Entomological Assessment of the Impact of Long-term Mass Administration of Ivermectin in Onchocerciasis – Endemic Communities in Kaduna State, Nigeria.

Background: Onchocerciasis constitutes a major public health burden in endemic communities and areas because of its dermal and ocular manifestations. Nigeria accounts for the highest burden of the disease globally. Long term mass distribution of ivermectin has been recommended by the World Health Organization as a potent strategy in halting the transmission of the disease in endemic communities. Aim: The aim of this study was to evaluate the prevalence of infective blackies after 17 years of community-based treatment with ivermectin in two onchocerciasis foci in Kaduna State, Nigeria. Method: Blood seeking female ies were collected using human landing capture from two breeding sites (River Galma and Gurara) in Lere and Kagarko LGAs of Kaduna State in 2010 and 2011. The y heads and bodies, in pools of 100, were analysed using Polymerase Chain Reaction (PCR)-ELISA specic for O. volvulus DNA. Results: A total of 24,937 blackies were caught during two-year catching period. The prevalence of infective ies reduced from 0.15% to 0% at River Galma and 0.08% to 0.04% at river Gurara in 2010 and 2011 catching period. Some pools containing the bodies of the ies were also positive for Onchocerca volvulus. Conclusion: The presence of O. volvulus in the heads and bodies of the black ies collected conrmed that there was ongoing transmission of onchocerciasis in Kaduna State, Nigeria during the period of survey. There is continued need for mass distribution of ivermectin in Kaduna in order to halt the transmission.

Results: A total of 24,937 black ies were caught during two-year catching period. The prevalence of infective ies reduced from 0.15% to 0% at River Galma and 0.08% to 0.04% at river Gurara in 2010 and 2011 catching period. Some pools containing the bodies of the ies were also positive for Onchocerca volvulus.
Conclusion: The presence of O. volvulus in the heads and bodies of the black ies collected con rmed that there was ongoing transmission of onchocerciasis in Kaduna State, Nigeria during the period of survey. There is continued need for mass distribution of ivermectin in Kaduna in order to halt the transmission.

Background
Onchocerciasis is a vector-borne parasitic disease caused by the larial nematode Onchocerca volvulus and transmitted by Simulium species. According to WHO, an estimated 217.5 million people live in areas at risk of onchocerciasis, the vast majority in sub-Saharan Africa. This estimate does not include low transmission areas that still need to be mapped [1]. The main complications of this infection are severe eye disease that lead to blindness and skin disease characterized by papular or hypo-pigmented lesions and intense itching. It is the micro lariae (mf) which drive this pathology.
The main strategy for combatting onchocerciasis is mass ivermectin (Mectizan®) distribution. Following the discovery of ivermectin and its subsequent donation by Merck and Co, African control programmes adopted mass ivermectin treatment as their main control strategy, using community-directed treatment (CDTi) to distribute [2]. Ivermectin (Mectizan®) is a potent micro laricide that has limited effect on the viability and reproductive capabilities of adult onchocercal worms. Thus, repeated treatment is needed in order to suppress the manifestations of the infection over time and limit transmission. In 2009 the rst evidence of elimination of the parasite from African foci using ivermectin alone emerged from Mali and Senegal [3]. This led to a wider evidence gathering effort by the African Programme for Onchocerciasis Control (APOC), and as evidence supporting the feasibility of onchocerciasis elimination in Africa using ivermectin alone mounted, countries switched the focus of their programmes from control to elimination.
In 2020, evidence of onchocerciasis elimination through annual mass drug administration was documented in Plateau and Nasarawa states in Nigeria [4].
In Nigeria, Onchocerciasis is endemic in 31 of the 36 States of the country including the Federal Capital Territory of Abuja. Repeated annual treatment with ivermectin commenced in 1990 and scaled up by 1997 in most of the endemic communities after undertaking rapid assessment of communities to be treated using Rapid Epidemiological Mapping of Onchocerciasis (REMO) [5]. The study was carried out in two onchocerciasis endemic foci, Kudaru (Lere LGA) and Gantang (Kagarko LGA) both located in Kaduna State "Additional le 1: Figure S1".

Ethical Clearance
Ethical approval was sought and obtained from the Kaduna State Ministry of Health before the commencement of the study (Ref No. MOH/ADM/122/VOLII/560). Verbal informed consent was obtained from the local government authorities, community leaders and y collectors. The y collectors were treated with ivermectin before and after completion of the study.

