Onchocerciasis

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GUIDELINES FOR STOPPING MASS DRUG ADMINISTRATION AND VERIFYING ELIMINATION OF HUMAN ONCHOCERCIASIS

CRITERIA AND PROCEDURES

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Annex 1. Key questions

Key question 1

Which test (and at which threshold and time-point) can be used to demonstrate interruption of transmission of onchocerciasis (and therefore stop ivermectin administration)?

Test Threshold Time point O-150 PCR in black flies (head) < 1/1000 (0.1%) parous Peak transmission flies or < 1/2000 (0.05%) in season all flies assuming a 50% parous rate. A 95% CI will be used. PCR in black flies (body) Optional, but protocol calls Peak transmission for switching to testing season heads at first positive pool Ov-16 serology in children (< 5 y) < 0.1% (CI: 95%); has been operationalized in children < 10 y Ov-16 serology in children (< 10 y) < 0.1%. A 95% CI will be In same quarter of used. year as flies are collected Annual biting rate NA (taken into account in Poolscreen) Annual transmission potential < 20. A 95% CI will be used Calculated from flies collected during peak transmission season as above for PCR Skin snips (microscopic evaluation) NA Skin snips (PCR) Only done on those As soon as possible children who test OV-16 after serological positive results are known DEC test (patch) NA DEC test (oral, Mazzotti test) NA Ultrasonography NA Antigen testing (dipstick for urine or tears) NA CI, confidence interval; NA, not applicable; Ov-16, IgG4 antibodies against Onchocerca volvulus 16 antigen; PCR, polymerase chain reaction; y, years

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Key question 2

Which test (and at which threshold and time-point) can be used to demonstrate elimination of onchocerciasis?

Test Threshold Time point O-150 PCR in black flies (head) < 1/1000 (0.1%) parous Peak transmission flies or < 1/2000 (0.05%) in season 3 years after all flies assuming a 50% cessation of MDA parous rate. A 95% CI will be used. PCR in black flies (body) See above Ov-16 serology in children (< 5 y) See note above Ov-16 serology in children (< 10 y) < 0.1%. A 95% CI will be Optional but at end used of 3–5-year PTS Annual biting rate NA Annual transmission potential < 20. A 95% CI will be used Calculated from flies collected as above for PCR Skin snips (microscopic evaluation) NA Skin snips (PCR) NA unless serology tested Optional but at end as option; only seropositive of 3–5-year PTS children would be snipped and tested DEC test (patch) NA DEC test (oral, Mazzotti test) NA Ultrasonography NA Antigen testing (dipstick for urine or tears) NA

CI, confidence interval; DEC, diethylcarbamazine citrate; Ov-16, IgG4 antibodies against Onchocerca volvulus 16 antigen; PCR, polymerase chain reaction; y, years

Other specific questions for panel experts

 Does it make sense to combine all populations worldwide (no – this will likely proceed as regional elimination for some time) or should we split the populations, for example: ▬ the general population of countries/regions of the Americas? Yes, this has been done as the foci in the Americas are very distinct and separate from each other with the exception of the one focus in the Yanomami Indians that is a cross border focus with the Bolivarian Republic of Venezuela and Brazil. ▬ the general population of countries/regions of Africa? In Africa, because a number of the foci (or transmission zones) are cross border and there will be fly movement within the zone, it will be necessary to validate that transmission has been interrupted in one or more countries before that region can undergo verification. The risk of reintroduction is also higher in the Africa setting and this will have to be taken into consideration. ▬ other countries/regions?

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 Can you give us any other feedback on the use of tests for onchocerciasis that may be difficult for us to obtain from the literature? For example: ▬ on the “gold standard”? This will most likely have the key words of “PCR on black flies” or “PoolScreen”. For serology, it is likely that most will have the key word “Ov- 16”, but could also simply use “serology”. ▬ on the timing of tests? This will come mostly from those published studies documenting interruption of transmission in specific foci, or the one article Guide to detecting a potential recrudescence of onchocerciasis during the post-treatment surveillance period: the American paradigm.1 ▬ in relation to the history of use of a test or the background of its development?

 Do you have any other comments or feedback for us?

1 Guide to detecting a potential recrudescence of onchocerciasis during the post-treatment surveillance period: the American paradigm. Res Rep Trop Med. 2012;3:21–33. doi.org/10.2147/RRTM.S30482.

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Annex 2. Summary of the evidence

Table of contents

1 Executive Summary ...... 4 2 Background ...... 5 2.1 Purpose of this supporting document ...... 5 3 Methods ...... 5 3.1 Topic refinement and review protocol ...... 6 3.2 Key questions & analytic framework ...... 6 3.3 Search strategy ...... 8 3.4 Eligibility criteria ...... 8 3.4.1 Population / setting ...... 9 3.4.2 Intervention (diagnostic tests) ...... 9 3.4.3 Control (DTA reference standard or other test) ...... 9 3.4.4 Outcomes & study designs ...... 9 3.5 Study selection ...... 10 3.6 Data extraction & risk of bias assessment...... 11 3.7 Data synthesis...... 12 3.8 Quality of body of evidence ...... 12 4 Results ...... 13 4.1 Results of literature searches ...... 13 4.2 Key points ...... 14 4.2.1 Summary of available evidence ...... 14 4.2.2 Summary of findings ...... 15 4.3 Description of included studies ...... 16 4.4 Detailed Synthesis ...... 25 4.4.1 Evidence for clinical pathway (overarching KQ1 & KQ2) ...... 25 4.4.2 Evidence from diagnostic studies for KQ1 & KQ2 ...... 31 4.5 GRADE profiles ...... 35 4.5.1 Evidence for clinical pathway (overarching KQ1 & KQ2) ...... 35 4.5.2 O-150 PCR of skin snips (index test) vs. skin snip microscopy (reference test) ...... 37 4.5.3 Ov-16 serology (index test) vs. skin snip microscopy (reference test) ...... 38 4.5.4 O-150 PCR [Poolscreen] (index test) vs. dissection of black flies (reference test) ...... 39 5 References ...... 40 6 Appendix A: Search Strategy ...... 44 7 Appendix B: Excluded studies at fulltext review level ...... 49 7.1 Ineligible outcome (n=51) ...... 49 7.2 Ineligible population(s) (n=4) ...... 52 7.3 Ineligible publication type (n=4) ...... 52 7.4 Ineligible or no diagnostic test (n=65) ...... 53 7.5 Ineligible comparisons (n=2) ...... 56 7.6 Study not obtainable (n=4) ...... 57 8 Appendix C: Data extraction sheets ...... 58 8.1 Evidence for clinical pathway (overarching KQ1 & KQ2) ...... 58 −

8.1.1 Traore 2012 ...... 58 8.1.2 Cruz-Ortiz 2012 ...... 60 8.2 Skin snip PCR (index test) vs. skin snip microscopy (reference test) ...... 62 8.2.1 Fink 2010 ...... 62 8.2.2 Boatin 2002 ...... 64 8.2.3 Pischke 2002 ...... 67 8.2.4 Vincent 2000 ...... 69 8.2.5 Toe 1998 ...... 71 8.2.6 Fischer 1996 ...... 73 8.2.7 Zimmerman 1994 ...... 75 8.3 Ov-16-serology (index test) vs. skin snip microscopy (reference test) [or different Ov-16- serology methods compared to each other] ...... 77 8.3.1 Golden 2013 ...... 77 8.3.2 Lipner 2006 ...... 79 8.3.3 Rodriguez-Perez 2003...... 80 8.3.4 Weil 2000 ...... 82 8.4 PCR in black flies (index test) vs. black fly dissection (reference test) [or different DNA extraction methods compared to each other] ...... 84 8.4.1 Rodriguez-Perez 2013...... 84 8.4.2 Gopal 2012 ...... 86 8.4.3 Rodriguez-Perez 1999...... 88 8.4.4 Yameogo 1999 ...... 89

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Table index Table 1: Abbreviations used in the report ...... 5 Table 2: Eligibility criteria ...... 9 Table 3: Ranking of the relative importance of the DTA outcomes by the panel ...... 10 Table 4: Characteristics of included studies ...... 17 Table 5: Risk of bias of observational study ...... 22 Table 6: Risk of bias and applicability assessment of diagnostic studies ...... 23 Table 7: Observational studies investigating operationalization of diagnostic tests ...... 27

Figure index Figure 1: Analytic framework: The clinical pathway and role of tests and relevant outcomes in relation to subsequent decisions and actions ...... 7 Figure 2: Disposition of the literature ...... 13 Figure 3: DTA values of O-150 PCR of skin snips (index test) vs. skin snip microscopy (reference test) ...... 32 Figure 4: DTA values of Ov-16 serology (index test) vs. skin snip microscopy (reference test) ...... 33

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1 Executive Summary

This document provides a summary of the evidence to support the task of the WHO guideline panel in updating the guideline “Verification of elimination of human onchocerciasis: criteria and procedures”. We based this evidence summary on a systematic review of the literature and applied the GRADE (Grading of Recommendations Assessment, Development and Evaluation) approach to synthesizing and rating the quality of the available evidence.

Human onchocerciasis, also known as river blindness, is a disease caused by infection with the parasite Onchocerca volvulus and leads to skin disease and blindness. Onchocerciasis is a vector- borne disease, where human beings are the only natural vertebrate host. The disease was previously endemic in many countries; however great progress has been made in interrupting transmission of Onchocerca volvulus. The aim of this systematic review was to investigate the validity and reliability of different tests to confirm interruption of transmission and at a later time point, elimination of the disease.

The evidence we have summarized should answer the question “which diagnostic tests or combination of tests can validly and reliably demonstrate the interruption of transmission of Onchocerca volvulus or elimination of the disease”. This is a complex question to answer because it involves elements of diagnostic test accuracy (DTA) and prognosis. Therefore we broke the concepts down into an analytic framework, which – using terminology from DTA research - we named the “clinical pathway”. Thus, we present the evidence and its relation to this framework/pathway separately – that for the entire chain, and that for the single DTA portions.

Overall, there was little direct comparative scientific evidence on the use of different tests for verification of elimination of human onchocerciasis. For example, we located multiple studies describing the operationalization of WHO criteria, but none with comparisons of approaches. The best available evidence was provided by two prospective observational studies, which provided data from interruption of transmission leading to cessation of treatment, towards elimination of onchocerciasis in terms of post-treatment surveillance data.

Similarly, we located multiple DTA studies comparing various tests, but these were generally done in populations with high prevalence of onchocerciasis (which does not reflect the situation close to interruption of transmission with a low prevalence of onchocerciasis). This 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 DTA studies were performed to assess test accuracy, but not to demonstrate interruption of transmission nor elimination.

In terms of demonstrating interruption of transmission or elimination of onchocerciasis, the quality of evidence for entomological assessment through O-150 PCR (Poolscreen) of black flies was rated high, whereas for epidemiological assessments such as Ov-16 serology, skin snip microscopy and ocular morbidity (slit lamp) it was low.

In terms of comparative diagnostic test accuracy and not directly connected with demonstration of interruption of transmission or elimination of onchocerciasis, O-150 PCR of skin snips and Ov-16

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serology show values at least as good as skin snip microscopy. One diagnostic study strongly suggests that in populations with low disease prevalence skin snip microscopy performs worse than the other tests. Therefore, in this context, O-150 PCR of skin snips and Ov-16 serology probably perform better than skin snip microscopy.

2 Background

2.1 Purpose of this supporting document

This report was commissioned by the World Health Organization (WHO) Department of Control of Neglected Tropical Diseases (NTD) in order to support their revision of the guideline entitled: “Verification of elimination of human onchocerciasis: criteria and procedures”. The aim of this report is to support the guideline panel in revising the guidelines by providing methodological summaries of the current evidence following the GRADE approach (Grading of Recommendations Assessment, Development and Evaluation). This report will include detailed methods and results sections without explanatory information in the form of an extended introduction or discussion of the results.

The following abbreviations will be used through this report:

Table 1: Abbreviations used in the report

ATP annual transmission potential DTA diagnostic test accuracy ELISA enzyme-linked immunosorbent assay FN false negative FP false positive GRADE Grading of Recommendations Assessment, Development and Evaluation KQ key question MDA mass drug administration MF microfilariae NPV negative predictive value Ov-16 Onchocerca volvulus antigen (signal peptide of 16 amino acids) PCR polymerase chain reaction PPV positive predictive value QoE quality of evidence

3 Methods

The PROSPERO (International Prospective Register of Systematic Reviews: http://www.crd.york.ac.uk/prospero) registration number of this review is: CRD42014010200

The main sections in this chapter reflect the elements of the protocol established for the review. All methods and analyses were determined a priori.

