Ongoing Transmission of Onchocerca volvulus after 25 Years of Annual Ivermectin Mass Treatments in the Vina du Nord River Valley, in North Cameroon

Background Recent reports of transmission interruption of Onchocerca volvulus, the causing agent of river blindness, in former endemic foci in the Americas, and more recently in West and East Africa, raise the question whether elimination of this debilitating disease is underway after long-term treatment of the population at risk with ivermectin. The situation in Central Africa has not yet been clearly assessed. Methods and findings Entomologic data from two former endemic river basins in North Cameroon were generated over a period of 43 and 48 months to follow-up transmission levels in areas under prolonged ivermectin control. Moreover, epidemiologic parameters of animal-borne Onchocerca spp. transmitted by the same local black fly vectors of the Simulium damnosum complex were recorded and their impact on O. volvulus transmission success evaluated. With mitochondrial DNA markers we unambiguously confirmed the presence of infective O. volvulus larvae in vectors from the Sudan savannah region (mean Annual Transmission Potential 2009–2012: 98, range 47–221), but not from the Adamawa highland region. Transmission rates of O. ochengi, a parasite of Zebu cattle, were high in both foci. Conclusions/significance The high cattle livestock density in conjunction with the high transmission rates of the bovine filaria O. ochengi prevents the transmission of O. volvulus on the Adamawa plateau, whereas transmission in a former hyperendemic focus was markedly reduced, but not completely interrupted after 25 years of ivermectin control. This study may be helpful to gauge the impact of the presence of animal-filariae for O. volvulus transmission in terms of the growing human and livestock populations in sub-Saharan countries.


Introduction
The interruption of transmission of Onchocerca volvulus, the causing agent of river blindness or onchocerciasis, has been confirmed for a growing number of endemic foci on the American continent [1,2,3] and in West [4] and East Africa [5,6,7]. The recent success in onchocerciasis control can be mainly attributed to the extensive and sustained mass treatment programs with the microfilaricide ivermectin, governed by institutions of the World Health Organization, like the African Programme for Onchocerciasis Control [8]. Long treatment rounds are necessary because the drug is only lethal to the larval stage and not the adult worm.
There are thus good prospects that elimination of onchocerciasis is well underway in the Americas [9] and may also have begun at different foci on the African continent [6,10,11]. However, currently there is a paucity of information on the actual situation in Central and Southern Africa, in particular with respect to vector transmission, albeit a significant proportion of these regions have been hyperendemic. Recent studies on the effects of ivermectin treatment on the epidemiology of O. volvulus in humans and the black fly vector Simulium damnosum sensu lato have been done in North and West Cameroon [12,13,14,15,16]. The caveat of the most recent studies is that the filarial species in the vector were not always correctly identified, and the prevalence of infective O. volvulus larvae and thus the transmission potential remains unknown. Local S. damnosum s.l. populations are vectors of at least two other species from the Onchocerca genus: O. ochengi, a common parasite of Zebu cattle Bos indicus [17] and O. ramachandrini, a filaria from the warthog Phacochoerus africanus [18]. The proportion of animal-filariae in the vector has direct and indirect consequences for parasite transmission to humans [19,20,21] rendering it an important factor to understand the epidemiology of river blindness. Furthermore, filariae closely-related to O. volvulus might repopulate the human host [22] posing a potential risk of infection, or they might transfer genes to O. volvulus which negatively affect the effectiveness of ivermectin, presently the sole drug intervention in use [23]. For this reason we combine microscopic differentiation of infective larvae with a PCR-based molecular approach which allows the detection of yet unknown filarial species and strains in addition to already known Onchocerca spp. [22].
This study presents the latest entomologic data of a longitudinal study in the Vina du Nord valley, North Cameroon, dating back to 1976 when ivermectin mass treatment had not yet commenced [24,25]. The impact on O. volvulus transmission after 25 years of annual community-directed treatment with ivermectin (CDTI) is demonstrated here. Furthermore, a second site endemic for onchocerciasis in an economically important cattle livestock region has been monitored since 1985. The epidemiologic data is also complemented with Onchocerca spp. transmitted by the same local vectors of the S. damnosum complex and discussed in light of their impact on transmission success of O. volvulus. We have not studied onchocerciasis transmission in regions where ivermectin treatment is contraindicated, such as co-endemic foci of Loa loa in the Central African rainforest, although they remain potential source-areas for reinvasion.

