Human onchocerciasis, a vector-borne disease, is endemic in 31 countries in sub-Saharan Africa, three countries in Latin America and in Yemen. WHO has verifi ed the elimination of transmission of the parasite in Colombia, Ecuador and Mexico respectively in 2013, 2014 and 2015 and declared them free of the disease. Th e infection is caused by Onchocerca volvulus, a fi larial nematode (1,2). Chronic infection causes itching and disfi guring lesions of the skin and produces eye lesions that can lead to irreversible blindness. Because the vectors (black fl ies of the genus Simulium) are insects that breed as larvae in fast-fl owing rivers and streams and bite humans near these sites, the disease is also known as river blindness. In the Americas, the disease is sometimes referred to as Robles' disease aft er Dr Rodolfo Robles, the Guatemalan physician who fi rst documented the causal relation of O. volvulus with vision loss and blindness.

# CONTROL AND ELIMINATION OF HUMAN ONCHOCERCIASIS
In Africa, the blindness and the severity of skin lesions have severe socioeconomic consequences. Historically, river blindness has led to the desertion of large areas of fertile land adjacent to vector breeding sites, impeding the economic development of aff ected countries (3). Control of morbidity from human onchocerciasis and interruption of transmission of the causative parasite have commanded attention and been addressed in several diff erent ways. Control strategies have included removal of nodules (nodulectomy), vector control and, more recently, mass drug administration (MDA) with ivermectin (4,5). Th ese interventions have varied among regions of the World Health Organization (WHO) in time and place,
with varying degrees of success; all have been well characterized and documented in the peer-reviewed literature (6). Currently, nearly all programmes use MDA with ivermectin, administered once, twice or four times a year (7,8).
In the WHO African Region, annual or semi-annual distribution of ivermectin is used to sustain the successes of the Onchocerciasis Control Programme in West Africa (OCP) and is distributed annually in community-directed country programmes. Communitydirected treatment with ivermectin is the main intervention of the African Programme for Onchocerciasis Control (APOC) established in 1995, which covers all of the non-OCP countries where the disease is endemic. Th ese programmes, initially aimed at controlling blindness, have operated under World Health Assembly resolutions WHA47.32 and WHA62.1 adopted in 1994 and 2009 respectively (9).
In the WHO Region of the Americas, semi-annual mass treatment using ivermectin with a minimum expected coverage of 85% of the eligible population in all endemic communities is the strategy adopted by all endemic countries; quarterly treatment is provided in selected areas. In 1991, resolution CD35.R14 of the XXXV Directing Council of the Pan American Health Organization called for the elimination of morbidity due to onchocerciasis by 2007 (10). Th e OEPA was established in 1992 to consolidate the eff orts of partner agencies with a view to eliminating the disease and providing technical and fi nancial assistance to national programmes. Th is goal was reaffi rmed in resolutions CD48.R12 and CD49.R19 endorsed in 2008 and 2009 respectively. Th e OEPA now operates under resolution CD49. R19, which calls for the regional elimination of ocular morbidity caused by onchocerciasis and interruption of transmission of the causative parasite by 2015 (10).

# ONCHOCERCIASIS ELIMINATION PROGRAMMES AND THEIR PHASES
Th e advent of ivermectin, an eff ective microfi laricide suitable for large-scale use in rural areas, has enhanced prospects for control or elimination of the disease in many areas, including Africa. Th e medicine is provided free of charge by Merck & Co., Inc. under the Mectizan Donation Program. Given as an oral dose, ivermectin temporarily lowers skin loads of microfi larial O. volvulus to levels below those required for eff ective transmission by Simulium species (black fl ies). Ivermectin also has a demonstrated microfi larial suppressant activity in adult female worms and a deleterious impact on adult worms, especially when given multiple times per year (11).
Studies in several endemic onchocerciasis foci in Africa and the Americas have shown that sustained, high level coverage with ivermectin is crucial for successful control of transmission and morbidity. Th erefore, an important criterion to trigger the initial evaluation of a country's control programme is evidence that broad, eff ective coverage with ivermectin has been achieved over a suffi ciently long period of time to eff ect interruption of transmission and reduce morbidity (5).
Onchocerciasis control programmes carrying out mass treatment with ivermectin have three phases (Figure 1).  (12,13). Treatment eventually leads to a suppression or near-suppression of the annual transmission potential of the vector and thus to a temporary suppression of transmission. However, the population of adult worms may still be at a point of potential recovery should treatment be withdrawn.
If eff ective MDA continues at the proper interval, this temporary suppression of transmission gives way to interruption of transmission. Th is phase typically lasts for at least 12-15 years in the case of annual treatment, corresponding to the reproductive lifespan of the adult parasite when exposed to drug pressure. At the end of the fi rst phase most if not all adult worms should have died or become infertile. Importantly, some adult worms and/or microfi lariae may persist, but the minimal level of transmission that may occur will not maintain the infection (that is, any transmission is below the threshold necessary to maintain the parasite population indicating that elimination of the parasite is imminent).
At some point during phase 1 the infection reaches a state of transmission interruption in which no evidence of ongoing transmission or new infections can be demonstrated. Th is is a critical time as the programme decides whether to suspend treatment.
Phase 2 Th e second phase -post-treatment surveillance -occurs when interventions are stopped. During this period, the national programme conducts periodic assessments to ascertain that transmission remains interrupted for a minimum period of 3 years, or up to 5 years depending on the specifi c parameters of each focus. Th is timeframe is critical in those areas at highest risk of recrudescence of infection, especially in (i) areas with historical prevalence of intensity of infection bordered by foci with ongoing transmission or (ii) areas with low coverage or few treatment rounds; and (iii) areas of political instability. Periodic assessments can be conducted during this phase by focus, by transmission zone or by country. Isolated foci or transmission zones in the same country may be evaluated at diff erent times if MDA activities are not implemented or progressing in a synchronized manner.