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3.1 Topic refinement and review protocol

This topic was nominated by the WHO and we, a team of methodologists with expertise in systematic reviews and the GRADE approach to guideline development at the Danube University Krems, designed the protocol prospectively in consultation with the guideline panel members.

In particular, we consulted the guideline panel via e-mail to provide input on:

1. Formulating the PICO-TS question (population, intervention, comparisons, outcomes, timing, setting) and key questions (KQs) 2. The relevant outcomes, including rating their relative importance 3. Relevant literature sources and databases 4. Tests currently in field use and therefore of practical relevance for the guideline

3.2 Key questions & analytic framework

The key questions of this systematic review are:

KQ1: In a population at risk for onchocerciasis, which diagnostic tests or combination of tests can validly and reliably demonstrate the interruption of transmission of Onchocerca volvulus after initiation of MDA (mass drug administration)?

a. What is the best point in time after initiation of MDA to apply these tests?

b. What are the best cut-off points to balance sensitivity and specificity?

KQ2: Can these tests reliably predict the elimination of human onchocerciasis in a population at risk after the cessation of MDA?

a. What is the best point in time after cessation of MDA to apply these tests?

b. What are the best cut-off points to achieve an optimal predictive validity?

The analytic framework and clinical pathway for this review is presented in Figure 1.

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Figure 1: Analytic framework: The clinical pathway and role of tests and relevant outcomes in relation to subsequent decisions and actions

ATP= annual transmission potential; FN= false negatives; FP= false positives; KQ= key question; MDA= mass drug administration; TN= true negatives; TP= true positives.

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3.3 Search strategy

To identify articles relevant to each key question, we searched Pubmed, EMBASE, the Cochrane Library, LILACS, and Africa-Wide Information up to May 2014 using analogous search terms (Appendix A presents the full search strategy.) We used a combination of medical subject headings (MeSH) and title and abstract keywords such as “onchocerciasis”, “Onchocerca volvulus”, “river blindness”, or “Robles disease”, focusing on terms to describe the relevant population. An experienced information scientist ran the searches. We also performed citation tracking and hand searches based on key literature. No language or publication date restrictions were used.

In addition to electronic searches, we manually searched reference lists of pertinent reviews, included trials, and background articles on this topic to identify any relevant citations that our searches might have missed and several guideline panel members provided further relevant references. We imported all citations into an EndNote®X5 electronic database.

3.4 Eligibility criteria

We specified our inclusion and exclusion criteria based on the population, intervention, comparators, outcomes, timing, and settings (PICO-TS) identified through the topic refinement process. We included original scientific publications (no letters to the editor, editorials, or narrative reviews) of tests to determine the interruption of transmission of Onchocerca volvulus or the elimination of human onchocerciasis in a region/population. In addition, we included primary research on the diagnostic test accuracy characteristics of tests (including harms, costs, and inconclusive results) used to evaluate the prevalence of Onchocerca volvulus in humans and black flies.

Eligibility criteria are summarized in Table 2.

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Table 2: Eligibility criteria PICO-TS Details  Any population at risk for onchocerciasis Population  Black flies in endemic regions for entomological evaluations  Skin snips (O-150 PCR or microscopy)  Ov-16 serology Intervention /  O-150 PCR (Poolscreen) in black flies Diagnostic intervention (test)  Dissection of black flies  Slit lamp (or other tests diagnosing ocular morbidity)  a known and validated reference standard or a comparison of Control two versions of a similar test  Recrudescence of onchocerciasis Outcomes  Diagnostic test accuracy (DTA)  Treatment (MDA) phase [KQ1] Timing  Post-treatment surveillance (PTS) phase [KQ2] Setting  Hypo-, meso- or hyperendemic regions with onchocerciasis Study designs  No restrictions on study designs; but only primary research Abbreviations: KQ = key question; L3 = infective stage of nematode larvae (in contrast to immature developing stages L1 and L2); MDA = mass drug administration; MF = microfilariae; O-150 PCR = polymerase chain reaction of O-150 DNA sequence; Ov-16 = Onchocerca volvulus antigen (signal peptide of 16 amino acids).

3.4.1 Population / setting Eligible populations for inclusion in our review were general populations at risk for onchocerciasis (i.e. persons living in hypo-, meso- and hyperendemic foci) as well as black flies in endemic regions. Because black flies (Simulium species) are the vector of Onchocerca volvulus transmission, they were considered a population when investigating entomological tests.

3.4.2 Intervention (diagnostic tests) We searched for studies of regions/populations where two methods of testing for the elimination of onchocerciasis had been implemented and compared. In addition, we searched for diagnostic studies of tests meeting the inclusion criteria (see Table 2).

3.4.3 Control (DTA reference standard or other test) We included evidence from studies with a known and validated reference standard or comparing two versions of a similar test.

3.4.4 Outcomes & study designs The key question of this review is to determine which diagnostic tests or combination of tests can validly and reliably demonstrate the interruption of transmission of Onchocerca volvulus after initiation of MDA (KQ1), or elimination of onchocerciasis after the cessation of MDA (KQ2). In this case the main outcome is that there is no recrudescence of onchocerciasis; for example, that a

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negative test at the time point of KQ1 predicts a negative test at the time point of KQ2. The negative test at the timepoint of KQ2 should be confirmed at a later time point by another negative test to verify elimination of onchocerciasis. Likewise, continued testing after a period of observation would be required to verify the validity of a certain test for determining elimination of onchocerciasis (see Figure 1). In this scenario the ideal study would compare two methods of testing for onchocerciasis in a population eligible for cessation of treatment and confirmation and verification of elimination.

In addition, we also sought primary evidence for DTA outcomes from diagnostic studies which provide limited information on long-term outcomes. In contrast, these studies directly compare two tests (in the same population or with a case-control design using known uninfected individuals as negative controls) and can therefore provide evidence on outcomes relevant for DTA decision making.

When selecting relevant outcomes for the diagnostic studies, we employed an iterative process where the experts ranked endpoints that are essential for decision making in practice. The panel rated the outcomes in terms of importance for decision making on a 9-point scale according to standard GRADE methodology (ratings of 7-9 are deemed critical for decision making, 4-6 important, and 1-3 not important).

The selection and ranking of the relative importance of the DTA outcomes by the panel are shown in Table 3:

Table 3: Ranking of the relative importance of the DTA outcomes by the panel Outcome Rating average for KQ1 Rating average for KQ2 True positive results 7.75 7.75 True negative results 6.33 6.67 False positive results 8.75 8.75 False negative results 4.67 4.33 Inconclusive results 4.25 3.25 (regardless of onchocerciasis status) Complications of test (harms) 3.25 3.00 Costs 4.25 4.00 Prioritization scale from 1 (least important) to 9 (most important); 1-3 = limited importance for decision making (not included in evidence profile), 4-6 = important, but not critical for decision making (included in evidence profile), 7-9 = critical for decision making (included in evidence profile). KQ = key question

In the course of this systematic review only critical or important ranked DTA outcomes were taken into account.

3.5 Study selection

We dually and independently reviewed all identified abstracts and fulltexts. After dually screening abstracts, two trained team members independently reviewed each fulltext article for inclusion or exclusion based on the eligibility criteria described above. If both reviewers agreed that a study did not meet the eligibility criteria, the study was excluded. If the reviewers disagreed, conflicts were resolved by discussion and consensus or by consulting a third member of the review team. All results

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were tracked in an EndNote®X5 database. We recorded the reason that each excluded fulltext publication did not satisfy the eligibility criteria. A bibliography of excluded studies and a reason for exclusion is presented in Appendix B.

3.6 Data extraction & risk of bias assessment

We extracted information regarding the population, setting and tests of all included studies into a structured Microsoft Word template. The template was piloted and modified after testing. In addition, we extracted data comparing the tests into a 2x2 table in Microsoft Excel and calculated DTA parameters, including sensitivity, specificity, positive (PPV) and negative predictive value (NPV).

Likewise, the risk of bias of the included studies was determined using pre-designed forms in Microsoft Excel, which were first piloted and modified after testing. Risk of bias assessment of observational studies are based on a publication from Deeks et al.1 The risk of bias forms for the diagnostic studies were developed with the QUADAS-2 (Quality Assessment of Diagnostic Accuracy Studies) tool for quality assessment of diagnostic studies,2 which includes an evaluation of the risk of bias as well as the applicability of diagnostic tests. It is important to note that applicability is part of the indirectness domain in the GRADE approach.3 Applicability/indirectness focuses on whether the results of studies are applicable to other populations than the study population (e.g. if the results of a study conducted in the Americas is applicable to an African region). In the case of onchocerciasis, there are substantial differences between populations in different regions, countries and continents - especially concerning the risk of recrudescence of onchocerciasis. In this report, the term “applicability” used in the QUADAS-2 tool to rate individual studies equates to the term “indirectness” used in the GRADE approach to determine the overall quality of the body of evidence. An overview of the signaling questions we used to rate risk of bias and applicability of the included studies are provided in Table 5 and Table 6.

We rated the risk of bias for each included study as low, unclear, or high risk of bias. In general terms, results of a study with low risk of bias are considered to be valid. Unclear risk of bias implies some confidence that the results represent true effects; the study is susceptible to some bias, but the problems are not sufficient to invalidate the results (i.e., no flaw is likely to cause major bias). A study with high risk of bias has significant methodological flaws (e.g., stemming from serious errors in design or analysis) that may invalidate its results. Ratings of risk of bias are not comparable across study designs. That is, a low risk of bias non-randomized observational study is not necessarily equal to a low risk of bias diagnostic study. We took limitations of certain study designs into consideration when we graded the overall quality of the evidence.

All data extraction and risk of bias assessments were dually and independently reviewed by a second team member. Any conflicts were resolved by discussion and agreement or by consulting a third team member. The detailed assessments of risk of bias and applicability judgements are provided in Table 5 and Table 6, data extraction forms in Appendix C.

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3.7 Data synthesis

Because of the heterogeneity of the included studies we were not able to conduct meta-analyses of results. We summarized the results qualitatively and provide ranges of results from the included studies. If possible, we present results graphically.

3.8 Quality of body of evidence

For the clinical pathway in this systematic review, no standardized or evaluated method is currently available. We followed proposed methodological solutions by Reitsma et al.4 and Schünemann et al.5 As the same diagnostic tests should demonstrate interruption of transmission and elimination of onchocerciasis at different time points and under varying circumstances (MDA, vector control measures, other factors influencing risk of recrudescence like political instability, migration etc.), overall evidence for the entire clinical pathway is built as a chain of linked bodies of evidence.

For the comparisons of different diagnostic tests in terms of DTA performance, the quality of the body of evidence (QoE) was assessed according to the methods proposed by the GRADE working group.6-8 During the guideline panel meeting (8-9 January 2015) we agreed on using the term “certainty of evidence” in the recommendation statements and decision tables. In this case “certainty of evidence” is equivalent terminology for the “quality of evidence” construct and we use the terms interchangeably.

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4 Results

In this section we will provide the results of the literature searches, the review of abstracts and full texts, and a summary of the included studies. The results answering the key questions of this review will be presented in subchapters based on the available direct comparisons.

4.1 Results of literature searches

We identified 3,011 citations through electronic literature searches and additional sources and screened 2,978 records, after removal of duplicates. Figure 2 documents the disposition of the 147 articles retrieved for fulltext review for this report. Overall, we included 17 studies, and excluded 130 articles for various reasons. Articles excluded during fulltext review are listed in Appendix B with reasons for exclusions.

Figure 2: Disposition of the literature

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4.2 Key points

In the following section, we summarize key points of our findings. First, we present an overview of the nature of the available evidence and its limitations. Second, we present a summary of the findings of the included studies with quality of evidence ratings.

4.2.1 Summary of available evidence

 We could not find any evidence that directly addressed our questions of interest, i.e. the comparative validity and reliability of tests at different time points to determine the interruption and elimination of Onchocerca volvulus. The best available evidence are two prospective observational studies that provide evidence for almost the entire pathway for skin snips, Ov-16 serology, and ocular morbidity in humans and O-150 PCR (Poolscreen) in black flies; tests were conducted at multiple time points, but not compared to other tests.9,10

 We could not find any evidence reporting test implementation in a comparative manner. Apart from the two prospective observational studies9,10 we are aware of case studies where tests were used to show interruption of transmission, but post-treatment surveillance data to confirm test results are missing or have not (yet) been published in a form that we could retrieve.

