Investigation of the occurrence of Mansonella ozzardi in the State of Rondônia , Western Amazonia , Brazil

Introduction: Mansonella ozzardi, a human filarial nematode transmitted by Diptera Simuliidae flies (black flies), is widely spread in the Amazon, while the occurrence of mansonelliasis in the State of Rondônia (Western Amazonia) has not been reported thus far. The existence of this parasitic disease in this state is investigated in this study. Methods: A total of 4,452 people who lived alongside the Madeira, Mamore, Guapore, Machado and Preto Rivers, in the State of Rondônia, were examined using the thick blood smear method. Alongside the Machado and Guapore rivers, 2,112 adult simuliid specimens were captured. Results: Infected people were not reported, although simuliids with vector potential such as Cerqueirellum pydanielli, C. argentiscutum and C. amazonicum were found. Conclusions: This study registered no infected people. This finding may be explained by the individuals’ low level of microfilaremia that is below the limit of detection of the diagnostic method used in the study or insufficient migration of people infected with M. ozzardi from endemic areas, making it impossible to introduce the disease into Rondônia. Further studies using more sensitive diagnostic techniques could shed light on this question.

Mansonella ozzardi microfilariae are found in the peripheral blood of human hosts and are one of the etiologic agents of mansonelliasis.In some countries, this parasite appears simultaneously with other filarial parasites, such as Onchocerca volvulus and/or Mansonella perstans 1,2 .In Brazil, this parasite was first observed in a nationwide survey of the occurrence of lymphatic filariasis in the City of Manaus in the Amazon region in 1949 3 .Three and a half percent (3.5%) of the population was examined by the thick blood smear method with positive results found in 0.6% (15 patients) for M. ozzardi.Among these patients, seven had never left the city of Manaus, which showed that M. ozzardi was autochthonous 3 .Later, some researchers observed individuals infected with M. ozzardi alongside the Solimoes, Purus and Negro Rivers in the State of Amazonas [4][5][6] .Recent data have indicated that this parasite is still found with high prevalence rates in the Amazonian territory [7][8][9] .
Outside of the State of Amazonas, this parasite has already been found in the State of Mato Grosso and Roraima 10,11 .In Brazil, only insects of the Simuliidae family transmit M. ozzardi: Cerqueirellum amazonicum, C. argentiscutum and C. oyapockense [12][13][14] and more recently C. pydanielli are potential vectors for mansonelliasis in the State of Amazonas 15 .
A survey on filariasis was conducted in the State of Rondônia in the 1950s as part of the national survey, and M ozzardi hosts were not found.Only lymphatic filariasis caused by Wuchereria bancrofti was detected in this State 16 .Since then, no other national survey has been performed in the region.However, a few local studies of M. ozzardi have been performed on the population in the Amazon region.No studies been have been conducted in the State of Rondônia despite the fact that some cases of mansonelliasis have been reported in the state, mainly in individuals that migrate from the State of Amazonas, especially from the municipalities of
The sampling of the population was non-random, and individuals older than five years of age (population at risk) who are exposed daily to simuliids stings were invited to voluntarily participate in the study.These patients were not only informed orally but also informed in written form of the low risks and procedures of the study.The hematologic survey was conducted using the thick blood smear method.Two to three blood drops (60μL) were obtained by finger prick and were then placed on a slide for microscopy, dried at room temperature, and washed with distilled water (12h after being prepared).The slides were then stained with Giemsa and examined with an optical microscope (10x and 40x magnification) in a blind, controlled trial performed by two microscopists.
In addition to the hematologic study, adult and immature (larvae and pupas) simuliids were collected along the Machado River in the communities of Tabajara on the middle of Machado River (8º 55' 29,68" S and 62º 04' 48,15" W) and Dois de Novembro (8º 54' 54.90" S and 62º 07' 21.11" W) and Guaporé River at São Francisco do Guaporé (12º 31' 43.53" S and 63º 32' 10.89" W).The adult simuliids were captured by suction traps upon landing in the morning (8 to 10 am), and in the afternoon (2 to 4 pm).The immature simuliids (larvae and pupas) were captured on the leaves and branches that had fallen into the rivers and water channels of the region using tweezers and scissors.All the collected simuliids (2,112 specimens) were soaked in 70% alcohol and taken to the laboratory of the National Institute of Research of Amazonia in the City of Manaus (State of Amazonas) for identification, according to Coscarón & Coscarón-Arias 18 .Larvae and pupas were identified for species, while 90% (ninety percent) of the adult simuliids were dissected into three parts (head, thorax and abdomen) using a stylet under a light microscope to determine infection with M. ozzardi microfilariae and/or larval stages (L 1 , L 2 and/or L 3 ).

Ethical considerations
The project was submitted to the Ethical Research Committee of the São Lucas College in Porto Velho (State of Rondônia), approved and recorded under the registry number 344/09.Altogether 4,452 individuals were examined alongside the major rivers of Rondônia, and all of these individuals were found to be negative for M. ozzardi microfilariae by hematologic examination (Table 1).

DISCUSSION
Only two simuliid species were found to have hematophagic activity, and Cerqueirellum pydanieli was more abundant in comparison to Chirostilbia pertinax (Table 2).
Ninety percent of the adult specimens were dissected, and none of them was found to be positive for M. ozzardi.The literature indicates that the Amazonian region is the only region in which M. ozzardi has been found in Brazil, and this region includes the States of Amazonas, Roraima, and Mato Grosso 10,11 .Rondônia is in the Amazonian region and was the subject of a study on the occurrence of mansonelliasis in the 1950s; however, there was no evidence of this parasitic disease 3,4 .In the present study, a large number of individuals were examined in several areas in the State of Rondônia and all of them tested negative for M. ozzardi microfilariae, confirming the results found in the previous decades.There is a question of whether the diagnosis method used in this study was not sensitive enough to detect low rates of microfilaremia.However, many studies carried out in the Amazon use the same method and show prevalence of up to 60% of microfilaremia [7][8][9] .Another hypothesis may be that there are not enough individuals infected with a high enough parasite load to disseminate this endemic disease in areas not affected by the infection.People with microfilaremia are a source of infection for insects that are vectors for the parasite and are responsible for perpetuating this endemic disease.Studies have shown that, in places with favorable conditions for other filarial diseases caused by W. bancrofti, the migration of individuals suffering from microfilaremia may be responsible for establishing new parasitic vector foci.In Sri Lanka, infected migrants introduced lymphatic filariasis into areas in which there were no previous parasitic diseases 19 .A similar situation was noted in areas previously free of filariasis such as the metropolitan area of the City of Recife (Brazil) that has since had cases of autochthonous lymphatic filariasis 20 .
In this work, no focus of mansonelliasis was found despite the presence of potential parasite vectors and environmental conditions similar to those existing in mansonelliasis endemic areas in the Amazon region that borders Rondônia to the north.Among the factors that influence the capability of the vector to be infected and transmit the infection is the level of microfilaremia of the vertebrate host.The infection rate of mosquitoes is proportional to the density

FIGURE 1 -
FIGURE 1 -Collection area of Simuliidae and blood samples.

TABLE 1 -Hematologic survey employing the thick blood smear method assessing Mansonella ozzardi microfilariae in individuals older than 5 years of age, State of Rondônia, 2008 to 2010. Total of the population older than 5 years of age who were submitted to the survey in relation to the local
*All negative for Mansonella ozzardi.