Introduction

Filariasis are tissue helminthiases caused by white filiform worms or nematodes transmitted by arthropods (diptera: mosquitoes, flies and midges) (1). Nine filarial species have been described in humans. Depending on their impact, they are divided into pathogenic filariases (onchocerciasis, loasis, brugiosis, wucheriosis or brancoftosis) and low- or non-pathogenic filarioses (M. perstans, M. rodhaini, M. ozzardi and M. streptocerca mansonellosis) (2). In Gabon, 5 species are encountered: L. loa, M. perstans, O. volvulus, M. streptocerca and M. rodhaini (3). Because of their chronicity, these filariases can become systemic diseases, affecting organs such as the heart, lungs, kidneys and liver, and leading to a dysregulation of the host's immune response (4). With almost a billion people exposed to these parasites and over 200 million affected, they are widespread throughout the world. Because of the disability they cause among African workers, the people living in high-risk areas have been estimated at nearly 14.4 million cfa francs (5). Loa loa filariasis is a parasitosis endemic to the tropical forests of Central and West Africa. It is transmitted by a hematophagous female fly of the genus Chrysops (6). More than 15-20 million people are infected with L. loa (7). In endemic countries such as Cameroon, Central African Republic, Congo, Democratic Republic of Congo, Gabon and Equatorial Guinea, Loa loa filariasis is a real public health problem. After malaria, it is the second most common cause of parasitological consultations. It is also found in endemic areas in southern Nigeria, Chad, Sudan and on its border with Kenya, as well as in Angola (8).

Loa loa filariasis has received little attention and is not included in the list of neglected tropical diseases such as lymphatic filariasis and onchocerciasis (6). It attracts attention mainly because of the embarrassing symptoms it manifests, such as pruritus, migration of adult worms into the eye and so-called Calabar edema (9). It contraindicates the treatment of "river blindness", also known as onchocerciasis, in cases of coinfection due to severe, even fatal, neurological reactions (10). However, in 2016, it was shown that this pathology is more dangerous than it appears. In fact, in infected people over the age of 24 with a parasitaemia greater than or equal to 30,000 mf/ml of blood, it would increase the risk of mortality (7). Also, in cases of hyperparasitemia of Loa loa filariasis, serious adverse effects can occur during treatment with diethylcarbamazine (DEC) and ivermectin, requiring close monitoring of treatment that prevents the mass administration of antifilarial drugs, aimed at controlling other filaria in areas where Loa loa is coendemic (11). Numerous epidemiological studies on Loa loa filariasis, have been conducted throughout the West and Central African forest block. They have indicated Cameroon as the most important focus of the disease, with a prevalence of over 50% in four localities (12). In Gabon, a country covered by an immense tropical forest, despite the existence of a study that revealed a prevalence of Loa loa filariasis of 22.4% in 4392 individuals aged at least 15 throughout the country (3) and a more recent one, which reported a prevalence of 9.27% (95% CI: [0.05. 0.15]) among pregnant women at the Sino-Gabonese Friendship Hospital in Franceville (13), the benign nature attributed to Loa loa filariasis contributes to making this disease a public health problem in the country. It is in this context that this study was undertaken, with the overall aim of determining the prevalence and risk factors associated with Loa loa filariasis in South-eastern Gabon.

Materials and Methods

Presentation of the study and sampling site:

Sampling for the present study was carried out at the Paul MOUKAMBI Regional Hospital in Koula-Moutou (PMRHKM) in the Mikoumou district of Koula-Moutou (Central-eastern Gabon). This healthcare institution is located in the MIKOUMOU district, at a latitude of .1.12724° or 1°7'38'' South and a longitude of 12.47764° or 12°28'40'' in the North-east of the town of Koula-Moutou, capital of the Ogooué-Lolo province. It is located at the entrance to the town from Lastourville, 800 m from the crossroads on the paved road. Inaugurated in 2002 by President OMAR BONGO, the PMRHKM comprises 15 buildings housing several departments, including the laboratory. Koula-Moutou is a semi-urban town characterized by alternating dwellings and equatorial vegetation. Human activities are based on administrative work, hunting, fishing, agriculture, trade and so on. The Koula-Moutou-Lastourville and Koula-Moutou-Pana axes are characterized by the presence of large expanses of equatorial vegetation all along the road, and human activities are based on forestry work, hunting, fishing, agriculture and so on.