 Fifteen diagnostic studies provided general information on diagnostic test accuracy (DTA) of various epidemiological and entomological tests compared to each other, in respect to diagnosing onchocerciasis in humans11-21 or infectivity in black flies.22-25

 Many studies had methodological limitations; three out of seventeen studies received ratings of high risk of bias12,13,17, eleven of unclear risk of bias9,10,14-16,18-20,22,24,25and three studies ratings of low risk of bias11,21,23. Risk of bias for the DTA studies was mainly increased by case-control design or by the fact that skin snip microscopy could be an imperfect reference test, especially in settings with low onchocerciasis prevalence due to MDA.

 Applicability concerns were low for all four diagnostic studies investigating O-150 PCR (Poolscreen) in black flies.22-25

 Applicability concerns regarding diagnostic studies in respect to diagnosing onchocerciasis in humans were unclear in seven out of eleven studies14,15,17-20, low in two11,16 and high in two other studies12,13. Applicability concerns were mainly increased by the fact that investigated populations had often high microfilarial prevalence, not reflecting populations with low microfilarial prevalence due to MDA.

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4.2.2 Summary of findings

 We used two distinct bodies of evidence for rating quality of evidence for each comparison – the observational evidence and the diagnostic study evidence.

 Based on two prospective observational studies, 9,10 skin snip microscopy, Ov-16 serology, ocular morbidity, and O-150 PCR (Poolscreen) in black flies reliably demonstrated the interruption of transmission of Onchocerca volvulus after initiation of MDA (KQ1) and elimination of onchocerciasis after the cessation of MDA (KQ2) [low QoE; except O-150 PCR (Poolscreen) in black flies with high QoE].

 Based on seven diagnostic studies,12,15,17-20 O-150 PCR of skin snips has DTA values at least as accurate as those of skin snip microscopy; O-150 PCR of skin snips might even have better sensitivity than skin snip microscopy, especially in settings with low onchocerciasis prevalence due to MDA* [low QoE].

 Based on four diagnostic studies,11,13,14,16 Ov-16 serology has DTA values at least as accurate as those of skin snip microscopy; Ov-16 serology may have better sensitivity than skin snip microscopy, especially in settings with low onchocerciasis prevalence due to MDA, whereas in settings with high onchocerciasis prevalence the performance is comparable* [low QoE].

 Based on four diagnostic studies,22-25 O-150 PCR (Poolscreen) in black flies was equally successful in detecting infective Onchocerca volvulus larvae as dissection of black flies [moderate QoE].

 Based on three diagnostic studies,22,23,25 initial costs for O-150 PCR (Poolscreen) in black flies are higher compared to costs of dissection of black flies, but subsequent costs are lower. In addition, increasing pool sizes through newer DNA extraction methods, while maintaining same sensitivity values, can lower the subsequent costs further [moderate QoE].

 We could not find any evidence on the best time points and cut-off points. Based on one prospective observational study, the best time point to demonstrate the interruption of transmission of Onchocerca volvulus after initiation of MDA (KQ1) differs between different foci.

* limitations of the study design of the included diagnostic studies, which used skin snip microscopy as the reference test, do not allow us to conclude that the comparator index test has better DTA characteristics, only that it is at least equally as good. For skin snip O-150 PCR vs. skin snip microscopy this was shown in one study and in unpublished data; for Ov-16 serology vs. skin snip microscopy only in unpublished data. Calculations of DTA values which are based on inverting index test and reference test are provided in Appendix C.

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4.3 Description of included studies

The included studies were conducted in Africa or the Americas, for example in provinces of Uganda or Ecuador. We found two prospective observational studies9,10 providing information on the clinical pathway and 15 diagnostic studies11-25 providing information on parts of the chain of linked bodies of evidence.

We located multiple publications that describe the operationalization of the previous (2001) WHO criteria for verification of elimination of human onchocerciasis. However, many of these studies did not provide longitudinal data or any comparison; therefore they did not fulfill the formal inclusion requirements for this report. These studies describe parts of the process towards verification of elimination in Uganda,29 ,31 Kenya,32 Ghana/Togo,33 Central African Republic,34 Cameroon,35 Nigeria,36- 38 Mali,39 Sudan,40 Guatemala,41,42 Ecuador,43,44 Mexico; 45-49 and Venezuela.50

We did not find any single study that directly addressed our questions of interest, i.e. the comparative validity and reliability of tests at different time points to determine the interruption and elimination of onchocerciasis. However, we found several studies that cover parts of the clinical pathway, for example, diagnostic studies on the diagnostic test accuracy (DTA) of different tests compared to one another or prospective observational studies without direct comparisons of tests. A full overview of the included studies is provided in Table 4, full data extraction sheets in Appendix C. Risk of bias and applicability ratings are shown in detail in Table 5 and Table 6.

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Table 4: Characteristics of included studies

Author, Year of data Number of Location Risk of Year of Study design collection / included persons / (continent; country; Index test Reference test bias publication testing black flies region; city/village)

Evidence for clinical pathway (overarching KQ1 & KQ2) Epidemiological evaluation: 2 skin snips from the KQ1: after 14-16 Epidemiological iliac crests; years of MDA evaluation: 29,753 microscopically Africa; Mali, Senegal; Prospective (2006) persons examined Traore et al., River Gambia focus, observational NA unclear 20129 River Faleme focus, study KQ2: 2-5 years Entomological Entomological River Bakoye focus after last MDA evaluation: evaluation: (2009-2011) 492,600 black flies O-150 PCR on black fly heads; Poolscreen with screening pools of 300 flies Epidemiological Epidemiological evaluation: 365 evaluation: KQ1: after 12 people Ov-16 serology and years of MDA (ophthalmological Americas; ophthalmological Prospective (2008) evaluation) and Cruz-Ortiz et al., Guatemala; evaluation observational 3118 children (6- NA unclear 201210 Huehuetenango study KQ2: 2-3 years 12y) focus Entomological after last MDA evaluation: (2010-2011) Entomological O-150 PCR Poolscreen evaluation: on black flies 266,224 black flies Skin snip O-150 PCR (index test) vs. skin snip microscopy (reference test) Fink et al., Diagnostic study April 1999- 129 persons Americas; US; Real-time O-150 PCR on Skin snip high

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201012 December 2009 (immigrants, Maryland, Bethesda skin snips microscopy expatriates or travelers) Skin snip microscopy Boatin et al., (Statistical models Diagnostic study February 2000 313 persons Africa; Guinea O-150 PCR on skin snips low 200221 used to calculate different reference standard values) Skin snips: 1993 and 1995 O-150 PCR (ELISA or Pischke et al., Africa; Western Skin snip Diagnostic study 127 persons rapid detection test unclear 200215 Uganda microscopy O-150 PCR: strips) on skin snips 2000 and 2001 Skin snip Africa; Cameroon; microscopy or South-West nodules (clinical province; Kokaka, Vincent et al., O-150 PCR (ELISA) of diagnosis) Diagnostic study NA 29 persons Kumba, Kurume, high 200017 skin snips (36/38 positive MF Bolo, Boa-Bukundu, in skin snips, 2 Bombanda, clinical diagnosis Bomanda only) 99 persons (children and Toé et al., Africa; Côte d’Ivoire; Skin snip Diagnostic study NA young adults) plus O-150 PCR of skin snips unclear 199818 Gnankoradji microscopy 42 negative controls from US) Fischer et al., November - Africa; Western Skin snip Diagnostic study 227 persons O-150 PCR of skin snips unclear 199619 December 1993 Uganda; Kigoyera microscopy

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province Americas; North- Skin snip Zimmerman et Diagnostic study NA 191 persons western Ecuador; O-150 PCR of skin snips microscopy unclear al., 199420 Esmeraldas Province (or non-exposure) Ov-16-serology (index test) vs. skin snip microscopy (reference test) [or different Ov-16 serology methods compared to each other] Africa; Ghana, Ov-16 serology (lateral- Liberia, Mali flow-based assay with sera; rapid tool) Americas; Golden et al., 449 serum and Confirmed positive Diagnostic study NA Guatemala, Ecuador, Ov-16 serology (lateral- low 201311 plasma samples microfilaria status US (travelers) flow-based assay with whole blood; rapid tool) Others: Cook islands, India OV-16 serology (ELISA) Africa; Burkina Faso; Linoguin, Wayen, Badone, Koumoon, Lipner et al., late 1999-early 1,511 persons (≥2 Ov-16 serology (rapid- Skin snip Diagnostic study Salimbor high 200613 2000 years of age) format card test; lgG4) microscopy

Africa; Côte d’Ivoire; Mafia, Zakpaberi Ov-16 serology (rapid-format card Rodriguez- Americas; Mexico; Ov-16 serology (ELISA; test; Perez et al., Diagnostic study April 2001 258 persons Southern Chiapas; mix of OvMBP7, unclear immunochromatog 200314 Las Golondrinas OvMBP11, OvMBP16) raphic test, ICT; lgG4) Weil et al., Africa; Ghana, Ov-16 serology (rapid- Skin snip Diagnostic study NA 228 serum samples unclear 200016 Cameroon, Sierra format antibody card (microscopy)

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Leone, others test; immunochromatographi Others (if Americas; Ecuador, c test, ICT; lgG4) microscopy was Guatemala negative, then O- 150 PCR, clinical symptoms, Mazzotti test) O-150 PCR in black flies (index test) vs. black fly dissection (reference test) [or different DNA extraction methods compared to each other] O-150 PCR Rodriguez- Americas; Mexico; (high-throughput O-150 PCR February-April Perez et al, Diagnostic study 13,400 black flies Chiapas; Las automated DNA- (phenol chloroform unclear 1994 201322 Golondrinas isolation; silica-coated DNA extraction) paramagnetic beads) Americas; Mexico; O-150 PCR (Poolscreen) O-150 PCR Chiapas; Jose Maria (DNA extractions with (Poolscreen) Morelos y Pavon Gopal et al., black flies specific oligonucleotide (DNA extraction Diagnostic study NA low 201223 (n = NA) based magnetic bead with silica-coated Africa; Burkina Faso; capture; pool size = up beads; pool size = Region des Cascades; to 200) 50-100) Bodajugu, Sakora 21,322 black flies Rodriguez- Americas; Mexico; (10,550 for O-150 Perez et al., Diagnostic study NA Chiapas; Las O-150 PCR Fly dissection unclear PCR and 10,772 for 199924 Golondrinas dissection) Africa; Ghana (Bielikpong on the Yameogo et al., Diagnostic study NA 25,548 black flies Kulpawn River), O-150 PCR (Poolscreen) Fly dissection unclear 199925 Burkina Faso (Nabere on the

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Bougouriba River and Leraba Pont on the Leraba River), Mali (Madina Diassa and N’zana, both on the Baoule River) Abbreviations: DNA = deoxyribonucleic acid; KQ = key question; MDA = mass drug administration; MF = microfilariae; NA = not available; Ov-16 = Onchocerca volvulus antigen (signal peptide of 16 amino acids); PCR = polymerase chain reaction.

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Table 5: Risk of bias of observational study

Were possible Were con- partici- founding pants at factors the dif- Were identified ferent methods and ade- Were any Were time equally Were quately partici- differ- points applied Was the Was an differ- ac- pants ences recruited at all interven- attempt ences counted Did a lot who Were between Is the from the time tion made to between (through of started outcome sub- study same points meas- blind the areas/foc study persons the trial measures groups design source (incl/excl ured outcome i taken design or refuse to excluded equal, ade- prospec- popula- criteria accu- assessors into statistical partici- from the valid and quately OVERALL Study tive? tion? etc.)? rately? ? account? analysis)? pate? analysis? reliable? tested? RATING

Traore 20129           NA 

Cruz- Ortiz           NA  201210 Legend:  = low risk of bias;  = unclear risk of bias;  = high risk of bias; NA = not applicable (no subgroups)

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Table 6: Risk of bias and applicability assessment of diagnostic studies Applicability Risk of bias Applicability concerns Risk of bias concerns Study Patient Index Reference Flow and Patient Index Reference OVERALL RATINGS selectiona testb standardc timingd selectione testf standardg Rodriguez-Perez 201322          Golden 201311          Gopal 201323          Fink 201112          Lipner 200613          Rodriguez-Perez 200314         

Boatin 200221          Pischke 200215          Weil 200016          Vincent 200017          Rodriguez-Perez 199924          Yameogo 199925          Toe 199818          Fischer 199619          Zimmermann 199420          Legend:  = low;  = unclear;  = high

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a = could the selection of patients have introduced bias?; b = could the conduct or interpretation of the index test have introduced bias?; c = could the reference standard, its conduct, or its interpretation have introduced bias?; d = could the patient flow and timing have introduced bias?; e = are there concerns that the included patients do not match the review question?; f = are there concerns that the index test, its conduct, or its interpretation differ from the review question?; g = are there concerns that the target condition as defined by the reference standard does not match the review question?