Collection of Black ies
The entomological evaluation was carried out over two wet seasons in 2010 and 2011 coinciding with the period of high density of ies and onchocerciasis transmission in Nigeria. Blood seeking female black ies were collected using the human landing method [7,8] from two main breeding sites in each location. Four y collectors worked simultaneously between 07.00hr and 18:00hr for three consecutive days weekly between June and October in 2010 and 2011. The samples were preserved in 30ml bottles containing 100% isopropanol and stored at 4 o C until use.

PCR-ELISA Pool Screening Assay
The heads and bodies were pooled (100 heads/bodies per pool) and subjected to DNA extraction and PCR-ELISA Pool Screening Assay using protocol earlier described by Unnasch [9]. The DNA extraction and PCR ampli cation targeting O-150 repeats were carried out as previously described by [9,10]. The ELISAbased detection of the ampli ed PCR products was carried out with a Spectra Max Microplate Reader Model Vmax (EMax Endpoint ELISA Microplate Reader). A pool on a particular plate is positive when the optical density of the well is greater than the mean plus 3 standard deviation of the 10 negative controls in the plate as read from micro plate reader [9].

Statistical Analysis
The results obtained from PCR -ELISA Pool screening Assay were subjected to statistical analysis to calculate the infective, infection and con dential intervals using PoolScreen software version 2.0 [11,12,13].

Result
The Biting rates of Simulium collected in Kudaru and Gurara foci A total of 24,973 Simulium damnosum adult ies were collected in two wet seasons of year 2010 and 2011. More ies were collected in 2011 (4, 691 in Kudaru; 10,600 in Gurara) as compared with 2010 (2,093 in Kudaru; 7,553 in Gurara). There is a no signi cant difference in the number of ies collected in both sites across the years (P > 0.05). However, the y population varied signi cantly (P > .05) with more ies collected around River Gurara (18,153) as compared with River Galma (6,784). The average hourly catch per site ranged from 0.87 to 4.42 "Additional le 2: Table S1".
The results of the monthly biting Monthly Biting Rate (MBR) are presented in "Additional le 2: Table S2 Entomological assessment has been adjudged as the most critical tool in estimating the real-time assessment of transmission in the endemic communities [14,15,16]. The positive pools of the bodies of the ies con rm that the ies have access to infected individuals in the community possibly with moderate to high micro larial load. The detection of L3 larvae in the head pools using O. volvulus speci c PCR-ELISA also negates the argument that the parasite found in the ies may be animal parasite (O. ochengi).
Entomological studies on cytotaxonomic identi cation of members of Simulium damnosum complex present in the study area had earlier con rmed localized breeding of the black ies in the area [17]. The high population density and biting rates recorded in the present results also corroborate the earlier report.
It is most unlikely that the ies collected during the study were migratory. Therefore, the transmission in the area is plausibly localized not migratory/importation. While the factors accounting for the discrepancies in the results of epidemiological and entomological surveys remained unclear and could not be established in the present study, the observation in the discrepancies justi es the need for both entomological and epidemiological surveys to be conducted simultaneously, most importantly in the current context of elimination. This will provide insightful and comprehensive data to guide the researchers and policy makers on decision on stop MDA.
Even though, the results of the Pool screening PCR Assay indicated drastic reduction in infective rates in both foci (to zero in Galma) in 2011 as compared with 2010, the fact the positive pools of body segment of the ies indicate that active uptake of micro laria by the ies from human population is ongoing in the study area. The results are consistent with results from other areas where MDA had been conducted for a similar period of time indicating reduction but not interruption in Cameroon [18,19].
The signi cant variation in population and biting densities of black ies in River Galma and River Gurara are expected. The proli c breeding of black ies in Africa has been associated with presence of rocks, vegetation, speed of the water and other physico-chemical parameters of the river [20.14,21]. The presence of the factors are more feasible at River Gurara and thus accounting for high productivity of the breeding site all year round. The number of ies caught along River Galma each year were below the recommended 6,000 per transmission zone, recommended by WHO for stop MDA surveys [4]. This nding is important for planning future monitoring and evaluation or onchocerciasis MDA in Kaduna; additional catching hours, sites, or time would be required to meet the 6,000 y threshold.