Fig 1: Study Site Map

Type, period and population of the study

Conducted from April 03 to July 31, 2023, this prospective, cross-sectional, analytical study involved randomly selected men and women aged at least 15 years, who had come during the study period to be diagnosed for Loa loa filariasis at the biomedical analysis laboratory of the PMRH in Koula-Moutou, and who met the inclusion criteria.

Inclusion and exclusion criteria for study participants:

Only people who

• Were at least 15 years old
• Had consented to participate in the study
• Had agreed to complete the survey questionnaire

People who did not wish to take part in the study, or whose examination results were unusable, were excluded.

Determining sample size

To estimate the prevalence and risk factors associated with Loa loa filariasis in the town of Koula-Moutou and surrounding departments, the study sample size was determined using the single-proportion population formula as used elsewhere, positing the formula:

n = (Z_{α / 2})2 X (P, (1 - P)² / (d)²) (14)

In this, n represents the number of the sample size, Z α /2 is the standard normal deviation (1.96) corresponding to a 95% confidence interval (CI), P is the prevalence of Loa loa filariasis. In the absence of P values obtained elsewhere, or in previous studies in the town of Koula-Moutou and surrounding departments, this P value was taken to be 50%. d is taken to be the precision/marginal error (d = 0.05) or 5%. Initially, the sample size determined for the study was 273. As it has been applied in two studies elsewhere, errors resulting from the probability of non-compliance or abandonment were minimized, the sample size was increased by 10% (15,16). Finally, the sample size used in the present study was 300 participants.

Sample collection

After signing the informed consent, venous blood samples (at the elbow crease) were taken in EDTA tubes between 11 am and 3 pm (due to daytime activity) at the laboratories of the PMRH in Koula-Moutou.

EDTA tube:

This is a purple-capped tube containing an anticoagulant: Ethylene Diamine Tetra Acetic (EDTA) and a calcium chelator (which lowers calcium). Plasma is obtained from the EDTA tube. This is the tube par excellence for CBC, CV, GE, RMF and PCR.

Parasitological diagnosis of Loa loa filariasis

Loa loa filariasis is diagnosed using two techniques: fresh blood and leucoconcentration. Detection of the parasite as an adult or in the microfilarial stage provides a definitive confirmation of infection. Adult filariae are rarely found; they can be found and extracted as they pass under the conjunctiva or skin. A blood sample is used to test for microfilariae.

Direct examination of fresh blood: 10 µL of whole blood is placed between a slide and a coverslip and observed with a light microscope at x10 and x40. Microscopic observation of fresh blood enables the presence of microfilariae to be detected thanks to their rapid movements. Indeed, Loa loa is a long worm with low mobility (diurnal) at the PMRH laboratories in Koula-Moutou.

Concentration techniques: Also known as the leucoconcentration technique, this is performed if the direct examination is negative and is carried out as follows:

Decantation

Whole blood (EDTA) is centrifuged at 2000 rpm for 5 minutes, then the supernatant (plasma) is removed.

Hemolysis 1

Add 2 mL saline (9% NaCl) and 1 mL saponin (2%), then invert the tube successively and leave for 10 minutes. Then centrifuge at 2000 rpm for 10 minutes and discard the supernatant.

Hemolysis 2

Add 2 mL saline (9% NaCl) and 1 mL saponin (2%), then successively invert the tube and centrifuge at 2000 rpm for 5 minutes, discarding the supernatant.

Washing

Add 3mL of saline (9% NaCl) to the pellet, successively invert the tube and centrifuge at 2000 rpm for 10 minutes, then discard the supernatant and drain.

Reading

Place the pellet between slide and coverslip and read under the microscope at x10 and x40.

Other parasitological diagnostics are also performed, such as:

• Thin smear and thick drop tests: These are used to identify microfilariae on the basis of characteristics specific to each species (size, coloring or absence of the sheath, appearance and size of nuclei, etc.).
• Blood microfilaria count: useful before starting treatment with a microfilaricide.

Survey questionnaire

Patients were interviewed to collect data on potential risk factors for Loa loa filariasis using a structured questionnaire, covering socio-demographic information, such as age, marital status, instruction level, professional status, residence (Urban or rural)), residence duration, and housing conditions. In addition, behavioral risk factors and medical history of study participants, such as tattooing, blood transfusion, surgery, clinical signs, were addressed

Quality assurance

Using standardized data collection tools, data quality was ensured by pre-testing questionnaires on 5% of participants, after appropriate training of staff in data collection and management of an integrated quality control system at the Paul MOUKAMBI Regional Hospital in Koula-Moutou (PMRHKM). All laboratory procedures were carried out in accordance with standard operating procedures.

