Imported Mansonella perstans in migrants, Spain

Sabino Puente ISCIII Mar Lago Instituto de Salud Carlos III Mercedes Subirats Instituto de Salud Carlos III Ismael Sanz-Esteban Universidad Europea de Madrid Marta Arsuaga Instituto de Salud Carlos III Belen Vicente Universidad de Salamanca Montserrat Alonso-Sardon Universidad de Salamanca Moncef Belhassen-Garcia (  belhassen@usal.es ) CAUSA https://orcid.org/0000-0002-2230-1256 Antonio Muro Universidad de Salamanca


Introduction
M. perstans is transmitted by biting midges (Culicoides). The life cycle is similar to that of other lariae. Micro lariae are responsible for the transmission of infection because they are taken up during the blood meal of the insect vector. The epidemiology of M. perstans has not been clearly de ned. Among the known human larial infections, mansonellosis is probably the most frequent lariasis in sub-Saharan Africa as well as a northern part of the Amazon rainforest stretching from equatorial Brazil to the Caribbean coast of South America [1]. It has been estimated that 114 million people may be infected and as many as 581 million people in 33 countries are at risk for M. perstans infection in Africa alone [2]. Many publications refer to mansonellosis as one of the most common human helminthiases in endemic areas, and it is more prevalent and more neglected than other larial diseases such as lymphatic lariasis, onchocerciasis, and loiasis. In endemic areas, the probability of infection increases with age, with the prevalence reaching 100% in highly endemic areas. However, the infection is the least studied and is likely one of the most neglected of all tropical diseases, subject to more neglect than schistosomiasis, taeniasis, echinococcosis, or rabies [1,3].
The adult parasites are thought to live in serous body cavities, and the female parasites release micro lariae into the blood [4]. The diagnosis of M. perstans infection is usually by detection and identi cation of the micro lariae that circulate in the blood. Because the micro lariae are present in the peripheral blood in almost equal concentrations during the day and night [5], blood samples for the diagnosis can be obtained at any time. The prevalence and intensity of micro laremia increase gradually with age. Few and old studies have reported on the clinical picture caused by infection with M. perstans nematodes because the parasite is widespread in remote areas [2,6]. Usually, infected people have other parasitic infections that could contribute to clinical manifestations [2,6]. Clinical manifestations seem to be related to adult parasites than to the micro lariae, and the symptoms are probably related to the migration of the worms, including transient subcutaneous swellings (similar to the Calabar swellings caused by Loa loa), pruritus, rash, urticaria, arthralgia, abdominal pain, eosinophilia, fatigue, pericarditis, pleuritis and in ammatory granulomatous nodules surrounding dead adult worms [2]. Currently, no standard treatment exists for mansonellosis and its handling is still debatable. Therefore, many drugs have been used alone or combined, such as diethylcarbamazine, ivermectin, mebendazole, levamisole, albendazole, doxycycline and thiabendazole [1,2,7].
Despite accumulating evidence of a high prevalence of human infections, no current large-scale lariasis control program has targeted mansonellosis. Mansonellosis is not listed among the neglected diseases of the World Health Organization, and no control strategy has been de ned against this human lariasis.
The health-related impact on individuals living with these lariae remains unknown, and evidence regarding treatment strategies is scarce. Like other neglected diseases, it mainly affects poor populations living in tropical and subtropical climates and it has not been associated with a clear and distinct clinical picture [2]. It can be considered one of the most neglected tropical infectious diseases [3].
In summary, the health-related impact on people living with these lariae remains unknown, and it can be considered one of the most neglected tropical infectious diseases [3]. Therefore, our study was aimed to describe the clinical patterns and treatment of imported M. perstans infection by migrants from Africa.