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4.4 Detailed Synthesis

4.4.1 Evidence for clinical pathway (overarching KQ1 & KQ2) We found two observational studies that provide evidence across almost the entire clinical pathway,9,10 Although these two observational studies do not present a comparison of two tests, they nonetheless represent the best available evidence for the clinical pathway, incorporating both KQ1 and KQ2. One overarching study is a prospective observational study including three endemic foci in Mali and Senegal and examined 29,753 persons and 492,600 black flies for the presence of Onchocerca volvulus at different time points.9 The other study is a prospective observational study conducted in Guatemala and examined 365 people ophthalmologically and 3118 children (between six and 12 years old) serologically. Furthermore, 266,224 black flies were analysed.10 The studies present data collected at the end of the treatment phase which led to cessation of MDA, as well as during the post-treatment surveillance which confirmed elimination phases. These are the only studies that specifically incorporated analyses at multiple time points and that used provisional test cut-off points in their analyses; both of these were sub-questions of KQ1 and KQ2.

In addition, we located multiple studies describing the application of tests to determine interruption of transmission of Onchcerca volvulus or elimination of human onchocerciasis in Uganda,29 ,31 Kenya,32 Ghana/Togo,33 Central African Republic,34 Cameroon,35 Nigeria,36-38 Mali,39 Sudan,40 Guatemala,41,42 Ecuador,44 Mexico, 45-48,51 and Venezuela.50 None of these studies fulfilled our inclusion criteria and were therefore not formally included in the evidence synthesis; however we present them in this report in Table 7.

In the included observational studies, epidemiological evaluation was made through skin snip microscopy (in persons above 1 year of age), Ov-16 serology (in children from 6 to 12 years of age), slit lamp, and entomological evaluation through O-150 PCR (Poolscreen) on black flies. Skin snip microscopy and O-150 PCR (Poolscreen) reliably demonstrated the interruption of transmission of Onchocerca volvulus after 1210 or 14-169 years of MDA (KQ1) and elimination of onchocerciasis 2-310 or 2-59 years after the cessation of MDA (KQ2). The quality of evidence, however, is low for skin snip microscopy, ocular morbidity, and Ov-16 serology due to indirectness and risk of bias or imprecision of evidence. The quality of evidence is high for O-150 PCR (Poolscreen) of black flies. For O-150 PCR (Poolscreen) of black flies, the quality of evidence is higher than for the other tests, as it is based on two observational studies from two different countries, which leads to a better applicability and less concerns regarding indirectness of evidence. There were no other serious concerns about the quality of evidence for O-150 PCR (Poolscreen) of black flies.

Specifically, the main weakness of these studies is the lack of a comparison group and some inconsistencies in the number of villages and individuals, which varied between the different time points. Similarly, one study addressing interruption of transmission and elimination through skin was conducted in three African foci; the applicability of results to other regions and countries in Africa and other regions of the world may be limited. Indeed, within this study the three foci received differing time plans for MDA cessation because one focus (River Faleme) did not meet provisional criteria for stopping treatment. On the other hand, results for Ov-16 serology obtained from foci in

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the Americas may not be applicable to other regions, countries, or continents. Furthermore, in that study Ov-16 serology and ocular morbidity showing interruption of transmission were verified during post-treatment surveillance with O-150 PCR testing in black flies and by another serological survey; therefore we also rated down the quality of evidence for risk of bias. A summary of the results of these studies is presented in a GRADE evidence profile (GRADE profile 1) and detailed results of risk of bias assessment is shown in Table 5.

Although WHO reports that certification of elimination has been achieved in several countries, we could not locate any reports where tests were implemented in a comparative manner; nor could we find published follow-up data from certification onwards in order to determine the reliability of that testing regime. Apart from the two observational studies included, which presented post-treatment surveillance data, we could not locate any other publically available reports presenting such data in order to assess if tests reliably demonstrated interruption of transmission of Onchocerca volvulus.

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Table 7: Observational studies investigating operationalization of diagnostic tests Country Focus Ref IDs Tests used Onchocerciasis status Onchocerciasis status according to literature according to WHO (various criteria used)§ (#9168 WHO 2013₸) Uganda Different foci (n=11) Oguttu, 201426 Serology Ov-16 in children Interruption (2009) - Uganda Itwara Lakwo, 201327 Skin snip microscopy Interruption (2010) - Skin snip PCR Elimination stated Serology Ov-16 in children (2010), because of vector elimination (2001) Uganda Kashoya-Kitomi Katabarwa, Skin snip microscopy Ongoing transmission - 2010*28,30 Fly assessment (2009) Uganda Kigoyera, Kicece Ndyomugyenyi, Skin snip microscopy Ongoing transmission - 200429 (2003)

Uganda Mount Elgon Katabarwa, 201430 Skin snip microscopy Interruption (2011) - Katabarwa, 2011*52 Serology Ov-16 in children Richards, 2009*53 Fly assessment Uganda Wadelai Katabarwa, 201231 Skin snip microscopy Interruption (2009) - Serology Ov-16 in children Fly assessment Kenya Kodera, Ngoina, Roberts, 196732 Fly assessment Vector elimination - Kakamega-Kaimosi Skin snips (1955) Eye diagnostics Ghana, Dayi River Cheke, 200833 Fly dissection Vector elimination - Togo Gban-Houa (Wawa) (1988) River Central Gami Yaya, 2014 34 Skin snip microscopy Ongoing transmission - African Eye diagnostics (2010) Republic Fly dissection Cameroon North Region Katabarwa, 201154 Skin snip microscopy Ongoing transmission -

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Katabarwa, 2010*55 Eye diagnostics (slit lamp) (2010) Fly dissection Cameroon West Region Katabarwa, 201335 Skin snip microscopy Ongoing transmission - Fly dissection (2012) Nigeria Plateu and Nasarwa Evans, 2013*36 Skin snip microscopy Interruption (2009) - states, Central Nigeria Richards 2010*37 Serology in children Fly assessment Nigeria Kaduna State; Tekle 201238 Skin snip microscopy Interruption (2008)¥ - Birnin Gwari, Kauru and Lere (LGAs) Mali, River Bakoye (Mali) Traore, 20129‡ Skin snip microscopy Interruption (2007) - Senegal River Faleme Diawara, 200956 O-150 PCR (Poolscreen) in black flies Elimination (2010) (Mali/Senegal border) River Gambia (Senegal) Mali Koulikoroni (Bamako Rougemont, 198039 Skin snip microscopy Ongoing transmission - region) Eye diagnostics (1977) Sudan Abu Hamed Higazi, 201340 Skin snip microscopy Interruption (2011) - Higazi, 2012*57 Serology Ov-16 in children Higazi, 2010*58 O-150 PCR (Poolscreen) in black flies Guatemala Escuintla Gonzalez, 200941 Serology Ov-16 in children Interruption (2007) Interruption (2007) O-150 PCR (Poolscreen) in black flies Elimination (2010) Eye diagnostics

Guatemala Santa Rosa Lindblade, 200742 Serology Ov-16 in children Interruption (2006) Interruption (2006) O-150 PCR (Poolscreen) in black flies Elimination (2010) Eye diagnostics Guatemala Huehuetenango Cruz-Ortiz, 201210‡ Skin snip microscopy Interruption (2008) Interruption (2008) Serology Ov-16 in children Elimination (2011) Elimination (2011) O-150 PCR (Poolscreen) in black flies Eye diagnostics

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Guatemala Central no evidence found Interruption (2011) Ecuador Esmeraldas Lovato, 201443 Skin snip microscopy Interruption (2008) Interruption (2009) Vieira, 200744 Serology Ov-16 in children Elimination (2012) O-150 PCR (Poolscreen) in black flies Eye diagnostics Mexico Northern Chiapas Rodriguez-Perez Skin snip microscopy Interruption (2006) Interruption (2007) 201045 Serology Ov-16 in children Elimination (2010) O-150 PCR (Poolscreen) in black flies Eye diagnostics Mexico Oaxaca Rodriguez-Perez, Skin snip microscopy Interruption (2008) Interruption (2008) 201046 Serology Ov-16 in children Elimination (2011) Rodriguez-Perez, O-150 PCR (Poolscreen) in black flies 200847 Eye diagnostics Mexico Southern Chiapas Rodriguez-Perez, Skin snip microscopy Interruption (2011) Interruption (2011) 200848 Skin snip PCR Rodriguez-Perez, O-150 PCR (Poolscreen) in black flies 201351 Eye diagnostics Colombia López de Micay no evidence found Interruption (2007) Elimination (2010) Venezuela North-central Convit, 201350 Skin snip microscopy Interruption (2010) Interruption (2010) Serology Ov-16 in children O-150 PCR (Poolscreen) in black flies Eye diagnostics Venezuela North-east Convit, 201350 Skin snip microscopy Interruption (2012) Interruption (2012) Serology Ov-16 in children O-150 PCR (Poolscreen) in black flies Eye diagnostics Venezuela South no evidence found Ongoing transmission Brazil Amazonas no evidence found Ongoing transmission LGA = local government area; § year in brackets refer generally to year of last test carried out, not the year of last administered treatment; * abstract only; ‡ included in systematic review; ₸ update of Richards et al. 201159 and WHO 201260; ¥ no entomological data

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4.4.2 Evidence from diagnostic studies for KQ1 & KQ2

4.4.2.1 O-150 PCR of skin snips (index test) vs. skin snip microscopy (reference test) We located seven diagnostic studies including 1,204 patients that directly compared O-150 PCR of skin snips to skin snip microscopy.12,15,17-21 None of the studies compared the DTA of different cut-offs or time points of testing. In general, the tests were conducted in Africa (Uganda,15,19 Cameroon,17 Guinea,21 and Ivory Coast18) and Ecuador.20 One study was conducted in the US and included travelers, immigrants or expatriates, which have been in endemic regions.12 All were endemic regions for onchocerciasis; however, there was variability in the timing of the administration of tests and as to whether MDA had been given in the tested region. Consequently, the underlying prevalence of onchocerciasis probably varied substantially across settings.

The reported sensitivity of O-150 PCR of skin snips using skin snip microscopy as the reference standard varied between 71% and 100%. Likewise, the specificity varied between 15% and 96% (one study had no participants without onchocerciasis, resulting in an invalid calculation of specificity of 100%).17

The DTA values (sensitivity and specificity) of O-150 PCR of skin snips compared to skin snip microscopy are presented graphically in Figure 3. The left upper corner represents the best DTA values (high sensitivity and high specificity), whereas the lower right corner represents the worst DTA values (low sensitivity and low specificity).

The overall quality of the evidence for this comparison is low. One study had low risk of bias,21 four studies had unclear,15,18-20 and two high risk of bias.12,17 The risk of bias was increased by the case- control design of several of the studies (one of which did not include persons without onchocerciasis17). Another factor that increased the risk of bias and weakens the quality of evidence of these studies is that it is likely that the O-150 PCR of the skin snips has more accurate DTA characteristics than skin snip microscopy, especially in settings with low onchocerciasis prevalence due to MDA,61 and therefore the results of these diagnostic studies using skin snip microscopy as the gold-standard reference test are unreliable. This fact is accounted for by the risk of bias rating (Table 6). One study estimated the upper and lower bounds of sensitivity and specificity of O-150 PCR and microscopy of skin snips and calculated values using statistical models with various prevalence levels. The authors produced a composite reference standard by combining the results of the three diagnostic tests together. Hence they attempted to compensate for the unreliability of skin snip microscopy as gold-standard reference test, especially in circumstances with low prevalence. Data of this study together with unpublished data suggests that the lower the prevalence, the better O-150 PCR of skin snips perform in terms of sensitivity compared to skin snip microscopy.21

Furthermore, we calculated DTA values of skin snip microscopy as index test against O-150 PCR of skin snips as reference test, which are provided in Appendix C. The concerns about applicability were unclear for six studies15,17-20 and high for one study12 (Table 6). The assessment of all GRADE domains is shown in GRADE profile 2.

