
 study area - blood and serum collections were carried out during four 
 expeditions , three of which were conducted in the rainy season , while one was carried 
 out in the dry season .
six riverside communities along the border of ac and am were 
 studied : mapinguari ( 0924'08"s 6808'51.6"w ) and andara ( 0917'29.8"s 

06745'44.0"w ) , on the antimary river , porto acre ( 935'18 's 6731'57''w ) and vila 
 antimary ( 0904'01.0"s 06723'50.1"w ) , on the acre river , and two localities located on 
 opposite edges of the purus river - monte verde ( 0868'13 ' 's 6739'94.8''w ) and praia do 
 gado ( 0874'12 ' 's 6739'94.4''w ) , in the municipality of boca do acre .
the number of 
 inhabitants in the selected communities were as follows : mapinguari , 142 , andara , 10 , 
 porto acre , 14,309 , vila antimary , 156 , praia do gado , 7,550 and monte verde , 236 [ data 
 provided by the technology foundation of acre , department of basic health actions / state 
 department of health of acre , national health foundation ( funasa)/boca do acre and 
 brazilian institute of geography and statistics in 2008 ] .
in these areas , with the 
 exception of porto acre and boca do acre , the inhabitants live in scattered , hidden and 
 very isolated communities .
most of these inhabitants are sustained by subsistence 
 farming and occasional hunting and in some communities they are provided with some basic 
 schooling and primary health care . 

survey procedure - a local radio station advertised the visit of a 
 research team to the area to the inhabitants of the selected communities from the 
 antimary and acre rivers as well as the purposes of the study and the risk factors for 
 simuliid - transmited diseases .
characteristic symptoms such as skin diseases ( pruritus , 
 erythema ) , articular pain , lymphadenopathy and headache were also provided .
the 
 communities along the purus river were contacted through the funasa / boca do acre and 
 clinical evaluations and blood collections were scheduled in advance .
the nature of our parasitological analysis and the methods involved were explained to 
 the population of each locality and individuals who agreed to be volunteers received and 
 signed a terms of consent form .
personal and clinical data , such as the presence / absence 
 of any of the described symptoms , were recorded during the clinical evaluations .
a 
 convenience sampling method was adopted in the area and the sample size was determined 
 by assuming an expected prevalence of 50% with an error of 5% and a confidence interval 
 ( ci ) of 95% .
a total of 355 volunteers from six different communities were enrolled in 
 the study .
all enrolled volunteers were 18 years of age or older and , in the case of 
 women , were not pregnant or breast feeding .
the study design was approved and registered 
 by the ethical committee of oswaldo cruz foundation ( fiocruz ) ( 281/05 ) .
data on the prevalence of m. ozzardi were obtained through venous blood 
 samples collections performed using 10 ml sterile , disposable syringes .
one millilitre 
 of venous blood was deposited in a polystyrene tube with 10 ml of a 2% formalin solution 
 ( knott 1939 ) . after a period of 12 h ,
thin 
 films were prepared with the deposited sediment , then fixed with methanol and stained 
 with giemsa . to circumvent problems due to the preparation of material in the field , two 
 samples consisting of thin films were prepared from each volunteer .
prevalence data were evaluated according to the age , sex and occupational activity of 
 the participants . to analyse the disease prevalence in terms of economic activity ,
the 
 study subjects were split into three different groups based on whether such activities 
 were carried out in primarily indoor , outdoor or mixed environments .
group a included 
 all volunteers who were enrolled in outdoor activities such as agriculture , cattle 
 raising and fishing , whereas group b consisted of those individuals whose daytime 
 activities consisted of primarily indoor activities , including nurses , policemen , cooks 
 and drivers .
participants involved in activities of a mixed nature , such as housewives , 
 motorboat drivers , teachers , students and salesmen , were placed in group c. the results 
 of clinical assessments of symptoms were compared between infected and uninfected 
 individuals .
elisa for o. volvulus proteins were performed according to the method 
 described by bradley et al .
the recombinant proteins 
 included in a cocktail for elisa were ovmbp/10 ( expressed during the l3 , l4 and 
 semi - adult stages ) , ovmbp/11 ( expressed in all developmental stages ) and ovmbp/16 
 ( expressed only in adult worms ) from o. volvulus .
the recombinant 
 proteins were provided by dr mario rodriguez - prez from the centre of genomic 
 biotechnology / national politechnic institute , mexico .
plasma samples were obtained from blood collected in heparin - treated tubes , followed by 
 centrifugation for 10 min at 400 g to separate the pellets containing 
 packed erythrocytes from the plasma .
a total of 355 plasma samples were identified and 
 kept in a container with liquid nitrogen until transportation to laboratory of 
 simuliidae and onchocerciasis / oswaldo cruz institute ( lso / ioc)-fiocruz in the state of 
 rio de janeiro ( rj ) , where they were stored at -20c .
ten plasma samples from 
 onchocerciasis and mansonellosis - nave donors from rj were selected and used as negative 
 controls in each experiment .
in addition , each elisa plate contained duplicates of blank 
 and reference positive ( n = 3 , strongly reacting plasma from onchocerciasis patients ) 
 and negative ( n = 3 , nave non - endemic donors ) controls .
the cut - off values for 
 seropositive samples were calculated as the mean optical density ( od ) plus three 
 standard deviations obtained from the sera of negative controls from rj ( n = 10 ; igg 
 cut - off value = 0.116 ) . to standardise the od data obtained in different experiments , an 
 od index was calculated as the ratio of the observed od / cut - off values .
the anti - maltose binding 
 protein from escherichia coli ( mbp ) responses of all analysed sera were 
 determined in parallel .
all elisa od values were corrected by subtracting the anti - mbp 
 od values from each serum specimen prior to determining the cut - off values . 