# DIAGNOSTIC TESTS
Any diagnostic tests used to monitor epidemiological progress towards transmission end-points during post-treatment surveillance should be statistically sound and include well established, validated numbers of required samples of the vectors and/or those of the aff ected human population. Th e samples should be examined, with identifi cation of correct target populations to be tested and the range of acceptable values including confi dence intervals.

# Entomological evaluation by O-150 PCR technique
Entomological evaluation by O-150 PCR is a diagnostic technique that aims to determine the level of infective-stage O. volvulus larvae in the vector population as analysed by polymerase chain reaction (PCR) technique based on amplifi cation using O. volvulusspecifi c DNA targeting probes O-150 repeat family sequence (14)(15)(16)(17). Th e fl ies must be collected during daylight hours when parous fl ies are most abundant (implying knowledge of their diurnal biting cycle for each species or cytoform concerned) and during the peak transmission season of the year (to optimize the collection of infected specimens).
Flies are pooled by collection site into pools containing no more than 200 individuals, and the heads and bodies are separated and examined individually. Th e bodies (thorax and abdomen of black fl ies), which may contain O. volvulus DNA from microfi laria or L2 stages, may be used to assess if any parasite remains in the human population using black fl ies as an alternative to skin snipping (xenodiagnosis). However, as bodies carry only immature larval stages, a positive result in bodies is not necessarily indicative of ongoing transmission, which requires the presence of infective-stage larvae (L3) in the head of the vector. Th us, if any evidence for parasite-vector contact is found in the analysis of body pools from a given area, all head pools from that area must be tested to gain as accurate an estimate of the prevalence of fl ies carrying infective-stage larvae as possible.
Th e criteria used for entomological assessment are:
- an upper bound of the 95% confi dence interval of the prevalence of fl ies carrying infective larvae (L3) in the head of less than 0.1% (< 1/1000) in parous fl ies; or - an upper bound of the 95% confi dence interval of the prevalence of L3 of less than 0.05% (< 1/2000) in all fl ies (assuming a parity rate of 50%).
Th ese criteria were fi rst applied by the former OCP in West Africa (18,19), then operationalized in the OEPA (20,21) and in recent APOC evaluations in West Africa (22,23).
For the sample size, suffi cient fl ies must be tested to ensure that the upper bound of the 95% confi dence interval for the prevalence of infective fl ies is less than 0.05%. Th is level of statistical confi dence requires that a minimum of 6000 fl ies must be collected from a transmission zone and all must be shown to be negative by PCR for parasite infective larvae. In areas where collection of such a large number of fl ies is not feasible, as many fl ies as possible should be collected over a period of time and tested to ensure that the upper bound of the 95% confi dence interval for the annual transmission potential or the seasonal transmission potential falls below an acceptable level.
Estimates of the annual transmission potential necessary to maintain the parasite have ranged from 2 to 54 L3/person/year using mathematical models (15,24), and from 8 to 18 L3/person/year using fi eld observations (25,26). Furthermore, previous estimates were developed before the advent of ivermectin, and the concept of few to no microfi lariae in the skin as a result of MDA with ivermectin would suggest that the data used are not entirely comparable to the present situation. Nonetheless, setting an annual transmission potential with an upper bound of the 95% confi dence interval of less than 20 has been successfully operationalized in the Americas where a variety of Simulium species with diff ering vectorial capacities serve as vectors; results to date indicate that this is a suitable cut-off point (20). However, such thresholds may be diff erent for African settings and further clarifi cation is awaited.
In the event that no fl ies are collected because the vector has been eliminated or has disappeared as a result of environmental changes, as in certain foci in Uganda where S. neavei was the vector (27), the absence of larval stages of S. neavei species on the phoretic crab in surveys and from fl ies collected in a defi ned focus is a substitute indication of interruption of transmission.

