ABSTRACT

Human filariasis is a group of tropical disease caused by eight species of nematodes which are located in blood, lymphatic tissue and other tissues. While in some area only one of the filaria species may be found, sometimes you may have two or three of them coexisting in some bioecological zone. Community directed treatment with Ivermectin has been carried out (through a strategy known as mass drug administration) in the Melong health district for 16 years. Questions are being raised concerning the feasibility of filariasis elimination using this strategy alone. As a result this study was aimed at assessing endemicity levels of filariasis despite these long term application of the strategy and which diagnostic tool can best be used to determine the actual prevalence of filariasis during and after such a program. We also aim to to establish the relation between ivermectin adherence and parasitological indices of onchocerciasis. In this study, a total of 1748 participants were recruited from whom skin snips and capillary blood was collected.DNA was then extracted from randomly selected 210 skin snip sample and Quantified using the Qubit^TM4 flouorometer. The DNA samples were then used to perform O-150 colorimetric LAMP assay and the OvwFtsZ/OvActin Duplex Real-time PCR. Amongst the 1748 participants from whom skin snips were collected, 85 (4.9%) were
found to be microfilaridermic for Onchocerca volvulus with 14.7% nodule prevalence, (2.9%) were microfilaremic for Loa loa   and 25 (1.4%) were microfilaraemic for Mansonella perstans microfilaria. Melong health district as a whole is still endemic for filariasis though with low level of endemicity <30% described as Hypoendemic. LAMP (sensitivity 84.4%) is more sensitive than qPCR (sensitivity 69.7%) whereas qPCR (specificity 96.6) is more specific than LAMP (specificity 93.8%). In this study those who have taken Ivermectin >15 were amicrofilaridemic for onchocerciasis.

Key words: Endemicity, ivermectin, Assessment, Filariasis Microfilaridemia, Microfilaraemia, Microscopy, Real-time PCR, LAMP,  Adherence.

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.1  Introduction

Human filariasis is a group of tropical disease caused by eight species of nematodes which are located in the skin, blood, lymphatic tissue and other tissues. They are transmitted by distinct species of insects (vectors) in the tropical and sub-tropical regions of the world. The distribution of filariasis is closely associated to the distribution of their vector. These vectors can be black flies, mosquitoes, tabanids or midges each of which have specific requirements for growth and developement. While in some area only one of the filaria species may be found, sometimes you may have four or five of them coexisting in the same bioecological zone (Languillon, 1957; Ripert et al., 1977).

Most filariae species are well known medically due to the striking pathological signs they induce in human. For instance, lymphatic filariasis due to Wuchereria bancrofti, Brugia malayi, and Brugia timori clinically manifest as enlargement of lymphatic vessels, lymphedema and Elephantiasis. Onchocerciasis due to Onchocerca volvulus has the most outstanding clinical pathologies due to blindness and severe dermatitis. Loiasis is caused by the filarial nematode Loa loa (Esum et al., 2001) and its stiking clinical manifestation are calabar swellings and sub conjunctival migration of adult worms (Pion et al., 2006; Boussinesq, 2006). Mansonellosis caused principally Mansonella perstans have statistically robust pathologies such as joint pains, enlarged lymph glandsand various abdominal Symptomes which are caused by countless pathogens that occur in Mansonellosis endemic region (Medeiros et al., 2008; Viana et al., 2012). Contrary to onchocerciasis and lymphatic filariasis, loasis has not yet been prioritized in the Wold Health Organisasion’s (WHO) list of Neglected Tropical diseases (NTDs) (Whittaker et al., 2018).

However, loiasis has recently emerged as a disease of public health importance because of its impact on the control of onchocerciasis in areas of Onchocerciasis/loiasis co-endemicity. Loa loa /onchocerca volvulus coinfection have been reported in Cameroon (Gardon et al., 1997; Esum et al., 2001 and Pion et al., 2006). Loa loa hypermicrofilaraemias are a significant risk factor for the development of severe adverse events (SAEs) in individuals treated with the microfilaricidal drug ivermectin during Mass drug administration (MDA) for filariasis elimination campaigns in Sub-Saharan Africa (Ducors et al., 1995; Chippaux et al., 1996; Gardon et al., 1997a and Boussinesq et al., 1998). The incidence of serious reactions is related to the intensity of pretreatment Loa loa microfilaraemia (Gardon et al., 1997). The relative risk of developing marked or serious reactions is significantly higher when Loa loa load exceeded 8000 microfilariae/mL (Gardon et al., 1997). Recent modeling predict that SAEs will occure is 10% of individuals with microfilaraemia loads above 50 000 microfilariae/Ml (Gardon et al., 1997 and Chesnais et al., 2020).

Other co-infections associated with filaria nematodes are similarly well known: L. loa and M. perstans dual infections have been recorded in Cameroon, (Wanji et al., 2003) Congo (Noireau et al., 1989), Gabon (Akue et al., 2011; Bouyou-Akotet al.,2014) and Nigeria (Agbolade et al., 2001) and Concomitant infections with three (or more) human filarial nematodes have also been reported (Wanji et al., 2003 and Uttah et al., 2011).

Detection and identification of microfilaria (MF) by microscopy is based on the morphological characteristics (Taylor et al., 1989; Thiele et al., 2016) of filarial parasites in skin snips is the gold standard for the diagnosis of filariasis in humans (Duke et al., 1962; Kale et al., 1974). However, microscopy may not be able to detect infection in low microfilaridemia (Taylor et al., 1989). The low sensitivity of microscopy may be significant in individuals treated with microfilaricides which further lower micrifilaria (mf) intensity (Taylor et al., 1989; Thiele et al., 2016). Molecular tools can augment sensitivity and provide a diagnostic confirmation of skin snips and will be useful in control and elimination programs (Thiele et al., 2016). Therfore we use two nucleic acid based assay targeting different genes of Onchocerca volvulus (OvwFtsZ/Ovactin-Duplex real-time PCR and 0-150 LAMP) to give a more exact prevalence of filariasis in the Melong health district.