Study
The La Paz-Carlos III Hospital in Madrid, Spain, is a tropical disease referral unit. Most patients voluntarily attend the emergency unit or are referred from primary care or general hospitals in Madrid. A very small percentage of patients come from other regions.
A retrospective study was conducted on the data regarding immigrants diagnosed with M. perstans infection over a 19-year period. The diagnosis of M. perstans infections was established with con rmed micro laremia or adult identi cation following extraction. The direct detection of circulating micro laria was performed on fresh venous blood obtained around midday with a thick lm and/or thin smear after Giemsa staining. Micro laremia was occasionally estimated on thin smears. The exclusion criteria included diagnosis in travelers, unspeci ed diagnosis methods (i.e., clinical data only), and medical records with missing data. The data included demographics (age, gender, nationality, time of the rst consultation) and clinical characteristics (symptoms and when the symptoms rst appeared). The eye examination results and analytical data regarding serologic tests for syphilis, HIV, hepatitis B and C, eosinophil counts, IgE levels and stool test results regarding ova and parasites were reviewed. Other laboratory test results were also recorded. Systematic ophthalmology exploration was performed in patients with a clinical suspicion of onchocerciasis. Hyper-IgE was de ned as an increase in peripheral blood IgE to more than 200 U/mL. Hyper-IgE was classi ed as being mild (>200-399 U/mL), moderate (>399-999 U/mL) and/or severe (>1000 U/mL). Relative eosinophilia was de ned as an elevated percentage of eosinophils (>5%) in individuals with <450×10 6 eosinophils/L. Absolute eosinophilia was de ned as an increase in the peripheral blood eosinophilic leukocytes to more than 450×10 6 eosinophils/L of blood. Mild eosinophilia was de ned as >450×10 6 eosinophils/L to 999×10 6 eosinophils/L. Moderate eosinophilia was de ned as >1,000×10 6 eosinophils/L to 2,999×10 6 eosinophils/L, and severe eosinophilia was de ned as >3,000×10 6 eosinophils/L.

Statistical analysis
Categorical variable results were expressed as percentages and as the mean and standard deviation (SD) for continuous variables. Chi-square test was used to compare the association between the categorical variables (i.e., clinical and demographic variables). The measured outcomes were expressed as the odds ratio (OR) with a 95% CI. The continuous variables were compared by Student's t-test or the Mann-Whitney test for two groups depending on their normal or non-normal distribution. The corresponding regression models were used for multivariate analysis considering p<0.05 for a statistically signi cant difference. The Statistical Package for the Social Sciences (SPSS 23.0®; IBM Corp., Armonk, New York, USA) was used to analyze all the data.

Clinical & Laboratory data
The main clinical and analytical patient data are described in Table 1 Table 1).  Table 3. Therefore, the drug most used, alone or associated, was mebendazole, in 407 patients. Most of them (n=382) received a single dose, 24 double doses and 1 triple dose. Corticosteroid therapy was administered concurrently with the anti-larial drug in 20 (4%) cases, and an antihistaminic drug was administered with the anti-larial drug in 38 (7.6%) cases. Figure 1 shows a signi cant decrease in eosinophilia before and after treatment (p<0.001).