# Serological evaluation by Ov-16
Th e O. volvulus Ov-16 antibody test (28,29) can be performed on fi nger-prick blood samples and is a proven, valuable and minimally invasive assay (9,41,42). Th e test determines the presence of IgG4 antibodies to the antigen Ov-16 and is useful for detecting the exposure to the O. volvulus parasite.
During the few past years this serological test has been operationalized in Latin America (11,20,21,(30)(31)(32)(33) and in Africa (27,34). Children under 10 years of age were included in the evaluation surveys, which determined exposure to the parasite with a suffi cient sample size to detect a prevalence of less than 0.1% at the upper bound of the 95% confi dence interval.
Generally, a sample size of 2000 children of less than 10 years is required for Ov-16 serology testing in order to meet this criterion (35). Children are selected by a multistage stratifi ed sampling method scheme applied to the local lower administrative unit level. For example, if the local lower administrative unit contains 20% of the total estimated population of the focus (transmission zone), a subsample of 400 children will be enrolled from that administrative unit.
In small foci where there are fewer than 2000 children under 10 years of age, the sample sizes must be tested to ensure that the prevalence of Ov-16 in the target population of children is equal to or less than 0.1% (Table 1).
Table 1 Proportion of fi nite target population that must be tested to conclude that the prevalence in the entire target population is  0.1% when none of the sample tested is positive When the eligible population of children is fewer than 1100, essentially all eligible children should be tested. In these situations, eff orts should be made to calculate the confi dence interval using statistical methods appropriate for fi nite populations. Sampling should be representative of the entire transmission zone, and analysis should allow for stratifi cation by age.
Because the Ov-16-based serological test may detect recent as well as historic exposure, other confi rmatory tests are still needed to distinguish new patent infections from exposure. Eff orts are under way to develop such tests, which should be incorporated into the evaluation process of programmes as they become available.

# Parasitological evaluation by skin snip microscopy and DEC patch test
Skin snips and the DEC patch test are useful tools for monitoring progress as an elimination programme is implemented and moves towards that goal during treatment (phase 1). A detailed description of the procedures and sample size is found in the APOC manual on evaluating the impact of community treatment with ivermectin using the skin snip method (36). However, a test capable of detecting any new patent infection with a high positive predictive value is still needed.

# Skin snip evaluation by PCR
A PCR test on skin snip is indicated in some limited situations where few serologically positive children (Ov-16 > 0.1%) are detected in order to confi rm actual infection rather than exposure to the parasite. Serologically positive children found negative by PCR testing of skin snips are considered negative for patent infection with O. volvulus and are accepted as not contributory to the 0.1% threshold calculation. Since these children would be considered as O. volvulus "exposed", ethically, the programme should re-examine them 1-1.5 years later to determine if they have developed patent infection. If so, they should be treated accordingly.
Th e original (2001) WHO guidelines for certifi cation of elimination of human onchocerciasis included the following criteria: (i) an absence or near absence of infectivestage O. volvulus in the vector as determined by the O-150 PCR (Poolscreen) test with a minimal sample size of 10 000 black fl ies; and (ii) an absence of detectable infection based on skin snip microfi lariae, DEC patch test, nodule detection and serological testing of untreated children reaching 5 years of age or in untreated new residents who have migrated into an endemic area where transmission has been interrupted. A fi ve-year cumulative incidence rate of less than 1 new case per 1000 susceptible children or individuals provided an acceptable sample size (37,38).
Since 2007, these criteria and the respective diagnostic tools have been operationalized in Latin America and in Africa.
In the Region of the Americas, elimination has been the main goal of the OEPA since its inception in 1991 and has been achieved in three formerly endemic countries since then (39). At the end of 2014, transmission is believed to have been interrupted in 11 of the 13 endemic foci, with only the twin foci on the Brazil-Venezuelan border remaining with active transmission. Consequently, WHO has verifi ed elimination of human onchocerciasis in Colombia in 2013 (40), Ecuador in 2014 (41) and Mexico in 2015 (42) on the basis of the original guidelines.
In the African Region, the shift from control to elimination programme in 2009 led to signifi cant operational changes and implications (12,13,43,44) as well as a renewed approach and criteria to monitor the progress, impact and outcome of the programme using the lessons learnt over the past decade in the Americas (11,(30)(31)(32)(33) and, more recently, in Africa (22,34,45,46).
In addition to these lessons learned during the operationalization of the criteria and procedures within the old WHO guidelines, there was a need to develop an evidenceinformed guideline using transparent and explicit methods for assessing the evidence and for formulating recommendations as stipulated in the WHO handbook for guideline development (47).
Th ese guidelines are therefore intended to update the criteria used in the WHO 2001 document and to adhere to WHO's standards and procedures for developing guidelines since 2007. By doing so, countries in which onchocerciasis is endemic will have appropriate tools at their disposal for guidance on when and how to stop MDA and to conduct posttreatment surveillance until confi rmation of the elimination of onchocerciasis, to be followed by verifi cation of elimination by WHO.
Th e purpose of these guidelines is to provide evidence-based recommendations that warrant the discontinuation of MDA and the verifi cation of elimination of transmission of O. volvulus. Information is provided on how to monitor and assess onchocerciasis in order to demonstrate that transmission of O. volvulus has been interrupted in areas previously identifi ed as endemic.
Th e objectives are:
- to provide national onchocerciasis elimination programmes and external agencies with the recommendations required to demonstrate and confi rm the interruption of transmission of O. volvulus over a specifi ed period of time; and - to inform those involved in national onchocerciasis elimination of the required procedures for verifying elimination of human onchocerciasis.