Ethical considerations:

Ethical authorization was obtained from the Director General of the Centre Hospitalier Régional Paul MOUKAMBI de Koula-Moutou, which maintains partnerships with the USTM Faculty of Sciences. An internship agreement was obtained from the Dean of the Faculty of Science to conduct the study in the said structure. Written informed consent was obtained from each participant, who was informed in advance of the right to terminate participation in the present study at any time. Confidentiality of information was maintained by coding and storage in a lockable cabinet. Clinicians were informed of the results for patient management.

Statistical analysis:

Data collected were entered into a Microsoft Excel 2013 spreadsheet, cleaned and then analyzed with R software version 3.6.1. Pearson's chi-square, Odds ratios, and 95% confidence intervals were used to find potential associations between risk factors and Loa loa filariasis. P-values were determined and considered significant when less than or equal to 0.05.

Results

Prevalence of Loa loa filariasis among study participants (N =300).

A total of 300 participants were registered for this study. With a sex ratio (M/F) of 0.60, women were in the majority. With a mean age of 26.7 ± 17.45 years, the majority of study participants, 60 (20%), were aged between 31 and 40 years, and 188 (62.67%) were women. Diagnosis for Loa loa filariasis indicated that 42 participants suffered from this disease, a prevalence of 14% (95% CI: [0.10. 0.18]), compared with 86%, or 258 negative results [Figure 2].

Figure 1 : Prevalence of Loa loa filariasis among study participants (N =300).

Prevalence of Loa loa filariasis according to socio-demographic characteristics of the study population, (N=300).

Statistically significant associations between dependent and independent variables were found in univariate analyses of the prevalence of Loa loa filariasis according to sociodemographic characteristics. Among Loa loa filariasis-positive patients in the study, those aged between 61 and 70 years (OR = 2.66, 95% CI : [1.09 ; 6.48] p =0.003*) , female (OR = 2.58, 95% CI : [1.33 ; 5.01] p =0.042* ), retired (e) (OR = 3.35, 95% CI : [1.08; 10.35] p =0.03*), living in rural areas (OR = 6.39, 95% CI: [3.06; 13.35] p <0.0001), living in average conditions (OR = 5.15, 95% CI: [2.54; 10.43] p <0.001), were statistically associated with Loa loa filariasis Further study using multivariate logistic regression of the variables indicated that women (adjusted OR= 56.9, 95% CI: [1.71; 1893.6] p =0.024*) were fifty-seven times more likely to suffer from Loa loa filariasis (Table 1).

History and clinical and paraclinical aspects associated with the prevalence of Loa loa filariasis in study participants. (N = 300)

To test the association between Loa loa filariasis exposure and the medical history and clinical signs of the study participants, crude and multivariate logistic regression analyses of the variables were carried out. It was found that only study participants with clinical signs such as pruritus, dermatitis or Calabar edema (OR = 0.16, 95% CI: [0.08; 0.32] p <0.0001) were at greater risk of developing Loa loa filariasis than other participants. Multivariate logistic regression, on the other hand, indicated no statistically significant association between participants' clinical signs or medical history and Loa loa filariasis Table 2.

Discussion

Highly endemic in Central African countries such as Gabon, and the cause of a high disease burden in these countries, Loa loa filariasis is a serious human parasitosis. In order to design, plan and evaluate appropriate intervention strategies against this disease, knowledge of its epidemiology, transmission, distribution and extent, as well as the associated risk factors, is required (17). With the main objective of determining the prevalence and risk factors associated with Loa loa filariasis: case of Koula-Moutou and surrounding departments, South-central Gabon, The present study reported a prevalence of Loa loa filariasis of 14% (95% CI: [0.10- 0.18]), This result is similar to that found in a previous study (18). However, it is higher than that obtained by a study elsewhere, which found an average prevalence of L. loa in the village of 6.3% (19), and lower than that of another study, conducted in Cameroon, which found an overall prevalence of loasis of 27.3% (20) and that of another study conducted in another region of Gabon, a prevalence of Loa loa microfilaremia of 22.4% was reported in one study (3). The variability of these results could be explained, on the one hand, by the number of participants recorded in each study and the diagnostic methods used. On the other hand, knowing that there is a periodicity of Loa loa microfilaremia (21), the difference in periods, regions and years of the studies may influence these results.