Study sites
Two S. damnosum fly catching sites at two foci in Northern Cameroon were visited between two to four times per month (Fig 1) [27]. Baseline and follow-up data of O. volvulus transmission to man before ivermectin mass treatments started is available for both foci [15,16,19,25,28], and publicly available via the project website www.riverblindness.eu (http://riverblindness.eu/epidemiology/fly-catchingsites-data/). Whereas S. damnosum sensu stricto and S. sirbanum are the predominant vector species at the Vina du Nord river, it is S. squamosum at the Vina du Sud river [29]. Fly catching was performed according to Duke et al. [30] with the following modifications. Blood meal-searching female Simulium flies were attracted on man by exposing the fly catcher's legs and trapped with sucking tubes as soon as they settled before starting to probe. Usually the catching period was from 7 am to 6 pm, interrupted by a one hour break at noon. Afterwards, the catches were transported to the research station in Ngaoundéré and stored at -15°C until dissection.

Fly dissection and identification of filarial species
Flies were sorted, counted, and a maximum of 100 female S. damnosum s.l. flies per site and day were dissected with needles under a stereomicroscope (Wild M5, Switzerland). The parous rate was determined by examination of the ovaries in the abdomen [31]. From parous flies infested with filarial worms the location (head, thorax or abdomen), molting stage and quantity was noted. Following the identification key of Wahl et al. [15], infective third-stage larvae (L3) were classified to species according to body length, measured by an ocular eye-micrometer attached to the stereomicroscope at 50x magnification, and shape of the anterior and posterior ends. For a subdivision of the L3 taken between February 2010 and February 2012, a molecular investigation of their mitochondrial DNA was conducted according to Eisenbarth et al. [22]. Briefly, single L3's were digested with 1 to 2 μl proteinase K (20 μg/μl stock) in 75 μl DirectPCR lysis reagent (Viagen Biotech, USA) at 55°C. Two μl of the crude extract was used for each 25 μl PCR reaction. Primer pairs of three mitochondrial loci (12S rRNA, 16S rRNA and coxI) that allow for the discrimination of filarial species were used. The amplified PCR products were sequenced on an ABI Prism 3100 genetic analyzer (Applied Biosystems, USA) following the manufacturer's protocol. For the comparison of the body lengths of L3's identified by molecular markers, a larger sample size was taken from flies caught in the same period at the Vina du Sud river about one kilometer downstream of the site near Galim. These flies were collected both from man and cattle.

Calculation of Annual Biting Rates and Transmission Potentials
The Annual Biting Rate (ABR) and Annual Transmission Potential (ATP) of Simulium damnosum flies were determined according to the literature [24,25,32]. First, the monthly biting rates (MBR) were calculated by the sum of the flies caught per month divided by the number of Overview map of the study sites (yellow circles) from two river basins in Northern Cameroon. The village Galim at the Vina du Sud river near Ngaoundéré is on the Adamawa highland plateau, an area above 900 m altitude (dark grey area) with intense cattle husbandry. The village Soramboum at the Vina du Nord river towards Touboro is located in the Sudan savannah (light grey area), a region not often frequented by cattle herds and with high wildlife density. Diagrams of hydrological data showing the mean monthly water discharge from three river basins is taken from Olivry [26]. CAR: Central African Republic. catching days and multiplied by the number of days per month. No correction was made for the missing hours due to rain, sandstorm, or any other reasons. The ABR is the sum of 12 MBRs per hydrological year, measured from April (beginning of rainy season) till March next year (end of dry season). For months during which no catches were attempted, the mean MBR value for the corresponding month and site over the respective decade was estimated by interpolation. The monthly infection rate was the sum of the infective L3 of O. volvulus, O. ochengi and O. ramachandrini from the head, thorax and abdomen found in all parous flies, divided by the sum of dissected flies. By multiplying the monthly infection rate with the respective MBR, the Monthly Transmission Potential (MTP) was determined. The ATP is the sum of 12 MTPs for one year, starting from April till March next year. Missing data points were extrapolated by the sum of all MTP divided by the number of months with data, and multiplied by factor 12. If the MTP data per year was below 3, the mean annual infection rate of proximate years multiplied with the respective ABR was used instead for the ATP calculation.

Statistical analysis
The statistical program Python version 3.4.1 was used for statistical analysis employing student t-tests. Results were considered statistically significant when the p-value was below 0.05. P-values were corrected for multiple testing by multiplying with the number of tests done. The effect size was calculated according to Cohen [33]. For depicting the distribution of the L3 body length from a random sample, violin plots, i.e. box plots with a rotated kernel density plot on each side, were used.