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Figure 3: DTA values of O-150 PCR of skin snips (index test) vs. skin snip microscopy (reference test)

Abbreviation: cd = collagenase digestion; cr2 = composite reference standard is skin snip microscopy + DEC patch; cr3 = composite reference standard is skin snip microscopy + skin snip PCR + DEC patch; si = saline incubation; sr = skin snip microscopy is reference standard

4.4.2.2 Ov-16 serology (index test) vs. skin snip microscopy (reference test) We located four studies containing 1,425 patients (partly blood and serum samples from biobanks) that directly compared Ov-16 serology to skin snip microscopy.11,13,14,16 In general, the participants and samples came mostly from Africa, South and Central America, and partly also from India and US travelers. The microfilariae positive (MF+) patients and samples all came from endemic regions; however there was variability in the timing of the tests and as to whether MDA had been in place. Consequently, the underlying prevalence of onchocerciasis probably varied substantially across settings.

The reported sensitivity of Ov-16 serology using skin snip microscopy as the reference standard varied between 81% and 98%. Likewise, the specificity varied between 89% and 98% (Figure 4). The quality of evidence for this comparison is low. One study had low,11 two studies had unclear,14,16 and one study had high risk of bias.13 To estimate specificity values, that reflect the reality in regions where other helminthic infections are common, patients positive for other helminthic infections are required in the control group. Two studies included such samples.11,16 In addition, it is possible that the Ov-16 serology has more accurate DTA characteristics than skin snip microscopy, especially in settings with low onchocerciasis prevalence due to MDA,61 and therefore the results of these

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diagnostic studies using skin snip microscopy as the gold-standard reference test are unreliable. This fact is accounted for by the risk of bias rating. Additionally, we calculated DTA values of skin snip microscopy as index test against Ov-16 serology as reference test, which are provided in Appendix C.

Furthermore, DTA values of Ov-16 serology depend on prevalence of onchocerciasis and differ between age-groups, as they do not discriminate between past or active infections. Ov-16 serology testing measures exposure to Onchocerca volvulus, and therefore detects the transmission in an indirect manner (indirectness of evidence).

The DTA values (sensitivity and specificity) of Ov-16 serology compared to skin snip microscopy are presented graphically in Figure 4. The left upper corner represents the best DTA values (high sensitivity and high specificity), whereas the lower right corner represents the worst DTA values (low sensitivity and low specificity).

The concerns about applicability were low (specimen from different regions worldwide leading to a better directness of the study results),11,16 unclear,14 or high (study conducted in a population with a high microfilarial prevalence; usually, regions where tests should be conducted, MF prevalence is low due to MDA).13 Detailed results of risk of bias and applicability assessment as well as the other GRADE domains are shown in Table 6 and in GRADE profile 3.

Figure 4: DTA values of Ov-16 serology (index test) vs. skin snip microscopy (reference test)

Abbreviations: a = focus 1; b = focus 2; lf-s = lateral flow with sera; lf-b = lateral flow with whole blood; E = ELISA

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4.4.2.3 O-150 PCR [Poolscreen] (index test) vs. dissection of black flies (reference test) We located four studies comparing O-150 PCR (Poolscreen) to dissection of black flies as the reference standard.22-25 Two studies directly compared O-150 PCR to dissection,24,25 and two studies compared different DNA extraction methods.22,23 The black flies were caught in different regions in Africa and Mexico (Chiapas), so different species of Simulium black flies were analysed. They all came from endemic regions; however, there was variability in vector control measures, timing of catching, and infectivity rates.

There was no statistically significant difference in diagnostic test accuracy between O-150 PCR (Poolscreen) and dissection of flies. Similarly, there were no differences between newer DNA extraction methods with larger pool sizes compared with traditional methods. The results of the included studies did not allow calculation of sensitivity and specificity.

The overall quality of the evidence from diagnostic studies for this comparison is moderate (see GRADE profile 4). One of the included studies had low23 and three unclear risk of bias.22,24,25. The studies were conducted in foci in Africa and Mexico which have different black fly populations (both differences in black fly species and vector control measures), which is accounted by low applicability concerns, as the O-150 PCR produced reliable results in the different black fly species. Furthermore, it is possible that the O-150 PCR has more accurate DTA characteristics than dissection of black flies and therefore the results of these diagnostic studies using dissection as the gold-standard reference test are unreliable. Furthermore, it is not possible to conduct a dissection and O-150 PCR on the same flies. Detailed results of risk of bias and applicability assessment are shown in Table 6.

In addition, one study assessed the costs of O-150 PCR (Poolscreen) compared to dissection of black flies. The initial expenditures for the O-150 PCR are much higher (~$45,000; specialized equipment) than those for dissection (~$2,000 for a dissection microscope). However, the long-term costs are much lower for O-150 PCR (~$3 per 100 black flies including supplies and labor) than for the dissection (~$15 per 100 black flies). These cost estimates were made in 1999.25 Increasing pool sizes can further reduce the total costs, as is shown for different DNA extraction methods.22,23 Importantly, because costs are highly wage-dependent this comparison may vary substantially between different countries.

4.4.2.4 Tests for ocular morbidity (index test) vs. other tests (reference tests) We could not find any comparative evidence for tests to diagnose ocular morbidity (e.g. slit lamp).

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4.5 GRADE profiles

4.5.1 Evidence for clinical pathway (overarching KQ1 & KQ2)

Question: In a population at risk for onchocerciasis, which diagnostic tests or combination of tests can validly and reliably demonstrate the interruption of transmission of Onchocerca volvulus and predict the elimination of human onchocerciasis?

Setting and tests used in included study: Three endemic study foci in Mali and Senegal: epidemiologic evaluation by skin snips (microscopy); entomological evaluation by examination of black flies via O-150 PCR (Poolscreen; screening pools of 300 flies). In the Huehuetenango focus of Guatemala: Ov-16 serology; ophthalmological evaluation; entomological evaluation by examination of black flies via O-150 PCR.

GRADE profile 1: Evidence for clinical pathway (overarching KQ1 & KQ2)

Quality assessment Summary of findings Quality/certainty No of Risk of Other Interruption of Elimination of Importance Design Inconsistency Indirectness Imprecision 3 of evidence studies bias considerations transmission onchocerciasis 2 (MDA stopped) Microfilarial prevalence (skin snip microscopy) prospective 1 4,5,7 4,8  1 observational none none serious serious 13 none CONFIRMED CONFIRMED CRITICAL LOW study

Antibody prevalence (Ov-16 serology)

prospective 1 12  1 serious11 none serious none none CONFIRMED NOT CONFIRMED11 CRITICAL observational LOW study Infectivity rate of black flies (F3H/1000; O-150 PCR, Poolscreen) prospective 6,9 6,10  2 observational none none nones none none CONFIRMED CONFIRMED CRITICAL HIGH studies

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Ophthalmological evaluation / Ocular infection (Microfilariae in the anterior segment of the eye)

prospective 1  1 serious11 none serious none none CONFIRMED12 NOT CONFIRMED11 CRITICAL observational LOW study 1 focus and population may not be not representative for other regions; problems of resources and accessibility can vary 2 after 12--16 years of MDA 3 2-5 years after ceasing MDA 4 below threshold of microfilarial prevalence <1% in 90% and <5% in 100% of sample villages 5 confirmed in 2 out of 3 foci (R. Faleme focus did not fully meet the threshold criteria; study design was modified and study prolonged by 1 year, then threshold criteria were fully met) 6 below threshold of 0.5 F3H/1,000 7 overall microfilarial prevalence 0.39% (69/17,890) 8 overall microfilarial prevalence 0.03% (3/9,365) 9 infectivity rates 0-0.07 (max. of upper 95%CI was 0.2/1000 flies; n=8,252+X; NA for one study) 10 infectivity rates 0-0.01 (max. of upper 95%CI was 0.471000 flies; n=246,499) 11 interruption of transmission indicated by Ov-16 serology or ocular morbidity was confirmed in and after post-treatment surveillance period through O-150 PCR Poolscreen in black flies (i.e., not using the same test) 12 no positive children with Ov-16 serology (0%; 95%CI 0-0,1%), Ocular morbidity 0% (95%CI 0-0,8%) microfilariae in the anterior segment 13 the measurements of skin snip microfilariae prevalence was performed in terms of percentages of villages with a defined prevalence of microfilariae and not on the individual level.

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4.5.2 O-150 PCR of skin snips (index test) vs. skin snip microscopy (reference test)

Sub-question: O-150 PCR of skin snips (index test) compared with skin snip microscopy (reference test)

GRADE profile 2: O-150 PCR of skin snips (index test) vs. skin snip microscopy (reference test)

Quality assessment Summary of findings Quality/ Interruption of certainty Other transmission (MDA of Importance No of studies Design Risk of bias Inconsistency Indirectness Imprecision DTA characteristics1 considerations stopped) or elimination evidenc of onchocerciasis e

Sensitivity & Specificity (True Positives, False Negatives, True Negatives, False Positives)

Sensitivity ranges from 71% to 100%6 7 studies (1204 diagnostic  serious2 none serious3 none none No direct evidence CRITICAL patients) studies LOW Specificity ranges from 15%5 to 100%7

No evidence for inconclusive tests, costs, and complications

1 Pooling of results not possible due to heterogeneity and high risk of bias, see figure below for a graphic representation of the ranges of sensitivity and specificity. 2 Many studies used a case-control system for identifying participants or did not include any participants without onchocerciasis, mostly studies at unclear or high risk of bias; in addition, skin snip microscopy is an imperfect reference standard, especially in low prevalence settings. 3 Some studies were conducted in populations with high prevalence of infection and this is unlikely to reflect the setting after long-term MDA (KQ1) and in post-MDA confirmation of elimination (KQ2). 5 This low result may be due to the conduct of the O-150 PCR test in this population and setting. 6 One study and unpublished data suggests that the lower the prevalence, the better O-150 PCR of skin snips perform in terms of sensitivity compared to skin snip microscopy. 7 Recorded in a population with no disease negative individuals on skin snip microscopy.

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4.5.3 Ov-16 serology (index test) vs. skin snip microscopy (reference test)

Sub-question: Ov-16 serology (index test) compared with skin snip microscopy (reference test)

GRADE profile 3: OV-16 serology (index test) vs. skin snip (reference test)

Quality assessment Summary of findings Quality/c Interruption of ertainty Other transmission (MDA Importance No of studies Design Risk of bias Inconsistency Indirectness Imprecision DTA characteristics1 of considerations stopped) or elimination evidence of onchocerciasis

Sensitivity & Specificity (True Positives, False Negatives, True Negatives, False Positives)

Sensitivity ranges from 81% to 98% 4 studies (1,425 diagnostic  serious2 none serious4 none none No direct evidence CRITICAL patients/samples) studies LOW Specificity ranges from 89% to 100%

No evidence for inconclusive tests, costs, and complications 1 Pooling of results not possible due to heterogeneity and high risk of bias, see results section for a graphic representation of sensitivity and specificity ranges. 2 Many studies used a case-control system for identifying participants or did not include any participants without onchocerciasis, mostly studies with unclear or high risk of bias; in addition, skin snip microscopy is an imperfect reference standard, especially in low prevalence settings. 3 Participants and serum samples from different regions, countries and both Africa and the Americas; may be representative. 4 Ov-16 serology testing measures exposure to O. volvulus, and therefore detects the transmission indirectly.