# PURPOSE AND OBJECTIVES
Th ese guidelines are primarily targeted at national neglected tropical disease or onchocerciasis elimination programme managers within health ministries in anticipation that the recommendations will be adopted as national policies by policy-makers. Th ey are intended also as guidance for those involved in verifying elimination of human onchocerciasis.

# DEVELOPMENT PROCESS
A Guideline Development Group comprising seven members from leading stakeholders with expertise of onchocerciasis in Africa, the Americas and Yemen was formed. In addition, two methodologists were commissioned to retrieve, synthesize and assess the evidence, a process preceded by formulating key questions and selecting outcomes in collaboration with the members of the Guideline Development Group and a WHO Steering Group. Th e methodologists reviewed the literature systematically and assessed the quality and certainty of evidence according to the GRADE approach (48). Th ey also prepared the decision tables, which guided members of the Guideline Development Group to formulate recommendations during a face-to-face meeting (Geneva, January 2015) co-chaired by a third independent methodologist familiar with WHO's procedures and standards for developing guidelines and one member of the Group. Th e draft guidelines were reviewed by fi ve external peers with diverse expertise and fi eld experience in onchocerciasis, lymphatic fi lariasis and malaria, and their comments helped to refi ne the fi nal document.

# GUIDELINE QUESTIONS
Two key questions were formulated as follows: Members of the Guideline Development Group contributed electronically to the development of these key questions by providing details of the type of tests to be used, their critical thresholds and the optimal time-point of their use as summarized in Table 2 and in Annex 1.

# SYSTEMATIC REVIEW
Th e commissioned methodologists conducted the review of the literature to answer two key questions (KQ1 and KQ2). Th e eligibility criteria comprised:
Population Th e eligible populations for inclusion in the review were human populations at risk for onchocerciasis as well as black fl ies in endemic regions. Black fl ies (Simulium species) were considered a population because they are the vector of O. volvulus.

# Intervention (diagnostic tests)
Th e search included studies on populations where diagnostic tests for measuring the elimination of O. volvulus have been implemented individually or in various combinations.

# Control (diagnostic test accuracy reference standard or other test)
Evidence from studies with a known and validated reference standard or comparison of two versions of a similar test was also included in the review.
Outcomes Th e main outcome was the absence of recrudescence of infection; that is, the negative test at the time-point of KQ1 predicts a negative test at the time-point of KQ2.
Timing Th e fi rst question (KQ1) determined the interruption of transmission, hence the end of MDA, whereas the second question (KQ2) confi rmed the end of post-treatment surveillance.
Areas endemic for onchocerciasis for which populations at risk have received MDA and are being evaluated in order to demonstrate or confi rm the interruption of O. volvulus transmission.
Th e details of the search strategy are included in Annex 2.