statistical analysis - all data obtained in this study were tested for 
 significance via ( 2-tailed ) fisher 's exact modification of the 2 x 2 chi - squared test 
 with a 95% ci and p < 0.05 indicating statistical significance .
the mantel - haenszel 
 test was applied using epi info7 software and the results are shown in 
 tables i , ii .
fischer 's exact test was performed with graphpad prism software , version 
 4.0 for windows ( graphpad.com ) . 

table iprevalence of mansonella ozzardi infections in six riverside communities of 
 antimary , acre and purus rivers bordering region of the states of acre and 
 amazonas , brazil ( 2006 - 2008 ) 

 m. ozzardi prevalence riversacre antimary purus 
  community   porto acre np / n ( % ) vila antimary
np / n ( % ) mapinguari np / n ( % ) monte verde np / n ( % ) praia do gado
np / n ( % ) total np / n ( % ) sex  males   4/39 ( 10.3 )   17/55 ( 31 )   1/6 ( 16.7 )   0/39 ( 0 )   14/19 ( 73.7 )   7/12 ( 58.3 )   43/170 ( 25.7 )  females   1/90 ( 1.1 )   6/33 ( 18.2 )   0/4 ( 0 )   0/29 ( 0 )   10/19 ( 52.6 )   5/10 ( 50 )   22/185 ( 11.7 )   total  community   5/129 ( 3.9 )   23/88 ( 26.1 )   1/10 ( 10 )   0/68 ( 0 )   24/38 ( 63.2 )   12/22 ( 54.5 )   65/355 ( 18.3 )  river 28/217 ( 13 ) 1/78 ( 1.3 ) 36/60 ( 60 )   - n : number of subjects examined ; np : number of positive subjects . 
 
table iidistribution of mansonella ozzardi infected individuals according to 
 economic activities in six riverside communities of acre and purus rivers 
 bordering region of the states of acre and amazonas , brazil ( 2006 - 2008 ) 
  occupation groups 
 m. ozzardi prevalence np / n ( % ) a   37/152 ( 24.3 )   b   4/54 ( 7.4 )   c   24/149 ( 16.1 )   total   65/355 ( 18.3 ) group a : individuals enrolled in outdoor activities such as agriculture , 
 fishing , cattle raising ; group b : individuals enrolled in indoor activities 
 such as nurses , policemen , cookers , drivers ; group c : individuals enrolled 
 in activities of mixed nature such as students , teachers , housewives , motor 
 boat drivers ; n : number of subjects examined ; np : number of positive 
 subjects . 
 