1.2  LITERATURE REVIEW

1.2.1 ONCHOCERCIASIS

Onchocerciasis is a filarial disease caused by infection with Onchocerca volvulus. The vector-borne parasitic nematode transmitted through the bite of an infected blackfly of the genus simulium. Modelling of data from the 2013 Global Burden of Disease Study suggests that at least 17 million people are currently infected with O. volvulus (Herricks et al., 2017). An estimated 198 million people live in areas where there is high potential for transmission of the parasite, although this number may increase as the mapping of areas of low transmission is finalized (WHO, 2016-2017). The disease is also known as river blindness because blackflies breed in and bite near fast-flowing highly oxygenated rivers and streams, also because the most devastating consequence is irreversible loss of vision.  High infection load are measured by the density of microfilariae (the larval progeny of adult worms) in the skin and it’s associated both with blindness incidence (Little et al., 2004) and excess human mortality which is due to blindness (Little et al., 2004; Walker et al., 2012).

Presently, the mainstay of onchocerciasis control is by annual mass drug administration (MDA) of the microfilaricidal drug ivermectin (Mectizan®), donated by Merck & Co.Inc. This drug is given endemic countries through the Mectizan Donation Program (Colatrella, 2008) for as long as needed. Ivermectin MDA in SSA started in the late 1980’s, and great strides in reducing morbidity have been made (Coffeng et al., 2014).  Questions are raised  concerning  the  feasibility  of  eradicability  based  on  annual ivermectin  MDA  alone(Dadzie et al.,2003; Borsboom et al.,2003;Winnen et al., 2002) , studies  in  some  foci  of  Mali  and  Senegal  (in  the  western  extension of the Onchocerciasis Control Programme in West Africa, OCP), indicated  that  local  elimination  may  be  achieved  after  15–17 years of ivermectin MDA (Diawara et al.,2009; Traore et al.,2012). Based on this, in 2010, the African Programme for Onchocerciasis Control (APOC) shifted its objdectives from elimination of the disease as a public health problem (EPHP), to elimination of transmission (EOT) (APOC, 2010).  This shift necessitates a drastic geographical extension of treatment. While EPHP focused on mesoendemic and hyperendemic areasfor ivermectin MDA, EOT at a Pan African scale requires that treatment be distributed alsoin hypoendemic areas.  EOT had challenges, particularlywhere loiasis (a filariasis caused by Loa loa) is co-endemic with onchocerciasis (espercial individuals heavily infected swith Loa loa) dueto severe adverse events (SAEs), including fatalities, that may resultfrom microfilaricidal treatment (Gardon et al., 1997).  In onchocerciasis, the original definition of endemicity levelscomprises three categories:  i) hypoendemic (microfilarial prevalence <30%), ii) mesoendemic (prevalence between 30–60%), and iii) hyperendemic (≥60%) (Prost et al., 1979); EOT should be achieved in all three.  A microfilarial prevalence ≥80% has also been used to indicate holoendemic (O’Hanlon et al., 2016). APOC had as objective to eliminate onchocerciasis as a public health problem (EPHP) was not quantitatively defined. The EPHP goal was defined as when prevalence is driven below the original baseline threshold required to launch the mass ivermectin treatment program, these threshold is an onchocercal nodule rate ≥ 20% or an mf rate ≥ 40% (Katabarwa et al., 2008)

1.2.1.1 Global Distribution of Onchocerciasis.

Geographical distribution 2017, Onchocerciasis occurs mainly in tropical areas. More than 99% of infected people live in countries in sub-Sharan Africa: Angola, Benin, Burkina Faso, Burundi, Cameroon, Central African Republic, Chad, Republic of Congo, Equatorial Guinea, Ethiopia, Gabon, Ghana, Guinea, Guinea Bissau, Kenya, Liberia, Mali, Mozambique, Niger, Nigeria, Rwanda, Senegal, Sierra Leone, South Sudan, Sudan, Togo, Uganda, United Republic of Tanzania as shown in figure 16.

1.2.1.2 Scientific Classification of Onchocerca volvulus

Domain – Eukarya

Kingdom – Animalia

Phylum-Nematoda

Class – Secernentea

Order – Spirurida

Superfamily – Filarioidea

Family – Onchocercidae/Filaridae

Genus – Onchocerca

Species – Onchocerca volvulus (Myers, 2014)

1.2.2 The life cycle of onchocerca volvulus and transmission of onchocerciasis.

Onchocerca volvulus is a parasite transmitted by blackflies of the genus Simulium. The flies breed in fast-flowing, highly oxygenated rivers and streams (Blacklock, 1926). An infected blackfly deposits one or more O. Volvulus infective larvae into the human host when it takes a blood meal. These larvae develop into mature adult worms in about a year. They commonly aggregate into fibrous nodules that lie under the skin usually over bony prominences. The adult female has a mean life span of 12–15 years with a reproductive life span of about 9 to 11 years (Habbema et al., 1992). When fertilized by a male worm, the female releases millions of embryos called microfilariae (mfs), which themselves live for about two years. The mfs that are released by the adult worm migrate from the nodules to swarm in the dermal layers throughout the body where they can be taken up by blackflies during a blood meal. The mfs develop into infective larvae in the Black fly from which they can be deposited in another human being with a subsequent bite, thus completing the life cycle of the parasite and the transmission for the infection as illustrated in Figure 1. Two different types of O. volvulus are found in West Africa; the savanna and the forest types, each associated with the different species of the Simulium damnosum complex (Crosskey, 1990).