Entomological data
During the study period, a total of 39,082 flies were caught on human fly catchers, and 21,897 (55.6%) of them were dissected: a total of 2096 L3 were found (Table 1). Depending on the catching site, a mean of 1.96 ±0.53 L3 (max. 20) were harvested per infective fly in Soramboum and a mean of 3.49 ±0.13 L3 (max. 23) in Galim. Near Soramboum at the Vina du Nord site the infection rate (flies carrying L1, L2 and L3) of parous flies in the rainy season was significantly higher than in the dry season (mean: 10.9 vs. 5.9%, t-value = -4.91, p < 0.001), as well as the infection rate with infective L3 stages (mean: 7.8 vs. 3.3%, t-value = -4.88, p < 0.001). The opposite was true at the Vina du Sud site near Galim (mean infection rate: 11.1 vs. 16.6%, tvalue = 2.36, p > 0.05; mean L3 infection rate: 4.3 vs. 7.1%, t-value = 1.89, p > 0.05). Moreover, the parous rate, which is a parameter of age structure, was on average 9.6% higher in the wet season than in the dry season in Soramboum (range: -18.3-19.3%, t-value = 5.31, p < 0.001), but in Galim on average 11.8% lower in the wet season compared to the dry season (range: 6.7-24.3%, t-value = 3.83, p < 0.001). The higher proportion of infective L3 to developing larvae during the rainy season in Soramboum (64.0% vs. 53.5%, t-value = -1.88, p > 0.05), but not in Galim (33.9% vs. 35.3%, t-value = 0.88, p > 0.05) is lacking statistical support. A generally higher proportion of L3 in Soramboum can be explained by a longer storage time of the caught flies at ambient temperature during the time (often days) until they were brought to the laboratory, 225 km away by public transport, so that more larval stages developed further.
Comparison of Simulium biting rates and Onchocerca transmission potentials pre-and post-ivermectin distribution With the exception of 1995 the ABR in Galim was higher than in Soramboum, on average by a . The yearly fluctuations were more pronounced in the Vina du Nord valley and followed a cyclical pattern (Fig 2A). In contrast, the ABR at the Vina du Sud fluctuated only mildly apart from intermittent dips, which reached previous levels in the following year (Fig 2B).  (Fig 3A), whereas in Galim the highest decline was within the peak of the dry season and the peak of the rainy season from February till August (Fig 3B).   Fig 3B). In contrast, Soramboum experienced an increase of L3 transmission after the early years of ivermectin mass treatments, from an average ATP of 1045 ±438 L3 per person and year in 1987-88 to 2286 ±1338 in 1993-98, which later returned to former levels, i.e. an ATP of 1242 ±741 in 1999-2012 (Fig 4A), although the pre-ivermectin control ATP from the adjacent Touboro site was much higher (4140 L3 per person and year, Fig 3A).
Species composition of the infective Onchocerca larvae from Simulium damnosum s.l.    [1985][1986][1987]1990 and 1997 in Galim have been extrapolated. In 1976 (color transition of white, light grey to dark grey) no species discrimination was done. ATP with available MTP data 80% have borders in bold; those with data 80% > x 30% have normal borders, and those with data < 30% have borders with a dotted line. The dotted line marks the beginning of ivermectin mass treatments. The asterisk indicates the introduction of PCR-based methods for species discrimination in the vector. Prior to 1990, the L3 species was determined according to the following body length criteria: O. volvulus 750 μm; 750 μm < O. ochengi < 900 μm; O. ramachandrini 900 μm. A Soramboum, Vina du Nord. Epidemiological data prior to 1998 was published before and modified to fit this graph. Data from 1976 was taken from Touboro, 30 km further downstream. B Galim, Vina du Sud. Epidemiological data prior to 1997 was published before and modified to fit this graph.  (Fig 5B), whereas in Galim only O. ochengi was found (Fig 5D). A recently discovered O. ochengi genotype called 'Siisa' [22,23,34] contributed to 8.6% and 10.7%, respectively, of the local O. ochengi L3 population in Soramboum and Galim (Fig 4B and 4D (mean: 180). Accordingly, the adjusted species proportion of the L3 population for these years were on average 6.3% O. volvulus, 82.2% O. ochengi and 11.5% O. ramachandrini (Fig 4A, right side).