group a : individuals enrolled in outdoor activities such as agriculture , 
 fishing , cattle raising ; group b : individuals enrolled in indoor activities 
 such as nurses , policemen , cookers , drivers ; group c : individuals enrolled 
 in activities of mixed nature such as students , teachers , housewives , motor 
 boat drivers ; n : number of subjects examined ; np : number of positive 
 subjects .
as shown in table i , a total of 355 blood films 
 prepared according to the knott ( 1939 ) test were 
 examined and 65 ( 18.3% ) were found to be positive for m. ozzardi 
 microfilariae .
monte verde , which is located on the left bank of this river , exhibited 63% 
 positive samples , whereas on the opposite side of the river , 54.5% of the volunteers in 
 the locality of praia do gado presented blood microfilariae of m. 
 ozzardi ( p > 0.05 ) .
only one volunteer from andara , located along the 
 antimary river , was positive for microfilariae and all of the individuals from 
 mapinguari presented blood samples that were negative for microfilariae .
vila antimary 
 and porto acre , situated along the acre river , both presented some infected individuals , 
 with vila antimary displaying a higher prevalence rate ( p < 0.05 ) . when the rates of 
 infection were compared between men and women ,
a slight difference could be detected 
 only in porto acre , whereas in all other communities , the rates of infections were 
 evenly distributed between the two sexes .
the prevalence rates were higher among individuals who primarily performed outdoor tasks 
 ( group a ) ( p < 0.05 ) compared to those performing mostly indoor activities ( group b ) 
 ( table ii ) .
no differences were observed 
 between groups a and c. all previously described clinical aspects were evaluated and it was found that symptoms 
 such as headache and articular pain were the prevalent symptoms in infected individuals 
 ( table iii ) . 

table iiicomparative analysis of clinical aspects of mansonella ozzardi infections 
 between infected and uninfected individuals from six riverside communities of 
 acre and purus river bordering region of the states of acre and amazonas , 
 brazil ( 2006 - 2008 ) 

 m. ozzardi infections 


 subjects positive ( n = 65 ) np ( % ) negative ( n = 290 ) np ( % ) porci  symptomatic individuals  coldness in legs   19 ( 29 )   80 ( 28 )   0.7623   1.084   0.5990 - 1.963  headache   43 ( 66 )   96 ( 33 ) < 0.0001   3.950   2.236 - 6.978  articular pain   37 ( 57 )   94 ( 32 )   0.0003  
2.755   1.591 - 4.772  lymphadenopathy   4 ( 6 )   52 ( 18 )   0.023   0.3001   0.1045 - 0.8623   asymptomatic individuals   21 ( 32 )   182 ( 63 ) < 0.0001   0.2832   0.1599 - 0.5018 ci : confidence interval ; n : total examined ; np : number of positive 
 individuals ; or : odds ratio . 
 
ci : confidence interval ; n : total examined ; np : number of positive 
 individuals ; or : odds ratio .
furthermore , division of the subjects according to age / sex revealed that among 
 volunteers under 40 years of age , the prevalence of infection was higher in males ( p 
 < 0.05 ) , whereas among those aged 40 - 50 and over 50 years , the prevalence of 
 infection was distributed evenly between the two sexes ( p > 0.05 ) .
m. 
 ozzardi infections were generally more prevalent among males in the study 
 area ( p < 0.05 ) ( table iv ) . 

table ivprevalence of mansonella ozzardi infected individuals according to age and 
 gender in six riverside communities of acre and purus rivers bordering region 
 of the states of acre and amazonas , brazil ( 2006 - 2008 ) age group   males np / n ( % ) females np / n ( % ) total np / n ( % ) < 40   20/95 ( 21 )   05/109 ( 4.6 )   25/204 ( 12 )   40 - 50   05/25 ( 20 )   07/41 ( 17 )   12/66 ( 18 ) > 50   18/47 ( 38 )   10/38 ( 26 )   28/85 ( 33 )   total   43/167 ( 26 )   22/188 ( 12 )   65/355 ( 18 ) n : number of subjects examined ; np : number of positive subjects . 
 
when the results regarding prevalence were compiled , it was possible to note a gradient 
 from the communities where m. ozzardi infections are more prevalent in 
 am to those located in the border regions of am and ac , where transmission foci may have 
 been recently established ( figure ) . the results obtained via elisa using the recombinant proteins ov10 , ov11 and ov16 
 coupled to mbp revealed that igg responsiveness to the cocktail was negative in 354 of 
 the 355 samples tested , with the one exception coming from porto acre .
the surveyed localities were chosen based on previous field studies revealing the 
 presence of simulium species such as s. amazonicum and 
 simulium oyapockense in this region , which are potential vectors of 
 filarial parasites ( nascimento et al .
2009 ) and 
 could serve as risk factors for simuliid - transmitted diseases in the population . 
 m. ozzardi infections are well known along the purus river and its 
 tributaries ( medeiros et al .
2008 , 2009 , 2011 ) , 
 but no epidemiological studies aimed at determining the presence and dispersion of this 
 species through strategic points on the border of am and ac have been performed 
 previously . the results of the present survey demonstrated varying rates of human 
 infection at the studied sites .
the thin films obtained from all volunteers from 
 mapinguari , which is situated along the antimary river , were negative , suggesting that 
 transmission was likely not occurring at this point along the river , although breeding 
 sites of simuliids had been detected ( nascimento et al . 