Discussion
Most symptoms ascribed to M. perstans infections in modern scienti c literature are based on symptoms that have been recorded in case study reports. Most of these reports have been based on the treatment of tourists and expatriate Europeans and North Americans returning home from endemic areas, and not on people who have lived all their lives in endemic areas [8]. It is unclear whether the symptoms reported from these studies can be used to compile a clinical picture that represents all or even most infections caused by mansonellosis. The identi cation of these infections is often complicated by coinfection with other infective agents [9]. Bassene et al. analyzed patients infected only with M. perstans and concluded that these infections had low pathogenicity because most individuals with M. perstans infection are asymptomatic [10,11]. When symptoms occur, they are predominantly related to the migration of the adult worms and include dermatological symptoms such as transient subcutaneous swellings similar to the Calabar swellings of Loa loa infection, serositis (i.e., pericarditis and pleuritis) [12,13], and ocular symptoms (granulomatous nodules in the conjunctiva, retinal lesions, and periorbital in ammation surrounding dead adult worms) [2]. Nonspeci c symptoms, including pruritus, urticaria, fever, pain in bursae and/or joint synovia, enlarged lymph glands, vague abdominal symptoms and fatigue, have also been attributed to M. perstans [2]. Headaches, neuropsychiatric manifestations, meningoencephalitis, and hepatitis have also been described. Nonspeci c but characteristic laboratory abnormalities include highlevel eosinophilia and elevated serum immunoglobulin IgE levels observed in some but not all patients with M. perstans infections, likely because of the body's reaction against the adult worm, rather than against micro lariae [14,15]. This phenomenon can also be observed in other helminthiasis such as strongyloidiasis and schistosomiasis [16][17][18]. In our series, all the described features are represented. Moreover, recent observations have suggested that mansonellosis infections can in uence the human immune system's response, which can in uence the development of secondary infections, such as malaria [19] Parasitological diagnosis is based on the detection and identi cation of sheathless Mansonella micro lariae in the skin or blood at any time of day or night. Additionally, the diagnosis is established by identifying the adult worm in tissues. Serologic tests based on crude larial antigens are useful but do not distinguish between active or past infection and show cross-reactivity among different larial species and with other nematode infections. Consequently, their usefulness is limited, although a negative result can exclude the possibility of infection. A recent study suggests that ELISA commercial kit can be useful to distinguish between active and past infection [20]. Given the limitations of serology, we do not apply this diagnostic technique and may underestimate the number of cases. Furthermore, laria Polymerase Chain Reaction (PCR) serves a special function in the differential detection of lariae in situations where species are co-endemic [1] and the use of PCR could improve the diagnosis of lariae infection.
Among the three types of human mansonellosis, that one caused by M. perstans is usually regarded as the most di cult to treat [1,2]. Our work shows great variability in the treatment of this disease. Therefore, human infection with M. perstans raises questions about treatment because of poor responses to standard anti larial drugs and limited ndings from controlled trials. In contrast to conventional anthelmintic treatments, doxycycline has proven to be excellent, effective, and safe in the treatment of M. perstans infections [21,22]. However, the course of treatment over 6 weeks that is necessary for this type of therapy probably makes it impractical for control programs, although it appears to be curative, making it a very desirable therapeutic for travel medicine [7,22]. M. perstans is relatively resistant to standard anti larial agents, including DEC, ivermectin, albendazole, and mebendazole [4]. The usefulness of doxycycline in the treatment of M. perstans varies according to geographic region [7,23]. An ideal drug treatment for M. perstans infections needs to be identi ed that is effective, fast acting, tolerable and easy to administer. The search for new treatments may include a more meticulous quantitative assessment of the above-mentioned drugs, both alone and in various combinations [2].
Our study had some limitations, which were caused mostly by the retrospective design. First, most patients visited our center because of symptoms or an increased eosinophil count. Thus, the proportion of symptomatic patients is not representative of the general population with M. perstans infection. Second, posttreatment follow up was available for only a few patients. Thus, we could not properly describe the response to treatment. Third the conclusions about the effectiviness of the treatment are weak, cause it´s not a clinical trial (randomized with placebo group).

Conclusions
In summary, a long series of M. perstans infections is presented in sub-Saharan immigrants. Mansonellosis should be included in the differential diagnosis with other helminthiasis in patients with pruritus or analytical alterations such as eosinophilia or hyper-IgE presentation. These patients also have a high number of coinfections with other microorganisms, the treatment of which needs to be protocolized.

Declarations
Ethics approval and consent to participate This was a retrospective analysis of data obtained over a 19-year period. The data were collected anonymously to ensure impartial analysis; therefore, written informed consent was not obtained from the individual participants. The research was approved by the La Paz-Carlos III Hospital's Ethics Committee.

Consent for publication
Not applicable Availability of data and materials Eosinophilia before and after treatment