# FORMULATION OF RECOMMENDATIONS
Th e experts commissioned by the Guideline Development Group conducted a systematic review of the literature using the GRADE approach to assess the quality of the evidence and to formulate the recommendations. Th e GRADE approach designates the body of evidence for each important outcome with a level of confi dence or certainty that the eff ect of a test or approach as measured or known is actually correct. Th us each recommendation is accompanied by an assessment of high, moderate, low or very low certainty that the supporting evidence provides the correct estimate of eff ect or association.
Th e co-chair led the Guideline Development Group through the process of formulating the recommendations. Members were asked to review each of the key questions and to formulate recommendations based on the evidence identifi ed by the systematic review. Th e discussion was structured around decision tables that were prepared a priori, and contained the following criteria: accuracy of the diagnostic tests; overall certainty of the evidence; resources required; cost; equity; feasibility; and acceptability of the test or approach. Aft er judging the various criteria, the members agreed on the balance of consequences, the direction and strength of the recommendation, and its wording.
A strong recommendation was formulated when members were confi dent that the desirable eff ects of adherence to the recommendation outweighed the undesirable eff ects (or vice versa). A conditional recommendation was formulated when desirable eff ects of adherence to the recommendation probably outweighed the undesirable eff ects (or vice versa).
Th e recommendations were formulated by consensus among the members. When consensus could not be reached, votes were taken by hand-raising at the Chair's discretion. A strong recommendation was adopted if supported by at least a two-thirds' majority. As members raised points that were relevant but not directly related to criteria that did not directly aff ect the recommendation, the co-chair attempted to classify them as conditions or key remarks to support the recommendation statement, implementation considerations, monitoring considerations or implications for future research. He off ered a neutral recommendation as a starting point for discussing the recommendation statement.
Th e draft guidelines underwent external peer review before fi nalization: no changes could be made in the recommendations during this process, however several clarifi cations were added.
All seven members of the Guideline Development Group and the three methodologists who participated in the face-to-face meeting completed and signed WHO Declaration of Interest forms (Annex 4).
Of the seven members with longstanding involvement in onchocerciasis control or elimination programmes or research activities, two reported having received research grants or consulting fees from either the Mectizan Donation Program or the Bill & Melinda Gates Foundation. Two members of the WHO Steering Group reviewed and assessed these two cases and concluded that these declared interests were not a signifi cant confl ict of interest and these individuals therefore participated fully in the guideline development process.
Th e two systematic reviewers and the guideline methodologist were neither voting members of the Guideline Development Group nor onchocerciasis experts; they declared no confl icts of interest. However, they participated in the meeting at which recommendations were formulated (the guideline methodologist was co-chair) to ensure that the evidence was evaluated objectively and that the recommendations accurately refl ected the evidence.

# DECLARATION AND MANAGE-MENT OF INTERESTS
Overall, there was little direct comparative scientifi c evidence on the use of diff erent tests for verifi cation of elimination of human onchocerciasis. Th e best available evidence was provided by two prospective observational studies that provided data from interruption of transmission leading to cessation of treatment. Multiple studies of diagnostic test accuracy comparing various tests were located but these were generally done in populations with high prevalence of onchocerciasis (which does not refl ect the situation close to interruption of transmission with a low prevalence of onchocerciasis). Th is is particularly important because the standard reference test used in these studies (skin snip microscopy) does not perform well in populations with low disease prevalence. Moreover, comparative studies of diagnostic test accuracy were performed to assess test accuracy, but not to demonstrate interruption of transmission or elimination.
For demonstrating interruption of transmission or elimination of O. volvulus, the quality of the evidence for entomological assessment through O-150 PCR (Poolscreen) of black fl ies was rated high, whereas for epidemiological assessments such as Ov-16 serology, skin snip microscopy and ocular morbidity (slit lamp) it was low. Th e systematic reviews and the assessments of the certainty of the evidence are included in Annex 2.
RESULTS OF EVIDENCE RETRIEVAL, SYNTHESIS AND ASSESSMENT Strong recommendation, high certainty of evidence 8.1.1.1 Background Onchocerciasis is a vector-borne disease, where humans are the only natural vertebrate host, and infection rates and intensity are determined by the degree of exposure to infected vectors. However, the epidemiology of onchocerciasis is not uniform throughout its distribution because diff erent disease patterns are associated with diff erent variants or strains of the parasite, with diff erences in the vector capacity and blood-feeding characteristics of local black fl y populations, with the seasonal abundance of the vector and with diff erences in the human host responses to the parasite. Th ese factors, together with those related to environmental, geographical, social and demographic infl uences, increase the complexity of the epidemiology of the disease in the diff erent areas of its distribution. Th e original WHO guidelines on the certifi cation of elimination of human onchocerciasis issued in 2001 (37,38) required a sample size of 10 000 black fl ies, which was operationalized at a minimum of 6000 black fl ies in Latin America and a few countries in Africa.
Summary of the evidence Th e systematic review conducted on the use of O-150 PCR (Poolscreen) testing in black fl ies to demonstrate the interruption of transmission of O. volvulus in a human population receiving MDA against onchocerciasis for the purpose of stopping MDA identifi ed two observational studies (33,46) and 15 studies of diagnostic test accuracy (29,49,(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62), which provide limited evidence on the comparative use of tests (Annex 3.1).
Based on those two observational studies, the O-150 PCR (Poolscreen) for black fl ies test was rated very accurate with a high certainty of evidence. Following historical data from Mali, this PCR test could distinguish O. volvulus from the cattle parasite O. ochengi, which could not be done by simple dissection of black fl ies.
- 1.1.3 Rationale for the recommendation Th e rationale for the strong recommendation was based primarily on the specifi city of the PCR testing, which is higher than that for the dissection of the black fl ies. In addition, larger pools of fl ies are more cost-eff ective than smaller pools for the overall costs of the programme.
Nevertheless, a few factors could aff ect the feasibility of implementing PCR in the community, including (i) the existence of a procurement and supply policy; (ii) support from the Ministry of Health; (iii) support from the frontline health facilities; and (iv) health workers' attitudes, motivation and outreach on the basis of one study in Africa (66). In this case, the value of properly informing and empowering communities to enlist them as crucial allies in disease control eff orts can facilitate the acceptability of the test by showing the benefi ts of the intervention.
Overall, the desirable consequences clearly outweighed any undesirable consequences in both Latin America and Africa.
Implementation considerations, including monitoring and evaluation Th e existence of regional laboratories serving the largest possible administrative area is the main prerequisite for implementation of this test.
Sampling should be appropriately conducted according to APOC standard operating procedures on entomological evaluation.
Laboratory quality control should be regularly monitored to ensure high-quality, reliable test results.
Th e test should be conducted annually up to the end of post-treatment surveillance.
Research priorities More investigations should be carried out to defi ne appropriate and standardized protocols for fl y catching.