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4.5.4 O-150 PCR [Poolscreen] (index test) vs. dissection of black flies (reference test)

Sub-question: O-150 PCR (Poolscreen) in black flies (index test) compared with dissection of black flies (reference test)

GRADE profile 4: O-150 PCR [Poolscreen] (index test) vs. dissection of black flies (reference test)

Quality assessment Summary of findings Quality/c Interruption of ertainty Risk of Other 1 Importance No of studies Design Inconsistency Indirectness Imprecision DTA characteristics transmission (MDA of bias considerations stopped) or elimination evidence of onchocerciasis Sensitivity & Specificity (True Positives, False Negatives, True Negatives, False Positives) No statistically significant differences between O-150 PCR (Poolscreen) and  diagnostic dissection. 4 studies serious4 none none none none No direct evidence MODERA CRITICAL studies No differences between TE newer DNA extraction methods with bigger pool sizes compared to traditional ones2 Cost O-150 PCR (Poolscreen) has higher initial expenditures,  diagnostic but lower following costs than 3 studies none none Serious3 none none No direct evidence MODERA IMPORTANT studies dissection. TE Increasing pool sizes can reduce total costs

No evidence for inconclusive tests and complications 1 Pooling of results not possible due to heterogeneity of results. 2 DNA extraction with specific oligonucleotide based magnetic capture with pool sizes up to 200 has the same performance as DNA extraction with silica-coated beads with pool size of 50 flies. High- throughput automated DNA extraction with silica-coated paramagnetic beads with pool size of 100 has the same performance as DNA extraction with phenol chloroform (PC) and pool size of 50 flies. 3 Total costs are depending on labor costs, which are differing between countries; cost estimates are from 1999. 4 Dissection of flies is an imperfect reference standard, as it is not possible to conduct dissection and Poolscreen on the same flies.

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5 References

1. Deeks JJ, D'Amico R, Sakarovitch C, F S, Petticrew M, Altman DG. Evaluating non-randomised intervention studies. Health Technol Assess. 2003;27(1366-5278 (Print)):1-173. 2. Whiting PF, A R, M W, et al. QUADAS-2: a revised tool for the quality assessment of diagnostic accuracy studies. Annals of internal medicine. 2011;155(1539-3704 (Electronic)):529-536. 3. Guyatt GH, Kunz R, Woodcock J, et al. GRADE guidelines: 8. Rating the quality of evidence-- indirectness. J Clin Epidemiol. 2011;64(1878-5921 (Electronic)):1303-1310. 4. Reitsma JB, Khan KS, Coomarasamy A, Bossuyt PM. A review of solutions for diagnostic accuracy studies with an imperfect or missing reference standard. J Clin Epidemiol. 2009;62(1878-5921 (Electronic)):797-806. 5. Schunemann HJ, R M, Brozek J. [Diagnostic accuracy and linked evidence--testing the chain]. Z Evid Forbild Qual Gesundhwes. 2012;106(1865-9217 (Print)):153-160. 6. Schunemann HJ, Brozek J, Glasziou P, et al. Grading quality of evidence and strength of recommendations for diagnostic tests and strategies. BMJ (Clinical research ed.). 2008;336(1756-1833 (Electronic)):1106-1111. 7. Brozek JL, Jaeschke R, Lang DM, et al. Grading quality of evidence and strength of recommendations in clinical practice guidelines: Part 2 of 3. The GRADE approach to grading quality of evidence about diagnostic tests and strategies. Allergy. 2009;64(1398-9995 (Electronic)):1109-1116. 8. Hsu J, Terracciano L, Kreis J, et al. Application of GRADE: making evidence-based recommendations about diagnostic tests in clinical practice guidelines. Implement Sci. 2011;10(1748-5908 (Electronic)). 9. Traore MO, Sarr MD, Badji A, et al. Proof-of-principle of onchocerciasis elimination with ivermectin treatment in endemic foci in Africa: final results of a study in Mali and Senegal. PLoS neglected tropical diseases. 2012;6(9):e1825. 10. Cruz-Ortiz N, Gonzalez RJ, Lindblade KA, et al. Elimination of Onchocerca volvulus Transmission in the Huehuetenango Focus of Guatemala. Journal of parasitology research. 2012;2012:638429. 11. Golden A, Steel C, Yokobe L, et al. Extended result reading window in lateral flow tests detecting exposure to Onchocerca volvulus: a new technology to improve epidemiological surveillance tools. PloS one. 2013;8(7):e69231. 12. Fink DL, Fahle GA, Fischer S, Fedorko DF, Nutman TB. Toward molecular parasitologic diagnosis: enhanced diagnostic sensitivity for filarial infections in mobile populations. Journal of clinical microbiology. Jan 2011;49(1):42-47. 13. Lipner EM, Dembele N, Souleymane S, et al. Field applicability of a rapid-format anti-Ov-16 antibody test for the assessment of onchocerciasis control measures in regions of endemicity. The Journal of infectious diseases. Jul 15 2006;194(2):216-221. 14. Rodriguez-Perez MA, Dominguez-Vazquez A, Mendez-Galvan J, et al. Antibody detection tests for Onchocerca volvulus: comparison of the sensitivity of a cocktail of recombinant antigens used in the indirect enzyme-linked immunosorbent assay with a rapid-format antibody card test. Transactions of the Royal Society of Tropical Medicine and Hygiene. Sep- Oct 2003;97(5):539-541. 15. Pischke S, Buttner DW, Liebau E, Fischer P. An internal control for the detection of Onchocerca volvulus DNA by PCR-ELISA and rapid detection of specific PCR products by DNA Detection Test Strips. Tropical medicine & international health : TM & IH. Jun 2002;7(6):526- 531.

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16. Weil GJ, Steel C, Liftis F, et al. A rapid-format antibody card test for diagnosis of onchocerciasis. The Journal of infectious diseases. Dec 2000;182(6):1796-1799. 17. Vincent JA, Lustigman S, Zhang S, Weil GJ. A comparison of newer tests for the diagnosis of onchocerciasis. Annals of tropical medicine and parasitology. Apr 2000;94(3):253-258. 18. Toe L, Boatin BA, Adjami A, Back C, Merriweather A, Unnasch TR. Detection of Onchocerca volvulus infection by O-150 polymerase chain reaction analysis of skin scratches. The Journal of infectious diseases. Jul 1998;178(1):282-285. 19. Fischer P, Rubaale T, Meredith SE, Buttner DW. Sensitivity of a polymerase chain reaction- based assay to detect Onchocerca volvulus DNA in skin biopsies. Parasitology research. 1996;82(5):395-401. 20. Zimmerman PA, Guderian RH, Aruajo E, et al. Polymerase chain reaction-based diagnosis of Onchocerca volvulus infection: improved detection of patients with onchocerciasis. The Journal of infectious diseases. Mar 1994;169(3):686-689. 21. Boatin BA, Toe L, Alley ES, Nagelkerke NJ, Borsboom G, Habbema JD. Detection of Onchocerca volvulus infection in low prevalence areas: a comparison of three diagnostic methods. Parasitology. Dec 2002;125(Pt 6):545-552. 22. Rodriguez-Perez MA, Gopal H, Adeleke MA, De Luna-Santillana EJ, Gurrola-Reyes JN, Guo X. Detection of Onchocerca volvulus in Latin American black flies for pool screening PCR using high-throughput automated DNA isolation for transmission surveillance. Parasitology research. Nov 2013;112(11):3925-3931. 23. Gopal H, Hassan HK, Rodriguez-Perez MA, Toe LD, Lustigman S, Unnasch TR. Oligonucleotide based magnetic bead capture of Onchocerca volvulus DNA for PCR pool screening of vector black flies. PLoS neglected tropical diseases. 2012;6(6):e1712. 24. Rodriguez-Perez MA, Danis-Lozano R, Rodriguez MH, Unnasch TR, Bradley JE. Detection of Onchocerca volvulus infection in Simulium ochraceum sensu lato: comparison of a PCR assay and fly dissection in a Mexican hypoendemic community. Parasitology. Dec 1999;119 ( Pt 6):613-619. 25. Yameogo L, Toe L, Hougard JM, Boatin BA, Unnasch TR. Pool screen polymerase chain reaction for estimating the prevalence of Onchocerca volvulus infection in Simulium damnosum sensu lato: results of a field trial in an area subject to successful vector control. The American journal of tropical medicine and hygiene. Jan 1999;60(1):124-128. 26. Oguttu D, Byamukama E, Katholi CR, et al. Serosurveillance to monitor onchocerciasis elimination: the Ugandan experience. The American journal of tropical medicine and hygiene. Feb 2014;90(2):339-345. 27. Lakwo TL, Garms R, Rubaale T, et al. The disappearance of onchocerciasis from the Itwara focus, western Uganda after elimination of the vector Simulium neavei and 19 years of annual ivermectin treatments. Acta tropica. Jun 2013;126(3):218-221. 28. Katabarwa MN, Lakwo T, Habomugisha P, et al. Has interruption of simulium neavei S.S. transmitted onchocerciasis been attained in the Kashoya-Kitomi focus? American Journal of Tropical Medicine and Hygiene. 2010;83(5):21. 29. Ndyomugyenyi R, Tukesiga E, Buttner DW, Garms R. The impact of ivermectin treatment alone and when in parallel with Simulium neavei elimination on onchocerciasis in Uganda. Tropical medicine & international health : TM & IH. Aug 2004;9(8):882-886. 30. Katabarwa M, Lakwo T, Habomugisha P, et al. Transmission of Onchocerca volvulus by Simulium neavei in Mount Elgon Focus of Eastern Uganda Has Been Interrupted. The American journal of tropical medicine and hygiene. Mar 31 2014. 31. Katabarwa MN, Walsh F, Habomugisha P, et al. Transmission of onchocerciasis in wadelai focus of northwestern Uganda has been interrupted and the disease eliminated. Journal of parasitology research. 2012;2012:748540. 32. Roberts JM, Neumann E, Gockel CW, Highton RB. Onchocerciasis in Kenya 9, 11 and 18 years after elimination of the vector. Bulletin of the World Health Organization. 1967;37(2):195- 212.

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33. Cheke RA, Fiasorgbor GK, Walsh JF, Yameogo L. Elimination of the Djodji form of the blackfly Simulium sanctipauli sensu stricto as a result of larviciding by the WHO Onchocerciasis Control Programme in West Africa. Medical and veterinary entomology. Jun 2008;22(2):172- 174. 34. Yaya G, Kobangue L, Kemata B, Galle D, Gresenguet G. [Elimination or control of the onchocerciasis in Africa? Case of Gami village in Central African Republic.]. Bulletin de la Societe de pathologie exotique (1990). May 10 2014. 35. Katabarwa MN, Eyamba A, Nwane P, et al. Fifteen years of annual mass treatment of onchocerciasis with ivermectin have not interrupted transmission in the west region of cameroon. Journal of parasitology research. 2013;2013:420928. 36. Evans D, Alphonsus K, Umaru J, et al. Transmission of onchocerciasis in central Nigeria: Ongoing transmission or disease elimination? American Journal of Tropical Medicine and Hygiene. 2013;89(5):444. 37. Richards F, Eigege A, Gonzales-Peralta C, et al. The challenges of the 'end-game' of african lf mda programs with respect to coendemicity of onchocerciasis: Must ivermectin treatment for onchocerciasis continue when lf transmission has been interrupted? American Journal of Tropical Medicine and Hygiene. 2010;83(5):236. 38. Tekle AH, Elhassan E, Isiyaku S, et al. Impact of long-term treatment of onchocerciasis with ivermectin in Kaduna State, Nigeria: first evidence of the potential for elimination in the operational area of the African Programme for Onchocerciasis Control. Parasites & vectors. 2012;5:28. 39. Rougemont A, Thylefors B, Ducam M, Prost A, Ranque P, Delmont J. [Treatment of onchocerciasis in hyperendemic communities in West Africa with small, gradually increasing doses of suramin. 1. Parasitological results and ophthalmological surveillance in a region where transmission has not been interrupted]. Bulletin of the World Health Organization. 1980;58(6):917-922. 40. Higazi TB, Zarroug IM, Mohamed HA, et al. Interruption of Onchocerca volvulus transmission in the Abu Hamed focus, Sudan. The American journal of tropical medicine and hygiene. Jul 2013;89(1):51-57. 41. Gonzalez RJ, Cruz-Ortiz N, Rizzo N, et al. Successful interruption of transmission of Onchocerca volvulus in the Escuintla-Guatemala focus, Guatemala. PLoS neglected tropical diseases. 2009;3(3):e404. 42. Lindblade KA, Arana B, Zea-Flores G, et al. Elimination of Onchocercia volvulus transmission in the Santa Rosa focus of Guatemala. The American journal of tropical medicine and hygiene. Aug 2007;77(2):334-341. 43. Lovato R, Guevara A, Guderian R, et al. Interruption of infection transmission in the onchocerciasis focus of Ecuador leading to the cessation of ivermectin distribution. 20140523 2014(1935-2735 (Electronic)). 44. Vieira JC, Cooper PJ, Lovato R, et al. Impact of long-term treatment of onchocerciasis with ivermectin in Ecuador: potential for elimination of infection. BMC medicine. 2007;5:9. 45. Rodriguez-Perez MA, Unnasch TR, Dominguez-Vazquez A, et al. Lack of active Onchocerca volvulus transmission in the northern Chiapas focus of Mexico. The American journal of tropical medicine and hygiene. Jul 2010;83(1):15-20. 46. Rodriguez-Perez MA, Unnasch TR, Dominguez-Vazquez A, et al. Interruption of transmission of Onchocerca volvulus in the Oaxaca focus, Mexico. The American journal of tropical medicine and hygiene. Jul 2010;83(1):21-27. 47. Rodriguez-Perez MA, Lizarazo-Ortega C, Hassan HK, et al. Evidence for suppression of Onchocerca volvulus transmission in the Oaxaca focus in Mexico. The American journal of tropical medicine and hygiene. Jan 2008;78(1):147-152. 48. Rodriguez-Perez MA, Lutzow-Steiner MA, Segura-Cabrera A, et al. Rapid suppression of Onchocerca volvulus transmission in two communities of the Southern Chiapas focus,