L3 body length as a parameter in species identification
In order to evaluate the reliability of body length as a characteristic trait that can be used for species discrimination of infective larvae, the body lengths of occurring Onchocerca spp. L3 in S. damnosum s.l. were compared with morphological and molecular identification methods (Fig 6). Whereas the inter-specific differences according to morphological criteria are significant (p < 0.001), no within-species length difference has been detected between morphological and molecular identification of O. volvulus and O. ramachandrini. A significant (p < 0.01) within-species difference has been found in the common genotype of O. ochengi sensu stricto, but with a low effect size (d = 0.598, n = 95); a significant difference (p < 0.001, d = 1.471, n = 19) was also found for the genotype O. ochengi 'Siisa'. Interestingly, a more than 4

Discussion
This study represents a comprehensive 4-year dataset of transmission from sites in two onchocerciasis-endemic river basins and re-evaluates data collected up to 36 years ago. Whereas we can observe a break of the transmission cycle on the Adamawa plateau, the decline of parasite transmission seems to be halted in the focus of the Sudan savannah despite ongoing treatment intervention. The treatment intervention in this focus has passed the estimated life expectancy of the worm (10 to 15 years) almost by factor two. Conjected that the residual transmission does not stem from invading infected flies from other endemic foci by wind drift, the elimination of O. volvulus in previous hyperendemic foci in North Cameroon by yearly-given CDTI appears to be difficult, even though the actual risk of skin-lesions and blindness due to onchocerciasis is presumably very low.
Despite the fact that the Mectizan treatment campaigns on the mesoendemic Adamawa plateau have started about 10 years later, the transmission of the parasites there seems to be disrupted. One reason might be that the transmission rates in the past were partly overestimated due to misidentification of infective larval stages derived from animals. However, several studies including this one emphasize the adequate discriminative power of morphological characters for species delimitation, in particular the body length (Fig 6) and shape of head and tail [15,19,35]. It is thus very likely that at least a fraction of the L3 found were correctly identified as O. volvulus. However, if the endemicity of a region reaches hypoendemic levels in an area of intensive transmission of filariae of non-human origin, such as in this case, the reliability of microscopic examination is limited. For confirmation of interruptions of O. volvulus transmission, molecular methods like PCR are necessary.

Decline of the Simulium damnosum biting rates of humans
Since 2006 there is a steady trend of lower ABRs, in particular at the Vina du Sud river, where biting rates before were with only one exception above 60,000 per man and year (Fig 2B). The vector transmission of filarial stages have also dropped during this time (Fig 4B), but to a lesser extent in the Sudan savannah (Fig 4A) due to a concomitant gain of the infection rate by bovine filariae. The reason for this vector decline could be the result of decreased availability of breeding sites or food for the aquatic Simulium larvae, and hence a drop in population size. A distinct increase of endoparasitic mermithids in human-biting nulliparous flies was evident at the Vina du Sud breeding sites (S1 table). It is, however, unlikely that these mermithids or other Simulium parasites are the main drivers for the massive decline in biting rates of recent years. A reduced longevity of adult flies was not observed, when comparing the current parous rate with those of flies at baseline [24]. Furthermore, a continuous rise in the pool of potential blood hosts, both human and livestock, may also contribute to lower individual biting frequencies. The regional impact of climate change cannot be excluded, either, although the water delivery of the investigated rivers have not changed drastically until 1980 [26].

Endemicity of Onchocerca volvulus in North Cameroon
The longitudinal monitoring in the Vina du Nord valley indicates that the average transmission of O. volvulus remained around 500 L3 per man and year for 20 years after the onset of ivermectin mass treatments (Fig 4A). This seems to contradict the reduction of onchocerciasispositive patients in the region as a result of control strategies with ivermectin [12]. One reason could be that there is a variable degree of misidentification of O. volvulus L3. In the most recent monitored years 2009-2012, when molecular detection methods were used, the degree of morphological misidentification was 72%. However, earlier epidemiological data from the Sudan savannah [25] showed ATP above 4000 L3 per man and year (Fig 4A, left side). Even though no differentiation of the species had been undertaken at that time, the proportion of animalfilariae in S. damnosum s.l. were likely low due to the lack of cattle as potential blood hosts [for explanation see 36]. Hence, only filariae from the warthog could have been co-transmitted, and the infection rate of the vector with O. ramachandrini has not changed considerably during the observation period. Another theory states that the regulation of parasite transmission may be density-dependent instead of linear. That means the effective reproductive ratio of filarial worms equals one even though the basic reproductive ratio is much higher. In the Vina du Nord river basin, Renz [25] compared the prevalence of onchocerciasis and burden of microfilariae with the L3 infection rate in the vector and found no linear correlation, but rather a dependency of fly infection rate with prevalence in the human population instead of the community's microfilarial load (mff/mg). A density-dependent mechanism has already been shown for the parasite acquisition in cattle when inoculated with infective larvae of O. ochengi [37,38].
The observed seasonal variations of the entomological parameters match well with baseline data from the Vina du Nord river [25], including the number of infective flies with L3. Nonetheless, the O. volvulus ATP has drastically reduced to 3.5% of the baseline value meaning that the majority of infective flies now carry filariae of animal origin. Additionally, the number of L3 per infective fly decreased moderately (from 3.2 to 1.8). The low but stable transmission level of O. volvulus could mean that the threshold for maintaining endemicity is perhaps lower than current mathematical models predict (ATP 100 in West Africa [39,40], but also ATP 54 in Central America [41]).

