyperplasia of the epidermis were more prominent on initial specimens (Figure 1A) than on follow-up specimens (Figure 1D). The thick collagen bundles observed in the dermis of initial biopsy samples (Figure 1A) were less obvious in specimens after 1 year of lymphedema management (Figure 1D). In the superficial dermis, substantial decreases were observed between the first (Figure 1B) and second (Figure 1E) biopsies, both in the prevalence of chronic perivascular infiltrate (100% to 59%, p = 0.0002, Table 3) and in the proportion of specimens with pronounced infiltrate intensity (37% to 11%, p = 0.03).

Perivascular fibrosis in the deep dermis was less common in follow-up biopsy samples (22%, Figure 1E) than in initial samples (52%, Figure 1B). Additionally, significant decreases were observed in the prevalence of chronic periadnexal infiltrate (70% to 33%, p = 0.007) and the percentage of specimens with pronounced infiltrates of lymphocytes and macrophages in periadnexal areas of the deep dermis (26% to 0%, p = 0.005, Table 3, and Figure 1C and F). The replacement of thick collagen bundles around adnexi in the first specimen (Figure 1C) with adipose cells in the second specimen (Figure 1F) was also noted in the subcutaneous tissue.

Figure 2 summarizes the histologic changes in each of the 27 patients with follow-up biopsies. Histopathologic regression or improvement was defined as the disappearance of a histopathologic characteristic on follow-up specimen, while histopathologic progression or worsening was defined as the appearance of a previously unnoted histopathologic characteristic on follow-up specimen. All but one patient (96%) showed histopathologic regression in one or more characteristics. Histopathologic improvement was observed among all stages of lymphedema. However, 4 (57%) of 7 legs with stage 3 lymphedema showed regression of chronic perivascular infiltrate in the deep dermis, compared to none of 20 legs with stage 1 or 2 lymphedema (p = 0.003). Eleven (41%) patients showed regression in four or more characteristics; they did not differ significantly from the other 16 patients with respect to sex, age, stage, or duration of lymphedema; duration of participation in the lymphedema program before the first biopsy; interval between biopsies; or ADLA incidence between biopsies (Table 4). Sixteen (59%) patients showed histopathologic progression in one or more characteristics. None of the 27 patients had progression of chronic perivascular infiltrate.