Prevalence of Mansonella ozzardi among riverine communities in the municipality of Lábrea , State of Amazonas , Brazil

Introduction: Estimate the prevalence of Mansonella ozzardi infection and calculate the parasitic infection rate (PIR) in simuliid black flies in the municipality of Lábrea, State of Amazonas, Brazil. Methods: Prevalence was measured using the thick blood smear method collected from the fingers and was related to age, sex and occupation. Simuliidae were collected with a suction apparatus, then stained with hematoxylin and dissected to verify the PIR. Results: The average prevalence rate of M. ozzardi among the 694 individuals examined was 20.7%. Infection was higher in men (27.6%) than in women (14.3%) (p < 0.001) and occurred in most age groups, with the highest prevalence in the following age groups: 38-47 (40%), 48-57 (53.1%) and >58 (60.5%). The highest prevalence rates were observed in the retired (64%), followed by farm workers (47.1%). Infection by M. ozzardi was only identified in Cerqueirellum amazonicum (Simuliidae) with a PIR of 0.6%. Conclusions: This study showed a high prevalence of M. ozzardi in the riverine communities of Lábrea due to the lack of policies regarding the treatment of microfilaremic individuals in the region and an abundance of competent vectors for M. ozzardi.


INTRODUCTION
Mansonella ozzardi is a human filarial parasite that is distributed in parts of Central and South America and some Caribbean islands.Adult worms have rarely been captured, but appear to live in the mesentery and peritoneal cavity, while the microfilariae circulate mainly in the blood.They are transmitted by Diptera insects of the families Ceratopogonidae and Simuliidae 1,2 .
The pathogenicity of M. ozzardi remains unclear.Some reports have associated the presence of filarial worm with clinical manifestation, such as fever, coldness in the legs, joint pain and headaches 3 , but the frequency of these manifestations in endemic areas has not been studied.In some situations the presence of fever and chills appears similar to malaria, but unlike malaria, the symptoms of M. ozzardi do not have specific schedules, can occur during the day and cause damage to individuals in their daily functions 4 .
The microfilariae of M. ozzardi were detected in Brazil in the 1940s and 1950s 5,6 .Even though endemic areas had already been identified during this period in the State of Amazonas, along the Solimões, Purus and Negro Rivers and their tributaries 7 , at least one study predicted the neglect of this filarial disease 8 .In the municipality of Lábrea, State of Amazonas, Brazil, studies between 1940s and 1980s reported prevalence rates varying from 0.4% to 4.4% 7,9 , while more recent studies have shown that prevalence continues increasing, reaching 30.2% among people living along the Ituxi river 4 .
As a part of our group's long-term study on the prevalence and distribution of filarial infection of humans in Brazil, a survey was conducted to determine the current status of M. ozzardi on the Purus river, in Lábrea.Thus, the study aimed to obtain data on human infection by M. ozzardi through comparison of prevalence rates between sexes and examine the relations between filarial infection and age and professional occupation.Identification of the simuliid species involved in the transmission of M. ozzardi and estimation of the parasitic infection rate (PIR) of these vectors was also performed.

Medeiros JF et al -Mansonella ozzardi infection in Amazonas
This work was conducted in riverine communities on the Purus river in the municipality of Lábrea (07 o 15S/64 o 51'W), State of Amazonas, Brazil (Figure 1).This municipality, with a population of 38,451 inhabitants, 80% of whom reside in urban areas 10 , is located in the southwest of the State of Amazonas.
The study was conducted in 23 communities selected at random along the Purus River:  1.The populations mainly practice subsistence farming (fishing and some vegetable and fruit farming, including cassava and bananas) and raising animals (cows, pigs and chickens) on a small scale.The average yearly rainfall in Lábrea fluctuates between 2,400 and 2,600mm.The temperature usually ranges between 24 and 26 0 C and relative humidity between 85 and 95%.
The population in the rural areas in Lábrea is approximately 7,690 (20% of the total population) 10 .The sample size was determined by assuming an expected prevalence of M. ozzardi of 30.2% 4 with an error of 12% and confidence interval of 95%.The population density is low, such that at least 20% in the communities was examined.The study was conducted in communities through convenience samples.Individuals that were present at the moment of study and informed of the purpose and scope of the study were invited to participate.A total of 694 individuals between 2 and 88 years-old (mean = 22.9±17.Estimation of M. ozzardi prevalence was performed using thick blood smears obtained by digital punctures.Two drops of blood (about 60µl) were collected from each individual and placed on a slide.After air-drying, the blood was dehemoglobinized, stained with Giemsa, placed under a microscope for identification of the species of microfilariae present 11 .
The evaluation of prevalence took into account the following data: sex; age group, 2-9, 10-18, 19-27, 28-37, 38-47, 48-57, 58-67 and ≥ 68 years-old; occupation, farm worker, housewife, student, teacher, retiree (man ≥ 65 and women ≥ 60 years-old) and those with not occupation (<5 years-old) 12 .Prevalences between the sexes were compared by the x 2 test and the differences between age groups were compared using the maximum likelihood Chi square G-test.Differences in microfilaremia between the sexes were assessed by the Mann Whitney U test and microfilaremiae counts and age were analyzed using Spearman's correlation.
Simuliidae were caught in the communities by a rapid entomological assessment technique using manual suction collectors.The flies were identified in the laboratory, stained with acid hematoxylin and dissected.The simuliidae collected were dissected into three parts (head, thorax, and abdomen) using a stylet, under a stereoscopic microscope 13 .When filarial worms were detected, they were identified and quantified regarding developmental stage (L 1 , L 2 , or L 3 ) 14 .The parasitic infection rate (PIR) was calculated by the number of females infected with stage L 1 , L 2 and/or L 3 M. ozzardi divided by the number of females dissected, multiplied by 100.