Molecular vs. morphologic L3 species identification
Even though molecular techniques of identification give higher accuracy, they are less suitable for high throughput analysis due to limitations of time, cost-effectiveness and local infrastructure. They are nonetheless very useful for the detection of unknown strains and species of filarial nematodes in vector and host, such as Onchocerca ochengi 'Siisa' [22,23,34]. Experimental infection studies from Togo [42] and Mali [43], where O. ochengi microfilariae were inoculated by the vector from infected cattle, revealed shorter L3 body lengths (Togo: 680 μm, 540-680; Mali: 647 μm, 540-810) than our observations (740 μm, 600-940) and previous studies from Cameroon [44]. These values, however, lie in proximity to the measurements for O. ochengi 'Siisa' (660 μm, 600-900) and may thus represent or morphologically resemble this strain. Ultimately the evolutionary relationships of Onchocerca parasites in humans, cattle and game animals can be compared and tested with genetic markers by generating phylogenetic trees [22,34].

Influence of closely-related animal filariae for the transmission of river blindness
Besides the climatic disparities of the two foci, which is mainly a result of different altitudes, they share similar conditions for their respective black fly populations. One major difference, however, is the disproportionately higher cattle stock density on the Adamawa plateau compared to the situation in the Sudan savannah (Fig 1). The cattle to human ratio around the Galim focus is approx. 10:1, whereas in the Soramboum focus it ranges between 1:1 and 2:1, and was even lower in previous years, because nomadic cattle were not allowed to enter the Vina du Nord basin until 1975, and the local villagers had not kept any livestock animals, either. Nowadays, an increasing number of vagrant Bororo herdsmen arrive with their herds of zebu cattle and become settled. The inherent difference in livestock density is both culturally inherited (migrating pastoralists of the North vs. settled cattle farmers of the South) and due to biologic conditions (water and food scarcity during the dry season in the Sudan savannah; absence of tsetse flies on the Adamawa plateau, which transmit bovine trypanosomiasis). Invading O. ochengi L3 elicit a humoral immune reaction in humans, which cross-reacts with O. volvulus L3 antigens, thereby reducing transmission success [19]. The protective effect of populations under repeated antigen exposure is called premunition and well known for malaria and other infectious diseases [45,46]. On the Adamawa plateau this effect seems to be strong enough to prevent or at least complicate the regional endemicity of O. volvulus. The advent of nomadic herdsmen and their cattle herds in the Vina du Nord valley is congruent with an increased transmission of animal-borne filariae, in particular O. ochengi (Fig 4A). This sudden jump of animal-filariae in the vector population implies the diversion of large quantities of local S. damnosum s.l. to take their blood meal from cattle, thereby reducing the vector pool for humans [16]. This phenomenon has been termed zooprophylaxis and acts also as a protective trait against onchocerciasis transmission [15,20].
The important question is how the low but stable rate of onchocerciasis transmission in the Sudan savannah can be further curbed or completely stopped. Altogether, five molecularly identified O. volvulus L3 from two infective flies (3.85% of total, 95% CI: 0.47-13.21%) were found in the dry season of 2010 (February) and the rainy season of 2011 (June). Since yearly CDTI application rounds are given at the end of July, the late time point of occurrence after ivermectin treatment may hint to an incipient reconstitution of skin microfilariae in humans infected with O. volvulus 12 months prior. This would be a strong argument for the continuation or even temporary intensification of the ivermectin control program [10,36,47]. However, current political instabilities in adjacent countries and the exclusion of certain patient groups in treatment intervention programs, like nomadic people, illegal immigrants and refugees, could impede the long-term success of such measures. Ongoing monitoring of vector transmission is therefore crucial for health policy in onchocerciasis-endemic countries.
Supporting Information S1 Table. Infection rate of female nulliparous flies and aquatic larvae of Simulium damnosum s.l. at two different sites in Northern Cameroon. Those infection percentages from mermithids, fungus and malpighian nematodes are given from nulliparous female flies and aquatic Simulium larvae, respectively, and those from planidium larvae from all flies dissected. (PDF)