Study design, diagnostic procedures and inclusion criteria
This retrospective study was conducted on imported loiasis cases admitted to the Beijing Friendship Hospital, Capital Medical University between July 2014 and July 2023.
The definitive diagnosis of loiasis was made based on the combination of clinical manifestations, traveling history in endemic areas and the parasite identification [19] as below:
a. Clinical manifestations: Patients present typical Calabar swellings (recurrent painful oedema of the extremities), skin pruritus, arthralgia, myalgia and hypereosinophilia (eosinophilic count > 0.52×109/L).
b. Parasitological identification: Patients with positive microfilaraemia or with documented migration of adult L. loa worm(s) in the eyelid, subconjunctiva or under skin (biopsy).
c. Molecular methods: PCR positive for L. loa DNA in patients’ blood.
d. Epidemiological evidence: Patients with history of visiting or living in endemic areas outside China, primarily in Africa.
Parasitological identification was made by the finding of microfilariae in peripheral blood smear or the presence of adult worms in the eyelid, subconjunctiva or under skin by biopsy examination. The load of microfilaremia was quantified by thick blood film technique using peripheral blood collected around midday (between 10 AM and 2 PM) reflecting the periodicity of the infection [20].
To exclude the possibility of cross-reactivity with other lymphatic filaria, the filarial antigenemia were detected by immunochromatographic card test (BinaxNOW; Alere Scarborough Inc., Scarborough, ME) used for immunological detection of soluble Wuchereria bancrofti antigens in peripheral blood [21].
To exclude the possible infection of other parasitic diseases co-endemic in the same areas, the microscopic and serological tests were also performed for the detection of Plasmodium, trypanosoma, leishmania spp., toxoplasma and Schistosoma parasites (Fig. 1).
Information collected
The medical information on the epidemiological data (age, gender, visited country before diagnosis, characteristics of travel), clinical presentation (reason of first consultation, description and duration of symptoms) and biological results (blood cells count, transaminases, and microfilaremia count) were extracted from each patient and organized into data tables. Additionally, etiological information of L. loa were initially collected.
Histopathology and immunohistochemistry of skin lesion
A patient had skin nodule located on the right forearm clinically suspicious for loiasis. Skin biopsies of the representative lesion were fixed in 4% formalin, embedded in paraffin, cut into tissue sections, and stained with hematoxylin-eosin (H&E). In immunohistochemical test, four μm thickness sections were stained with immunohistochemical markers including anti-CD4, anti-CD8, anti-CD19 and anti-CD56 (Becton Dickinson, San Jose, California).
PCR amplification and phylogenetic analysis
A regular polymerase chain reaction (PCR) was used as a sensitive molecular biology technique to detect DNA of L. loa microfilaria in blood [22]. Briefly, DNA was extracted from peripheral blood samples using a DNA extraction kit (TIANGEN, DP705, Beijing, CHN). The primers were designed (Table 1) based on the internal transcribed spacer region 1 (ITS1) region of the ribosomal RNA gene from L. loa [23]. The amplification of housekeeping gene GAPDH was used as positive control. The cycling conditions for PCR were 95 °C for 9 min, then 30 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 45 s followed by 1 cycle of 72 °C for 10 min. The PCR products were analyzed by electrophoresis in 2% agarose gel (Conda, Spain).
For phylogenetic analysis, the obtained sequences of ITS-1 were compared with those sequences deposited in the NCBI GenBank. Then they were aligned by the ClustalW method in MEGA software (version 11) [24] and generated the phylogenetic tree in the neighbour-joining (NJ) clustering approach with one thousand bootstrap values.
Treatment regimen and follow-up
Given the risk of serious adverse events after diethylcarbamazine (DEC) or ivermectin (IVM) treatment which are the essential drugs to treat filariasis, the diagnosed patients were given a course of albendazole (ABZ, 400 mg thrice daily for 10 d) to reduce the load of Loa microfilaremia before starting with DEC treatment (6 mg/kg/d divided in two to three doses for 21d). Repeated DEC therapy is required for patients with severe manifestations, high eosinophilia, or positive for parasite detections as above. The clinical symptom and laboratory parameters were followed up regularly in these patients before starting additional treatment cycle.
Statistical analysis
Statistical analyses were conducted using SPSS version 21.0 (IBM SPSS Statistics 22; Armonk, NY). Continuous variables were described as mean ± standard deviations while categorical variables were expressed as frequencies and percentages.
Ethics review
A written consent letter was signed by patients who participated in this research. The patients’ IDs were never disclosed. The study protocol was approval by the Ethics Committee of Beijing Friendship Hospital, Capital Medical University for Human Research (Beijing, China) with the number 2023-P2-358.