a city bordering the acre river , low but detectable 
 rates of m. ozzardi infection were observed .
indeed , as porto acre is 
 moderately developed compared to other riverine communities , most of the volunteers from 
 this site were involved in indoor activities such as nursing , teaching and domestic 
 care , reducing their exposure to simuliid bites .
the geographical proximity of riverine 
 communities to this particular city makes it very attractive in terms of economic 
 activities such as trade , fishing and agriculture .
porto acre is still very important to 
 individuals searching for medical assistance , resulting in frequent human migration to 
 the city .
the results obtained in monte verde and praia do gado support the contention 
 that m. ozzardi infections are scattered along the purus river and that 
 the infected individuals in these areas exhibit high parasitic loads on average ( data 
 not shown ) .
indeed , the infection rates observed in monte verde were much higher than 
 have previously been described ( medeiros et al . 

2009 ) , possibly due to differences in methodology , such as the use of the 
 knott ( 1939 ) method , which improves 
 sensitivity .
it is of note that the rates of m. ozzardi infection in this area ( the 
 purus river ) have been the subject of various studies and appear to be increasing with 
 time . in 1975 , shelley studied riverine communities along the purus river and reported 
 an index of 4.4% .
( 2009 , 2011 ) found prevalence rate of 20% to almost 40% depending of the 
 locality . in general , m. ozzardi infections are observed at prevalence 
 rates of approximately 20 - 47% in endemic areas such as those in bolivia , venezuela and 
 colombia ( kozek et al .
the present work was conducted in localities 
 bordering the antimary , acre and purus rivers , which have never been surveyed 
 previously .
these areas were presumed to display prevalence rates of approximately 50% , 
 which is the value employed when there is an absence of conclusive data .
instead , the 
 actual global prevalence rate recorded was 18.3% and by examining each community , the 
 highest rates were shown to occur along the purus river . in studies conducted in 
 communities along the pauini and purus rivers as well as in lbrea , medeiros et al .
as some localities along the purus river are very important economically , 
 they appear to be exporting infected individuals to previously uninfected areas , such as 
 areas of ac . when the prevalence of m. ozzardi infections was compared between males 
 and females , the frequency of infection
was observed to be higher in males , in agreement 
 with previously reported data ( tavares 1981 , 
 medeiros et al . 2011 ) .
indeed , our data showed 
 that for individuals involved in outdoor and mixed indoor - outdoor activities , there was 
 an enhanced probability of exposure to infective bites .
farmers , fishermen , housewives 
 and teachers are examples of occupations that are frequently reported to exhibit high 
 prevalence rates ( batista et al .
1960b , medeiros et al . 2009 , 2011 ) . additionally , the 
 infection rate increased with age and became more evident from 40 years of age onward , 
 with very similar rates being detected between men and women , as reported for other 
 areas ( moraes et al .
1978 , medeiros et al . 2008 ) . since 1960 , when batista et al .
described the most frequent symptoms presented by 
 infected individuals with a high parasitic load , mansonellosis , as a disease , has been a 
 subject of controversy .
briefly , restrepo and ochoa 
 ( 1986 ) carried out clinical trials and observed symptomatology similar to that 
 reported previously in brazil . in argentina ,
jorg 
 ( 1983 ) described a unique case of mansonellosis in an immunocompromised 
 patient .
however , in studies carried out in bolivia , no correlation was found between 
 infections and clinical signs ( bartoloni et al . 