# Ov-16 serology testing in children less than 10 years of age
Th e Ov-16 serology test should be used in children less than 10 years of age to demonstrate the interruption of transmission of O. volvulus in a human population receiving MDA against onchocerciasis for the purpose of stopping MDA.

# Strong recommendation, low certainty of evidence
Background In areas where onchocerciasis is endemic, infected individuals harbour both adult (macrofi lariae) and immature (microfi lariae) worms. Following an appropriate period of MDA with ivermectin (that is, corresponding to the lifespan of the O. volvulus parasite under recurrent treatment), infected subjects are believed to be free of any microfi lariae in their skin or eye. Consequently, children born by the end of MDA implementation are not exposed to O. volvulus parasites, justifying the indirect method of determining the interruption of transmission of onchocerciasis. Towards that end, the Ov-16 serology test aims to determine the level of detectable onchocercal infection in children and migrant individuals in an endemic area during the post-treatment surveillance phase as stipulated in the 2001 WHO guidelines for certifi cation of elimination of human onchocerciasis (37,38).
Summary of the evidence Th e systematic review conducted on the use of the Ov-16 serology test in children to demonstrate the interruption of transmission of O. volvulus in a human population receiving MDA against onchocerciasis for the purpose of stopping MDA identifi ed the same two observational studies (33,46) and 15 studies of diagnostic test accuracy (29,49,(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62) above, which provide limited evidence on the comparative use of tests for the determination of interruption of O. volvulus (Annex 3.2).
Th e Guideline Development Group rated the Ov-16 serology test as accurate, with a low certainty of evidence. Studies were mostly performed in populations with high onchocerciasis prevalence, and the reference standard used (generally skin snip microscopy) is an imperfect test in low prevalence settings at this stage of the programme (63). In addition, interruption of transmission demonstrated by Ov-16 serology testing was not confi rmed during and aft er post-treatment surveillance using the same test.
Rationale for the recommendation Apart from the costs of a plate reader for ELISA testing, estimated at approximately US$ 5000, Ov-16 serology testing can easily be used in the fi eld with strong community participation (64)(65)(66)(67)(68) and support from nongovernmental development organization partners alongside other programme assessments such as transmission assessment surveys for lymphatic fi lariasis (69). One ELISA test costs about US$ 0.15, which is cheaper than the cost of continuing MDA.
Compared with skin snipping the ELISA (fi nger-prick) test is considered to be minimally invasive and hence more acceptable in some settings.
Th e Guideline Development Group concluded that the desirable consequences clearly outweighed any undesirable consequences in most settings.
Implementation considerations, including monitoring and evaluation Despite the simplicity of the Ov-16 serology test, challenges include the need for 2 days to test an average acceptable sample, which sometimes is diffi cult to obtain. Th erefore, Ov-16 serology should be used in conjunction with the PCR test in black fl ies. Research priorities Two main topics need to be addressed. First, the sero-reversion of Ov-16 responses by age and over time should be investigated. Second, the new Ov-16 Rapid Test should be validated as a possible replacement of the standard test.

