Occurrence of Mansonella ozzardi diagnosed using a polycarbonate membrane in a riverside population of Lábrea in the Western Brazilian Amazon

Introduction: Mansonella ozzardi is a widely distributed fi laria worm in the Amazon region. This study aimed to determine the prevalence of M. ozzardi infection in riverine communities of Lábrea municipality, Amazonas State, Brazil. Methods: A diagnostic blood fi ltration method in a polycarbonate membrane was used. Results: M. ozzardi was found in 50.3% of the sample, with the highest prevalence in farmers/fi shermen (69.4%; χ 2 = -19.14, p<0.001). The prevalence was higher in longerterm residents (≥11 years; 60.2%). Conclusions: M. ozzardi infection rates are high near the Purus River, much greater than those previously reported based on diagnosis using thick blood smears.

An endemic region for M. ozzardi in Brazil is in the municipality of Lábrea, in the Purus River region, Amazonas, where it has been detected since the fi rst survey in the 1950s (1) .Subsequent studies conducted in the 1970s and 1980s confi rmed the wide distribution of M. ozzardi in this region (8) (9) .More recently, the prevalence of the infection had reportedly increased in riverine communities located near the Purus and Ituxi Rivers (2) (3) (4) .
In the Purus River region, only Adami et al. (10) utilized the concentration method by Knott to diagnose mansonelliasis.Therefore, the present study aimed to determine the prevalence of M. ozzardi infection in riverine communities of the Lábrea municipality using a diagnostic blood fi ltration method in a polycarbonate membrane, which is more sensitive than thick blood smears (11) .
This study was conducted in the riverside communities of the Purus River (Cassianã, Bacural, Jucuri, Buraco, Santa Rosa, Jurucuá and Samaúma), located approximately 200km from the municipality of Lábrea communities (S: 07º15' 34" and W: 64º47'59"), State of Amazonas, Brazil (Figure 1).The sample consisted of volunteer men, women, and children aged 5-60 years selected using convenience sampling.Data and blood samples were collected after signing the free and informed consent form by the individual or the legal guardian.Of the estimated 350 inhabitants in the included communities, 171 (48.6%) inhabitants were present at the time of the study and agreed to participate.
To estimate the prevalence of Mansonella ozzardi, blood was filtrated in a polycarbonate membrane (12) .First, 1mL venous blood was collected from each individual, diluted in 10mL 0.9% saline, and fi ltered in a polycarbonate membrane (Nucleopore Corporation, Pleasanton, CA, USA) with a 3µm pore diameter.Then, the membranes were removed from the fi lters and mounted on microscope slides, fi xed in methanol, stained with a Panoptic kit ® , and examined under an optical microscope with a 200 magnifi cation to search for M. ozzardi microfi lariae.
The prevalence of microfi laremia was compared between sexes and length of residence (1-5, 6-10, and ≥11 years) using Chi-squared tests and between age groups (5-15, 16-30, 31-45, and 46-60 years) and occupations (farmer/fisherman, housewife, student, and others) using linear generalized models, followed by a contrast analysis to verify the specifi c groups between which differences existed.In addition, trend analysis was conducted for length of residence (13) .Furthermore, odds ratios (ORs) and 95% confi dence intervals (CIs) were determined for each group using OpenEpi.The data were adjusted to a binomial distribution for errors and analyzed with a signifi cance level of 5%.
To the best of our knowledge, this is the fi rst epidemiological survey to use blood fi ltration in a polycarbonate membrane to diagnose M. ozzardi in the Purus River region.Our data support the high prevalences (>50%) of mansonelliasis in the riverine population of Purus reported in previous studies (10) (14) .However, the rate (50.3%) of M. ozzardi infections in the present study was higher than that (20.7%) in another study conducted in the Purus River region in Lábrea that used thick blood smears (4) .In other Purus River municipalities, such as Pauini and Boca do Acre, prevalences of 24.8% and 24.1%, respectively, were previously reported (3) (14) .Therefore, lower prevalences were detected using the thick blood smear method, indicating lower sensitivity of this method and consequently under-reporting of the prevalence of M. ozzardi microfi laremic individuals in the Purus River region.Using the method by Knott, high parasitic loads were reported in the communities of the Purus River: 63.3% in Monte Verde and 54.5% in Praia do Gado, both in the Boca do Acre municipality (10) .In addition, the infection rates observed in Monte Verde in the present study were much higher than previously described (33.3%) (14).Polymerase chain reaction (72.3% positive) is also reportedly more sensitive than thick blood smear (42.6% positive) for the diagnosis of M. ozzardi (10) (15) .The slightly higher prevalence in men, although not signifi cant, is possibly attributed to the increased contact of men with insect vectors in their day-to-day activities and is consistent with the observation of other studies conducted in Amazonas State (3) (4) (5) (9)   .This degree of exposure to Simuliidae bites also explains the higher prevalence in individuals that work in fi elds, such as farmers, and in those of older age (3) (4) (5) (10) .In this particular scenario, women are highly exposed to vectors due to working in the fi elds with their husbands, leading to a high infection rate.
Longer residence duration in the communities (≥11 years of residence) was also associated with higher rates of infection, indicating that the length of residence in an endemic area and consequent exposure to vectors are crucial for the maintenance of M. ozzardi infection.This is possibly one of the factors responsible for accumulation of parasites in individuals, maintaining several adult worms of different ages with continuous production of microfi lariae, infection, and transmission by insect vectors (3) (5) .There are no previous reports of residence duration and prevalence rates of mansonelliasis; however, this relationship possibly also occurs in other regions.
Another important fi nding was that blood fi ltration using a polycarbonate membrane was able to detect a high number of parasitized children and adolescents aged 5-15 years (32.4%); this rate was higher than studies that used the thick blood smear method, with prevalences of 20.7%, 9.7%, and 10.2%, respectively, in children aged 10-18 years (2) (3) (4) .
The present study showed that M. ozzardi infection was present in 50.3% of the local sample, suggesting that the prevalence of this parasite in the region is higher than those recorded in recent surveys (2) (3) (4) .Furthermore, blood fi ltration using a polycarbonate membrane can be used in epidemiological surveys, especially in regions of low microfi lariae density or in post-treatment control programs, during which this method can be performed in the fi eld with minimum requirements to store blood for later analysis.Finally, an appropriate control model should be structured of this Amazonian endemic disease, which is currently neglected by the health authorities.

Ethical approval
The study was approved by the Ethics Committee of the Faculdade São Lucas, Porto Velho City, State of Rondonia (344/09 registry).All patients signed the free and informed consent term.

BasanoFIGURE 1 -
FIGURE 1 -Municipalities of the Lábrea region in State of Amazonas, Brazil.

TABLE 1 -Prevalence of Mansonella ozzardi microfi laremia, diagnosed using blood fi ltration in a polycarbonate membrane, according to riverine community, sex, age group, and occupation in the Purus River, Lábrea municipality, Amazonas State, Brazil, 2009.
*Different letters a b indicate statistical differences (p < 0.05).CI: confi dence interval; OR: odds ratio.