additionally , the suspected involvement of m. ozzardi 
 in ocular impairment was studied in volunteers from a endemic area along the solimes 
 river in am ( cohen et al .
2008 ) and , more 
 recently , an association with corneal lesions was found ( vianna et al .
the clinical analysis indicated a 
 significant relationship between infection and two of the traditional symptoms described 
 in the literature : headache and articular pain . the physiological reasons for these 
 symptoms
have not been well studied , but circulating immune complexes associated with 
 m. ozzardi infections have previously been described and could serve 
 as an explanation for the articular pain observed in infected individuals ( godoy 1998 ) . 
 furthermore , asymptomatic individuals were present in significant numbers , some of whom 
 exhibited high parasitaemia levels ( data not shown ) , which may be a result of a 
 well - balanced host / parasite relationship .
finally , 40% of the serum samples collected previously from subjects in vila antimary 
 and tested at lso / ioc - fiocruz were found to be positive when subjected to elisa against 
 a cocktail of recombinant proteins from o. volvulus ( shelley et al .
2001 ) , which is considered suggestive 
 of exposure to this parasite ( bradley & unnasch 
 1996 ) .
the use of non - invasive methods such as serological tests has 
 increased , but although such methods are more sensitive , extensive cross - reaction 
 between different nematodes has been observed , compromising their specificity ( cabrera et al .
controversial findings and 
 variations in sensitivity and specificity depending on the geographical area and the 
 parasite proteins employed have been reported ( bradley 
 & unnasch 1996 ) . the antigens used in the present work were selected due 
 to showing promising results in other studies and despite one inconclusive positive 
 result from an uninfected volunteer from porto acre , the elisa applied here using the 
 ov10 , ov11 and ov16 recombinant proteins showed high specificity ( 99.7% ) , with no 
 cross - reactivity with m. ozzardi - infected individuals being detectable . 

2001 ) in which cross - reaction with serum samples from 
 m. ozzardi - infected individuals was detected .
however , these 
 previous assays employed ov29 rather than ov16 . furthermore , blood microfilarias similar 
 to those of o. volvulus in the cephalic space , but with a posterior 
 region morphology similar to m. ozzardi have been found in the study 
 area ( adami et al .
it is possible that the 
 presence of these parasites in blood samples could be responsible for the previously 
 observed elisa cross - reactivity .
however , even in infected volunteers with atypical 
 microfilarias , the serological profile has not changed .
atypical microfilarias have also 
 been found in peru and a molecular analysis performed on these samples clustered the 
 atypical microfilarias into the mansonella group , which is 
 indistinguishable from m. ozzardi ( arrspide et al .
other factors , such as the preparation of materials and variations in 
 protocols , may have had some effect on the obtained results , as a different combination 
 of recombinant proteins was employed in our cocktail . in conclusion , epidemiological surveillance should be sustained through border regions 
 of am and ac to prevent the critical dispersion of m. ozzardi 
 infections to non - endemic areas where simuliid species are present and can act as 
 competent vectors .
as long as mansonellosis remains a poorly understood filarial disease 
 and scattered infections continue in human populations situated along the purus river 
 and some of its tributaries , further studies will be needed to increase our 
 understanding of its pathology and to develop safe treatment strategies .
 
 mansonella ozzardi infections are common in the riverside 
 communities along the solimes , negro and purus rivers in the state of amazonas ( am ) . 
 
 however , little is known about the presence of this parasite in communities located 
 in regions bordering am and the state of acre . the prevalence rate of m. 
 ozzardi infections was determined in blood samples from volunteers 
 according to the knott method . 
 a total of 355 volunteers from six riverine 
 communities were enrolled in the study and 65 ( 18.3% ) were found to be infected with 
 m. ozzardi . 
 as expected , most of the infections ( 25% ) occurred in 
 individuals involved in agriculture , cattle rearing and fishing and an age / sex group 
 analysis revealed that the prevalence increased beginning in the 40 - 50-years - of - age 
 group and reached 33% in both sexes in individuals over 50 years of age . 
 based on the 
 described symptomatology , articular pain and headache were found to be significantly 
 higher among infected individuals ( 56 and 65% prevalence , respectively , p < 0.05 ) . 
 
 sera from volunteers were subjected to elisa using a cocktail of recombinant proteins 
 from onchocerca volvulus to evaluate the specificity of the test in 
 an endemic m. ozzardi region . 
 no cross - reactions between m. 
 ozzardi - infected individuals and recombinant o. volvulus 
 proteins were detected , thus providing information on the secure use of this 
 particular cocktail in areas where these parasites are sympatric . 
SUBJECTS, MATERIALS AND METHODS
RESULTS
DISCUSSION