# Skin snip microscopy
Skin snip microscopy should not be used to demonstrate the interruption of transmission of O. volvulus in a human population receiving MDA against onchocerciasis for the purpose of stopping MDA. Skin snip microscopy may be used in a transition to using Ov-16 serology; during such transition, skin snip microscopy and Ov-16 serology should be used in parallel. Skin snip microscopy, if used, should be applied with a sample size providing adequate statistical certainty that programmatic goals have been reached.

# Conditional recommendation, low certainty of evidence
Background Th e Ov-16 serology test and skin snip microscopy were both listed in the original (2001) WHO guidelines for certifi cation of elimination of human onchocerciasis as diagnostic tests to be used in determining the absence of detectable O. volvulus parasites in humans (37,38). Since 2005, skin snip microscopy has been extensively used to follow up trends towards elimination in several APOC projects using community-directed treatment with ivermectin across Africa.
Summary of the evidence Th e systematic review conducted on the use of skin snip microscopy to demonstrate the interruption of transmission of O. volvulus in a human population receiving MDA against onchocerciasis for the purpose of stopping MDA recorded two observational studies. Th e Guideline Development Group was divided on the accuracy of the evidence: three members voted that skin snip microscopy is very inaccurate, two voted that it is inaccurate and two considered the test accurate. Although specifi city is 100%, the main challenge with skin snip microscopy at this stage of the programme is its low sensitivity (estimated at 20%). Additionally, skin snipping of humans seems to be redundant in the presence of O-150 PCR testing in black fl ies. Consequently, a majority of the GDG (fi ve "yes" votes out of seven) concluded that the test was not relevant based on the low certainty of evidence (Annex 3.3).
Rationale for the recommendation Currently, skin snip microscopy is widely used in Africa as a monitoring and evaluation tool to assess progress towards onchocerciasis elimination. Although skin snip microscopy is 10 times less expensive than O-150 PCR testing on skin snips, its sensitivity substantially decreases as the programme reaches the end of the treatment phase, making it inappropriate for use at this stage. Furthermore, a high rate of refusal has been observed in some communities as the manifestations of the disease decrease with the number of years of MDA implementation. However, as the test has been performed for many years, its acceptability is high in some settings.
Given the initial lack of consensus among the Guideline Development Group on the balance of desirable and undesirable consequences of skin snip microscopy for demonstrating the interruption of transmission aft er MDA, a vote was held. Five out of seven members supported a conditional recommendation against skin snip microscopy. Th e voting results allowed the adoption of a conditional recommendation against skin snip microscopy.
Implementation considerations, including monitoring and evaluation Skin snip microscopy is currently used as a tool for monitoring and evaluation during phase 1, which is an appropriate context as sensitivity is still relatively high because the prevalence is still also high. Th erefore, the use of skin snip microscopy was considered acceptable as a transitional test while Ov-16 serology testing is being introduced.
Research priorities Th e acceptability of skin snip microscopy in settings of low prevalence should be investigated, as a high rate of refusal has been observed in some settings.

# Ocular infection
Assessment of ocular infection (that is, the presence of microfi lariae in the anterior chamber) should not be used to demonstrate the interruption of transmission of O. volvulus in a human population receiving MDA against onchocerciasis for the purpose of stopping MDA.

# Strong recommendation, low certainty of evidence
Background Identifi cation of microfi lariae in the anterior chamber during ophthalmological assessment is a pathognomic sign of onchocerciasis and was included among the diagnostic tests to be used in the original (2001) WHO guidelines for certifi cation of elimination of human onchocerciasis in order to determine the absence of detectable O. volvulus parasite in humans (37,38). It requires an experienced ophthalmologist with good knowledge of onchocerciasis-related eye lesions using slit lamp biomicroscopy.
Summary of the evidence As for other diagnostic tests, the systematic review conducted on using ophthalmological assessment to demonstrate the interruption of transmission of O. volvulus in a human population receiving MDA against onchocerciasis for the purpose of stopping MDA recorded two observational studies, of which only one study allowed the evidence for this diagnostic test to be assessed. Testing for ocular infection was considered inaccurate, with a low certainty of evidence (Annex 3.4).
Rationale for the recommendation In addition to the high cost of the equipment needed for this diagnostic test, the Guideline Development Group noted the diffi culties involved in implementing ophthalmological assessments in the fi nal phase of elimination, particularly in settings of low endemicity, given the specialized technical expertise required. Aft er considering its high cost and the need for specialized personnel to conduct the assessment, ocular infection assessment was considered inappropriate despite the fact that local communities appreciate the delivery of eye examination most of the time at no cost. Consequently, the undesirable consequences of ocular infection assessment clearly outweigh any desirable consequences, justifying this recommendation.  Rationale for the recommendation As is the case for the O-150 PCR test used to confi rm the interruption of transmission, the evidence assessment of the Ov-16 serology test as well as its implementation considerations and research priorities are the same at the beginning and at the end of post-treatment surveillance. Nevertheless, it is used only under certain conditions, justifying the conditionality of this recommendation. Th e fi rst condition is when the result of PCR equals the threshold, meaning one positive infective black fl y out of 1000 parous fl ies or 2000 total fl ies examined. Th e second condition is when the result of PCR is near that threshold, meaning 2 or 3 positive infective black fl ies out of 1000 parous fl ies or 2000 total fl ies examined.