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Mexico, achieved by quarterly treatments with Mectizan. The American journal of tropical medicine and hygiene. Aug 2008;79(2):239-244. 49. Richards FO, Jr., Eigege A, Miri ES, Jinadu MY, Hopkins DR. Integration of mass drug administration programmes in Nigeria: The challenge of schistosomiasis. Bulletin of the World Health Organization. Aug 2006;84(8):673-676. 50. Convit J, Schuler H, Borges R, et al. Interruption of Onchocerca volvulus transmission in Northern Venezuela. Parasites & vectors. 2013;6(1):289. 51. Rodriguez-Perez MA, Dominguez-Vazquez A, Unnasch TR, et al. Interruption of transmission of Onchocerca volvulus in the Southern Chiapas Focus, Mexico. PLoS neglected tropical diseases. 2013;7(3):e2133. 52. Katabarwa MN, Lakwo T, Habomugisha P, et al. Elimination of onchocerciasis transmission in Mt. Elgon focus of eastern Uganda has been attained. American Journal of Tropical Medicine and Hygiene. 2011;85(6):179-180. 53. Richards F, Lakwo T, Habomugisha P, et al. Prospects for the interruption of onchocerciasis transmission in Mount Elgon focus, Eastern Uganda. American Journal of Tropical Medicine and Hygiene. 2009;81(5):236. 54. Katabarwa MN, Eyamba A, Nwane P, et al. Seventeen years of annual distribution of ivermectin has not interrupted onchocerciasis transmission in North Region, Cameroon. The American journal of tropical medicine and hygiene. Dec 2011;85(6):1041-1049. 55. Katabarwa MN, Eyamba A, Nwane P, et al. Seventeen years of annual distribution of ivermectin has not interrupted onchocerciasis transmission in North Region, Cameroon. American Journal of Tropical Medicine and Hygiene. 2010;83(5):64. 56. Diawara L, Traore MO, Badji A, et al. Feasibility of onchocerciasis elimination with ivermectin treatment in endemic foci in Africa: first evidence from studies in Mali and Senegal. PLoS neglected tropical diseases. 2009;3(7):e497. 57. Higazi TB, Mohamed H, Zarroug I, et al. Interruption of onchocerciasis transmission in the abu hamed focus, northern sudan. American Journal of Tropical Medicine and Hygiene. 2012;87(5):195. 58. Higazi T, Zarroug I, Ali H, et al. Onchocerciasis elimination in abu hamed focus, Northern Sudan: A 2007 entomological survey. American Journal of Tropical Medicine and Hygiene. 2010;83(5):65. 59. Richards FO, Sauerbrey M, Flores GZ, et al. Update on the onchocerciasis elimination program for the Americas (OEPA). American Journal of Tropical Medicine and Hygiene. 2011;85(6):188. 60. Progress towards eliminating onchocerciasis in the WHO Region of the Americas in 2011: interruption of transmission in Guatemala and Mexico. Releve epidemiologique hebdomadaire / Section d'hygiene du Secretariat de la Societe des Nations = Weekly epidemiological record / Health Section of the Secretariat of the League of Nations. Aug 17 2012;87(33):309-314. 61. Boatin BA, Toe L, Alley ES, Dembele N, Weiss N, Dadzie KY. Diagnostics in onchocerciasis: future challenges. Annals of tropical medicine and parasitology. Apr 1998;92 Suppl 1:S41-45.

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6 Appendix A: Search Strategy

Pubmed 19.05.14

Search Query Results Search (“Onchocerciasis"[Mesh] OR "Onchocerciasis”[tiab] OR river #1 blind*[tiab] OR “Robles disease”[tiab] OR “Onchocerca”[Mesh] OR 4,948 “Onchocerca”[tiab]) Search ("Neglected Diseases/prevention and control"[Mesh] OR "Endemic Diseases/prevention and control"[Mesh] OR "Communicable #2 11,369 Diseases/prevention and control"[Mesh] OR "Disease Transmission, Infectious/prevention and control"[Mesh]) Search (((control[tiab] OR controlled[tiab]) AND (transmission[tiab] OR disease[tiab])) OR interruption[tiab] OR interrupted[tiab] OR #3 823,475 elimination[tiab] OR eliminated[tiab] OR suppression[tiab] OR suppressed[tiab] OR "end of treatment"[tiab]) Search ("Communicable Disease Control"[Mesh] OR "Insect Control"[Mesh] #4 299,988 OR "Program Evaluation"[Mesh]) Search ("Simuliidae/drug effects"[Mesh] OR #5 314 "Simuliidae/parasitology"[Mesh]) Search (“PCR” OR "Polymerase Chain Reaction"[Mesh] OR Polymerase Chain #6 Reaction[tiab] OR “Polymerase Chain Reactions”[tiab] OR pool screen[tiab] 578,927 OR poolscreen[tiab]) Search (“Ov16”[tiab] OR “ov-16”[tiab] OR "Serology"[Mesh] OR “Serology”[tiab] OR "Immunoassay"[Mesh:NoExp] OR "Enzyme-Linked #7 419,105 Immunosorbent Assay"[Mesh] OR “ELISA”[tiab] OR "Blotting, Western"[Mesh] OR western blot*[tiab]) #8 Search (transmission potential*[tiab] OR ATP[tiab]) 137,757 Search (biting rate*[tiab] OR bite rate*[tiab] OR ABR[tiab] OR "Bites and #9 6,775 Stings/epidemiology"[Mesh]) #10 Search skin snip*[tiab] 275 #11 Search slit lamp[tiab] 5,726 #12 Search (#2 Or #3 OR #4 OR #5 OR #6 OR #7 OR #8 OR #9 OR #10 OR #11) 2,064,124 #13 Search (#1 AND #12) 1,650 #14 Search ("Onchocerciasis/prevention and control"[Mesh]) 599 #15 Search (#13 OR #14) 1,881 Search (#15 NOT ("Editorial" [Publication Type] OR "Comment" [Publication #16 1,847 Type] OR "Ephemera" [Publication Type]))

EMBASE 19.05.14

No. Query Results 'onchocerciasis'/exp OR 'onchocerca'/exp OR onchocerciasis:ab,ti OR #2 5,902 'onchocerca volvulus':ab,ti OR 'river blindness':ab,ti OR 'robles disease':ab,ti #3 [embase]/lim NOT [medline]/lim 6,141,858 #4 #2 AND #3 864 'disease control'/exp OR 'prevention and control'/exp OR 'infection control'/exp OR 'insect control'/exp OR control:ab,ti OR controlled:ab,ti OR #5 4,735,690 interruption:ab,ti OR interrupted:ab,ti OR elimination:ab,ti OR eliminated:ab,ti OR suppression:ab,ti OR suppressed:ab,ti OR

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prevalence:ab,ti OR prevention:ab,ti 'polymerase chain reaction'/exp OR pcr:ab,ti OR ('polymerase chain' NEAR/1 #6 605,565 reaction*):ab,ti OR poolscreen*;ti,ab OR (pool NEXT/1 screen*):ab,ti ov16:ab,ti OR 'ov 16':ab,ti OR 'serology'/exp OR 'enzyme linked #7 immunosorbent assay'/exp OR 'immunoassay'/exp OR 'western blotting'/exp 753,294 OR elisa:ab,ti OR (western NEXT/1 blot*):ab,ti #8 'simuliidae'/exp AND ('drug effect'/exp OR 'epidemiology'/exp) 215 #9 'skin snips':ab,ti 173 #10 ((biting OR bite) NEXT/1 rate*):ab,ti OR abr:ab,ti 5,163 #11 (transmission NEXT/1 potential*):ab,ti OR atp:ab,ti 151,038 #12 'slit lamp':ab,ti 6,667 #13 #5 OR #6 OR #7 OR #8 OR #9 OR #10 OR #11 OR #12 5,756,370 #14 #4 AND #13 464 #15 editorial:it OR letter:it 1,283,448 #16 #14 NOT #15 441

Cochrane Library 19.05.14

ID Search Hits #1 [mh Onchocerciasis] or [mh Onchocerca] 132 Onchocerciasis or "Onchocerca volvulus" or "river blindness" or "Robles #2 183 disease" #1 or #2 in Other Reviews, Trials, Methods Studies, Technology Assessments #3 180 and Economic Evaluations

LILACS 19.05.2014

Query Items found (mh:(Onchocerciasis OR Onchocerca)) OR (ti:(Onchocerciasis OR "Onchocerca volvulus" OR "river blindness" OR onchocercal OR "Robles disease")) OR (ab:(Onchocerciasis OR 149 "Onchocerca volvulus" OR "river blindness" OR onchocercal OR "Robles disease"))

Africa-Wide (Ebsco) 19.05.2014

No. Query Limiters/Expanders Last Run Via Results Database - Africa-

Wide Information Search Screen -

Advanced Search (DE (Onchocerciasis OR "Onchocerca Volvulus")) OR (KW Search modes - Interface - EBSCOhost S1 4,655 (Onchocerciasis OR Boolean/Phrase Research Databases "Onchocerca Volvulus")) Database - Africa-

Wide Information Search Screen -

Advanced Search

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TI ( "river blindness" OR "robles disease" OR Onchocerciasis OR "Onchocerca Volvulus") Search modes - Interface - EBSCOhost S2 OR AB ( "river 491 Boolean/Phrase Research Databases blindness" OR "robles disease" OR Onchocerciasis OR "Onchocerca Volvulus") Database - Africa-

Wide Information Search Screen -

Advanced Search Search modes - Interface - EBSCOhost S3 S1 OR S2 5,675 Boolean/Phrase Research Databases Database - Africa-

Wide Information Search Screen -

Advanced Search TI ( (control OR controlled) AND (disease OR transmission) ) OR AB ( (control OR controlled) AND (disease OR transmission) ) OR TI ( interruption OR interrupted OR elimination OR Search modes - Interface - EBSCOhost S4 eliminated OR 48,653 Boolean/Phrase Research Databases suppression OR suppressed OR "end of treatment" ) OR AB ( interruption OR interrupted OR elimination OR eliminated OR suppression OR suppressed OR "end of treatment" ) Database - Africa-

Wide Information Search Screen -

Advanced Search (TI ("PCR" OR "Polymerase Chain Reaction" OR Search modes - Interface - EBSCOhost S5 "Polymerase Chain 25,069 Boolean/Phrase Research Databases Reactions" OR "pool screen" OR poolscreen)) OR (AB ("PCR" OR

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"Polymerase Chain Reaction" OR "Polymerase Chain Reactions" OR "pool screen" OR poolscreen)) Database - Africa-

Wide Information Search Screen -

Advanced Search (TI (ov16 OR ov-16 OR serology OR "Enzyme- Linked Immunosorbent Assay" OR ELISA OR (western N1 blot*))) OR Search modes - Interface - EBSCOhost S6 18,888 (AB (ov16 OR ov-16 OR Boolean/Phrase Research Databases serology OR "Enzyme- Linked Immunosorbent Assay" OR ELISA OR (western N1 blot*))) Database - Africa-

Wide Information Search Screen -

Advanced Search (TI (transmission N1 potential* OR ATP)) OR Search modes - Interface - EBSCOhost S7 3,181 (AB (transmission N1 Boolean/Phrase Research Databases potential* OR ATP)) Database - Africa-