Contrary to studies that reported that the 18-28 age group was statistically more associated with filariasis than other age groups (22), or that the maximum load of Loa loa microfilariae, was found in individuals aged 35-49 years (20), univariate analyses of the prevalence of Loa loa filariasis according to sociodemographic characteristics, history, medical, and clinical signs of study participants, indicated that being aged between 61 and 70 years, was significantly associated with Loa loa filariasis. This finding is in line with a study that indicated that the prevalence of Loa loa microfilaremia was highest (14.3%) in people aged between 65 and 84 years (18). This may reflect this age group's greater exposure to chrysop bites than younger groups. Most elderly people are also exposed to a number of factors which may contribute to their predisposition to infections, such as impaired immune function (23).

Contrary to the results obtained in previous studies, which reported that men were likely to have a higher prevalence of Loa loa filiarisis than women (24), a statement also supported by epidemiological data from central Cameroon, where the prevalence of Loa loa filariasis was significantly higher in men than in women (25), in the present study, he reported that factors such as female gender, widow, retired, living in rural areas, housed in average conditions, were significantly associated with Loa loa filariasis. This result, confirmed by multivariate logistic regression analysis, is in line with a study carried out elsewhere, in which a female was more likely to report a history of eyeworm than a male subject (26). This can be justified by the fact that, in the African context, even a widow, the woman is the pillar of the family, and the domestic chores performed by the latter constitute so many challenges to their health that weigh on their perception of it. They may also deteriorate their functional health, due to the physical efforts required (27). The women in the present study, residing in rural areas and housed in average conditions, were significantly associated with Loa loa filariasis. Now retired, their main activities were farming and fishing, which are carried out in forests. This result, although contrary to that of a study on the subconjunctival and intraocular presence of adult Loa loa in populations living in urban centers (28), is however, in line with a study that indicated that Loa loa and other filariases are established diseases observed in villages and rural communities in endemic areas (12). In addition, most homes are surrounded by vegetation or forest, potential reserve for Chrysops, the loase vector (22). In the present study, it was reported that only study participants with clinical signs such as pruritus, dermatitis or Calabar edema were more likely to develop Loa loa filariasis than other participants. This result is in line with that of a study conducted in Burkina Faso, in which transient angioedema (Calabar edema) and pruritus were observed in a patient diagnosed with Loa loa filariasis (29). Multivariate logistic regression showed no statistically significant association between clinical signs or medical history of participants and Loa loa filariasis.

Study limitations

Despite the contributions made, the present study has certain limitations that should be highlighted for future studies. Within the conceptual framework of this study, a number of factors were identified upstream of the living context of the Koula-Moutou populations and the socio-demographic characteristics likely to influence Loa loa filariasis. Firstly, clinical signs based on a questionnaire are highly subjective. Blood smear staining and concentration techniques used in this study are limited. Detection of Loa loa filariasis in peripheral blood is insensitive, as only 30% of individuals are microfilaremic, while 70% are amicrofilaremic with a variety of clinical signs (30). In the presence of an apparently healthy individual with occult infections, the negative serological screening test could be biased. The application of nucleic acid-based detection techniques, such as polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), loop-mediated isothermal amplification and lateral flow assays (LFA), or the detection of biomarkers, such as immunoglobulin 4 (IgG4), antibodies directed against Loa loa antigens, would have been appropriate to prevent infection in the participants of the present study.

Conclusion and Outlook

By providing key information, the results of the present study show very active transmission of Loa loa filariasis in Koula-Moutou and surrounding departments, as suggested by the prevalence rate. The latter was clearly associated with socio-demographic variables of the study participants, such as being aged between 61 and 70, female, retired, living in rural areas, living in average conditions and presenting clinical signs such as pruritus, dermatitis or Calabar swelling. These results could guide Gabonese health authorities in the control and prevention of Loa loa filariasis. To implement appropriate intervention strategies against Loa loa filariasis, Gabonese health authorities should introduce molecular (qPCR) and immunological diagnostic methods, such as the use of biomarkers, which could help identify the true profile of Loa loa infection, beyond pathognomonic signs such as the presence of microfilariae or attested ocular passage, specific but not sensitive enough to detect all clinical cases; in this case, biomarkers may be useful in areas where this condition is endemic.

Acknowledgments

We would like to thank all the participants of this study, the Regional Health Department of the Centre-East, in Koula-Moutou. We also thank, the management of the Paul Moukambi regional hospital in Koula- Moutou, and the staff of the medical analysis laboratory, for their availability.

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