Ethical considerations
This study was approved by the Research Ethics Committee of the National Institute of Amazonian Research (Instituto Nacional de Pesquisas da Amazônia, INPA), Manaus, Amazonas, Brazil, under process no.043/2005.Free, informed consent was obtained from all adult participants during meetings with the community and from parents in the case of minors.Of the 694 individuals examined, 144 (20.7%) were M. ozzardi positive, with prevalence that varied from 8.4% to 62.5% in different communities (Table 1).
The distribution of individuals with M. ozzardi according to sex and age is presented in Table 2. Men exhibited significantly higher prevalence than women (27.6% vs 14.3%; Chi square p < 0.001).The lowest prevalence was observed in the group of individuals between 2 and 9 years-old (4.9%).The percentage of microfilaremic individuals increased linearly with age (r = 0.96, p < 0.001; Spearman's correlation), with the highest indices of infection in individuals over 58 years of age (men = 66.7% and women = 55%).With the exception of individuals between 2 and 9 years of age, in all the other age groups, men presented higher prevalence than women, with significant differences verified for the age groups 19-27, 28-37 and 38-47 (p < 0.001) (Table 2).
Microfilaremia was higher in men than in women, but this difference was not significant (32.01 vs 19.36 mf/40µl blood; Mann Whitney p = 0.27); while the highest concentrations were verified in retirees and farm workers (Table 3).Overall microfilaremia was low, with most individuals (86 -59.7%) presenting between 1 and 9mf/40µl and was higher in individuals over 38 years of age (r = 0.44, p < 0.001) (Table 4).

DISCUSSION
This work confirms that M. ozzardi prevalence has increased in Lábrea: currently registering 20.7%, which represents a 41.5-fold increase in relation to that reported by Lacerda & Rachou (0.4%) 7 in 1956 and 4.7-fold in relation to that reported by Shelley (4.4%) 9 in 1975.This increase also was observed on the Ituxi River 4 , where a 5.1-fold increase was verified in relation to that reported by Tavares (5.9%) 15 in 1981.
The present results show that prevalence increases linearly with age, a result that is in agreement with other studies 12,[15][16][17][18] .Older individuals with greater exposure to open air contract the infection with greater regularity, whereas younger individuals whose daily agenda includes closer proximity to home and school are apparently less exposed.The greater exposure to the vectors explains the higher infection rates in men than in women, also confirmed in other studies 12,17,19 .In certain age groups (19-27, 28-37, 38-47), men showed much higher prevalence than women, possibly explained as a by-product of different occupations.Whereas men have greater exposure to vectors due to their daily activities, women are less exposed as they typically maintain day-to-day activities close to home.
Observation verified that among men and women over the age of 48, the rates of prevalence of infection were very similar.Some evidence exists that during the in individuals 50 years-old or over, prevalence for M. ozzardi in both sexes becomes virtually the same 18 .The similarity in prevalence rates between older men and women could be an artifact of the changing daily habits of men with age, as they tend to live a more sedentary lives, similar to the daily routine of women, thus farther removed from direct exposure to vectors.
The microfilariae load was highest among those aged over 38 years-old.These observations indicate that the high microfilaremia level determined in those over 40 is probably due to a continuum of overlapping infections.These findings are in agreement with observations reported in Bolivia, where the microfilaria loads using the finger prick method were lowest in children and increased with age (> 100mf/20µl blood observed in people >44 years-old) 18 .Monkeys were used as experimental hosts to monitor the microfilaremia of M. ozzardi.After a mean prepatent period of 163 days, microfilaremia increased, peaking at around 20 weeks, and then decreased, stabilizing at low levels for up to 48 weeks 20 .Microfilariae density was significantly higher in males.These results are similar to those verified among inhabitants of Colombia, based on a Knott´s sample (mf/ml) 21 and also in studies with Wuchereria bancrofti using polycarbonate membrane filtration in Brazil 22 .It should be noted, however, that mf-counts are highly variable between smears from an individual due to variations in blood sampling time 23 and short-term variation in mf density [23][24][25] .
The highest prevalence was determined in retirees, suggesting that these individuals could have suffered a continuous succession of overlapping infections that remained untreated during their lifetime.Farm workers represent the second most microfilaremic group, again confirming that individuals working in agricultural fields, in contact with the forest environment, or along river margins, are more exposed to simuliidae and therefore present greater prevalence of infection than other occupations.Most epidemiological studies of M. ozzardi report that individuals who work in the open air show higher prevalence rates 9,12,26 .

Medeiros JF et al -Mansonella ozzardi infection in Amazonas
6) were examined by Rapid Epidemiological Mapping for microfilariae (mf) of M. ozzardi in April 2006 and August 2007.

TABLE 2 -Prevalence of infection by Mansonella ozzardi microfilariae according to age and sex in communities along the Purus River, municipality of Labrea, State of Amazonas, Brazil.
ex: examined, pos: positive, prev (%): prevalence (%), *correspond to statistical significant difference (p < 0.001) between males and females in different age groups, using G-test.

TABLE 3 -Prevalence of infection by Mansonella ozzardi according to the occupation of individuals in communities along the Purus River, municipality of Lábrea, State of Amazonas, Brazil.
*children (2-5 years old), **three healthcare professionals, one blind person, two fisherman and two teachers.