# Ocular infection
Ocular infection (that is, the presence of microfi lariae in the anterior chamber) should not be used to confi rm the interruption of transmission of O. volvulus.

# Strong recommendation, low certainty of evidence
Ocular infection (that is, the presence of microfi lariae in the anterior chamber) should not be used to confi rm the interruption of transmission of O. volvulus.
Strong recommendation, low certainty of evidence.
Rationale for the recommendation As indicated for the rejection of ocular infection assessment at the beginning of posttreatment surveillance to determine the interruption of transmission of O. volvulus for the purpose of stopping MDA, its implementation to confi rm elimination of human onchocerciasis is inappropriate at the end of post-treatment surveillance (see also recommendation 8.1.4).
Once an endemic country has been completely mapped, with all the endemic communities identified and stratified according to their level of endemicity (hypoendemic, mesoendemic, hyperendemic or non-endemic), the national programme typically launches an elimination intervention mainly based on MDA during a number of years. This is known as phase 1 or the intervention (treatment) phase (Figure 1). Theoretically, this phase lasts 12-15 years, corresponding to the lifespan of the adult female worm whose death will lead to the permanent interruption of transmission.
At the end of mapping, sentinel communities are selected and monitoring and evaluation activities are regularly conducted to assess the impact of the programme after baseline data have been determined. These in-depth parasitological (skin snip), entomological and serological surveys are done every 4-5 years according to the existing procedures of regional onchocerciasis programmes (36,70,71). The programme should continue treatment with ivermectin with at least 80% coverage of the eligible population each year (assuming MDA is performed annually) until the infection has been interrupted. At this stage, the country will consider stopping the intervention (MDA mainly) and start post-treatment surveillance.

# POST-TREATMENT SURVEILLANCE (PHASE 2)

# Step 1
Before initiating post-treatment surveillance, the health ministry establishes a national oversight committee to review programme data and validate that the criteria for interruption of transmission have been met. The committee should be independent from the national programme and comprise national and international experts, in accordance with practice in some countries in Africa (72). This committee can be embedded in any existing national committee for neglected tropical disease activities or onchocerciasis-specific matters.

# Step 2
During an annual meeting, the national oversight committee advises its respective health ministry to stop MDA according to the recommendations contained in these guidelines.  Th e 3-5 years of post-treatment surveillance can be extended in areas where lymphatic fi lariasis is coendemic and ongoing treatment for this disease may continue aft er the decision to stop MDA for onchocerciasis has been justifi ed (73). Furthermore, because the risk of reintroduction of infection exists it is likely that within a single country, or among WHO regions, interventions may stop at diff erent times. Th e two most relevant risk factors for reintroduction of infection are dispersal of infected fl ies, which may be transported via wind currents from endemic areas, and infected persons emigrating from endemic areas with active transmission to cleared areas. Referring to migration, other factors such as political instability in neighbouring countries may also increase the risk of recrudescence of parasite transmission, highlighting the need to address cross-border issues.
Under normal circumstances, post-treatment surveillance ends in 3-5 years. Th is supports the three recommendations on confi rmation of the interruption of transmission (see sections 8.2.1-8.2.3). Th is is based on O-150 PCR testing. In the case of insuffi cient or absence of fl ies, then the Ov-16 serology test should be used.

# Step 3
At the end of post-treatment surveillance, the independent national oversight committee reviews all the data assembled by the country programme, either countrywide or by individual foci or transmission zone. Once the committee has made its assessment and is satisfi ed that its fi ndings agree with the present verifi cation guidelines and that the risk of re-introduction or recrudescence of infection no longer exists, the secretariat of the national programme prepares a country report (dossier) according to the format in Annex 5 and contacts WHO to begin the verifi cation process.

# Step 4
Th e Ministry of Health submits the dossier to WHO through the Country Offi ce and the Region, in order to start the verifi cation process by an international verifi cation team.

# THE INTERNATIONAL VERIFICATION TEAM
Aft er receipt of the country report, WHO constitutes an international verifi cation team to independently assess the dossier on its behalf, including a country fi eld visit, and recommends to WHO that it either supports or rejects the national assessment of transmission interruption and therefore the national claim of interruption of transmission of the onchocercal parasite.