Wide Information Search Screen -

Advanced Search (TI (((biting OR bite) N1 rate*) OR ABR)) OR (AB Search modes - Interface - EBSCOhost S8 744 (((biting OR bite) N1 Boolean/Phrase Research Databases rate*) OR ABR)) Database - Africa-

Wide Information Search Screen -

Advanced Search (TI skin N1 snip*) OR Search modes - Interface - EBSCOhost S9 248 (AB skin N1 snip*) Boolean/Phrase Research Databases Database - Africa-

Wide Information Search Screen -

Advanced Search (TI "slit lamp") OR (AB Search modes - Interface - EBSCOhost S10 221 "slit lamp") Boolean/Phrase Research Databases Database - Africa-

Wide Information Search Screen -

Advanced Search

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S4 OR S5 OR S6 OR S7 Search modes - Interface - EBSCOhost S11 90,575 OR S8 OR S9 OR S10 Boolean/Phrase Research Databases Database - Africa-

Wide Information Search Screen -

Advanced Search Search modes - Interface - EBSCOhost S12 S3 AND S11 1,055 Boolean/Phrase Research Databases Database - Africa-

Wide Information Search modes - Search Screen -

Boolean/Phrase Advanced Search Limiters - Data Contributor: .....African Index Medicus, .....Bibliography of Filariasis, .....Community Health Information Database, Interface - EBSCOhost S13 S12 .....Healthlit, .....Medicines 136 Research Databases Information Centre Database, .....SAMED, .....Nexus - Database of Current & Completed Research in South Africa Database - Africa-

Wide Information Search modes - Search Screen -

Boolean/Phrase Advanced Search Limiters - Data Contributor: Interface - EBSCOhost S14 S12 45 .....African Journals Online Research Databases Database - Africa-

Wide Information Search Screen -

Advanced Search Search modes - Interface - EBSCOhost S15 S13 OR S14 178 Boolean/Phrase Research Databases Database - Africa-

Wide Information Search Screen -

Advanced Search TP comment OR letter Search modes - Interface - EBSCOhost S16 44,003 OR editorial Boolean/Phrase Research Databases Database - Africa-

Wide Information Search Screen -

Advanced Search Search modes - Interface - EBSCOhost S17 S15 NOT S16 178 Boolean/Phrase Research Databases

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7 Appendix B: Excluded studies at fulltext review level

The following fulltext trials were considered for inclusion but failed to meet the criteria for this report.

7.1 Ineligible outcome (n=51) 1. Adami YL, Rodrigues G, Alves MC, Moraes MA, Banic DM, Maia-Herzog M. New records of Mansonella ozzardi: a parasite that is spreading from the state of Amazonas to previously uninfected areas of the state of Acre in the Purus River region. Memorias do Instituto Oswaldo Cruz. 2014;109(1):87-92. Epub 2013/10/22 2. Awadzi K, Edwards G, Opoku NO, Ardrey AE, Favager S, Addy ET, et al. The safety, tolerability and pharmacokinetics of levamisole alone, levamisole plus ivermectin, and levamisole plus albendazole, and their efficacy against Onchocerca volvulus. Annals of tropical medicine and parasitology [Internet]. 2004; 98(6):[595-614 pp.]. Available from: http://onlinelibrary.wiley.com/o/cochrane/clcentral/articles/886/CN-00490886/frame.html 3. Amazigo UV, Brieger WR, Katabarwa M, et al. The challenges of community-directed treatment with ivermectin (CDTI) within the African Programme for Onchocerciasis Control (APOC). Annals of tropical medicine and parasitology. Mar 2002;96 Suppl 1:S41-58. 4. Bradley JE, Atogho BM, Elson L, Stewart GR, Boussinesq M. A cocktail of recombinant Onchocerca volvulus antigens for serologic diagnosis with the potential to predict the endemicity of onchocerciasis infection. The American journal of tropical medicine and hygiene. 1998;59(6):877-82. Epub 1999/01/14 5. Buttner DW, Awadzi K, Opoku NO. Histological studies of onchocercomata from an area with interrupted transmission in Ghana. Acta Leidensia. 1990;59(1-2):49-50. 6. Cho-Ngwa F, Zhu X, Metuge JA, Daggfeldt A, Gronvik KO, Orlando R, et al. Identification of in vivo released products of Onchocerca with diagnostic potential, and characterization of a dominant member, the OV1CF intermediate filament. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases. 2011;11(4):778-83. Epub 2010/08/18 7. Convit J, Schuler H, Borges R, Olivero V, Dominguez-Vazquez A, Frontado H, et al. Interruption of Onchocerca volvulus transmission in Northern Venezuela. Parasites & vectors. 2013;6(1):289. Epub 2014/02/07 8. Cruz-Ortiz N, Gonzalez RJ, Lindblade KA, Richards FO, Jr., Sauerbrey M, Zea-Flores G, et al. Elimination of Onchocerca volvulus Transmission in the Huehuetenango Focus of Guatemala. Journal of parasitology research. 2012;2012:638429. Epub 2012/09/13 9. Dafa'alla TH, Ghalib HW, Abdelmageed A, Williams JF. The profile of IgG and IgG subclasses of onchocerciasis patients. Clinical and experimental immunology. 1992;88(2):258-63. Epub 1992/05/01 10. Diawara L, Traore MO, Badji A, Bissan Y, Doumbia K, Goita SF, et al. Feasibility of onchocerciasis elimination with ivermectin treatment in endemic foci in Africa: first evidence from studies in Mali and Senegal. PLoS neglected tropical diseases. 2009;3(7):e497. Epub 2009/07/22 11. Eyo JE, Onyishi GC, Ugokwe CU. Rapid epidemiological assessment of onchocerciasis in a tropical semi-urban community, Enugu State, Nigeria. Iranian journal of parasitology. 2013;8(1):145-51 12. Foster JM, Zhang Y, Kumar S, Carlow CK. Parasitic nematodes have two distinct chitin synthases. Molecular and biochemical parasitology. 2005;142(1):126-32. Epub 2005/05/05 13. Gomez-Priego A, Cruz-Gutierrez LE, Paniagua-Solis JF, Ruenes-Meza MT. DIG-ELISA test in

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onchocerciasis. Serum antibodies and probability of infection. Archives of medical research. 1993;24(4):353-9. Epub 1993/01/01 14. Guevara AG, Vieira JC, Lilley BG, Lopez A, Vieira N, Rumbea J, et al. Entomological evaluation by pool screen polymerase chain reaction of Onchocerca volvulus transmission in Ecuador following mass Mectizan distribution. The American journal of tropical medicine and hygiene. 2003;68(2):222-7. Epub 2003/03/19 15. H A. Procedure of definitive cessation of larviciding in the onchocerciasis control program in West-Africa - Entymoogical post-control studies. Parasite-Journal De La Societe Francaise De Parasitologie. 1995;2(3):281-8 16. Higazi TB, Mohamed H, Zarroug I, Mubarak WE, Deran TC, Hashim K, et al. Interruption of onchocerciasis transmission in the abu hamed focus, northern sudan. American Journal of Tropical Medicine and Hygiene. 2012;87(5):195 17. Higazi TB, Zarroug IM, Mohamed HA, Elmubark WA, Deran TC, Aziz N, et al. Interruption of Onchocerca volvulus transmission in the Abu Hamed focus, Sudan. The American journal of tropical medicine and hygiene. 2013;89(1):51-7. Epub 2013/05/22 18. Hoppner J, Perbandt M, Betzel C, Walter RD, Liebau E. Crystallization of the major cytosolic glutathione S-transferase from Onchocerca volvulus. Acta crystallographica Section D, Biological crystallography. 2004;60(Pt 8):1496-7. Epub 2004/07/24 19. Katabarwa MN, Eyamba A, Nwane P, Enyong P, Yaya S, Baldiagai J, et al. Seventeen years of annual distribution of ivermectin has not interrupted onchocerciasis transmission in North Region, Cameroon. The American journal of tropical medicine and hygiene. 2011;85(6):1041-9. Epub 2011/12/07 20. Katabarwa MN, Eyamba A, Nwane P, Enyong P, Yaya S, Yougouda A, et al. Seventeen years of annual distribution of ivermectin has not interrupted onchocerciasis transmission in North Region, Cameroon. American Journal of Tropical Medicine and Hygiene. 2010;83(5):64 21. Katabarwa MN, Eyamba A, Souaibou M, Enyong P, Kuete T, Souleymanou Y, et al. Does transmission take place in hypoendemic areas for onchocerciasis? A study in North Region of Cameroon. American Journal of Tropical Medicine and Hygiene. 2009;81(5):294-5 22. Katabarwa MN, Walsh F, Habomugisha P, Lakwo TL, Agunyo S, Oguttu DW, et al. Transmission of onchocerciasis in wadelai focus of northwestern Uganda has been interrupted and the disease eliminated. Journal of parasitology research. 2012;2012:748540. Epub 2012/09/13 23. Katholi CR, Unnasch TR. Important experimental parameters for determining infection rates in arthropod vectors using pool screening approaches. The American journal of tropical medicine and hygiene. 2006;74(5):779-85. Epub 2006/05/12 24. Keating J, Yukich JO, Mollenkopf S, Tediosi F. Lymphatic filariasis and onchocerciasis prevention, treatment, and control costs across diverse settings: A systematic review. Acta tropica. 2014;135C:86-95. Epub 2014/04/05 25. Kisinza WN, Kisoka WJ, Mutalemwa PP, Njau J, Tenu F, Nkya T, et al. Community directed interventions for malaria, tuberculosis and vitamin A in onchocerciasis endemic districts of Tanzania. Tanzania journal of health research. 2008;10(4):232-9. Epub 2009/05/01 26. Korenaga M, Tada I, Mimori T. Enzyme-linked immunosorbent assay (ELISA) in the detection of IgG antibodies in onchocerciasis using blood collected on filter paper. Japanese Journal of Parasitology. 1983;32(4):347-55 27. Lecaillon JB, Godbillon J, Guderian R, Guevara A, Cascante S, Poltera AA. Concentrations of amocarzine in plasma of 71 Ecuadorian patients of two different races receiving 3 mg/kg b.i.d. and 5 mg/kg o.d. oral postprandial doses for 3 days. Tropical medicine and parasitology : official organ of Deutsche Tropenmedizinische Gesellschaft and of Deutsche Gesellschaft fur Technische Zusammenarbeit (GTZ) [Internet]. 1991; 42(3):[291-3 pp.]. Available from: http://onlinelibrary.wiley.com/o/cochrane/clcentral/articles/585/CN-00082585/frame.html 28. Lindblade KA, Arana B, Zea-Flores G, Rizzo N, Porter CH, Dominguez A, et al. Elimination of Onchocercia volvulus transmission in the Santa Rosa focus of Guatemala. The American journal

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of tropical medicine and hygiene. 2007;77(2):334-41. Epub 2007/08/11 29. Marchon-Silva V, Caer JC, Post RJ, Maia-Herzog M, Fernandes O. Detection of Onchocerca volvulus (Nematoda: Onchocercidae) infection in vectors from Amazonian Brazil following mass Mectizan distribution. Memorias do Instituto Oswaldo Cruz. 2007;102(2):197-202. Epub 2007/04/12 30. Meredith SE, Lando G, Gbakima AA, Zimmerman PA, Unnasch TR. Onchocerca volvulus: application of the polymerase chain reaction to identification and strain differentiation of the parasite. Experimental parasitology. 1991;73(3):335-44. Epub 1991/10/01 31. Ngu JL, Nkenfou C, Capuli E, McMoli TE, Perler F, Mbwagbor J, et al. Novel, sensitive and low- cost diagnostic tests for 'river blindness'--detection of specific antigens in tears, urine and dermal fluid. Tropical medicine & international health : TM & IH. 1998;3(5):339-48. Epub 1998/06/12 32. Oguttu D, Byamukama E, Katholi CR, Habomugisha P, Nahabwe C, Ngabirano M, et al. Serosurveillance to monitor onchocerciasis elimination: the Ugandan experience. The American journal of tropical medicine and hygiene. 2014;90(2):339-45. Epub 2013/12/18 33. Park J, Dickerson TJ, Janda KD. Major sperm protein as a diagnostic antigen for onchocerciasis. Bioorganic & medicinal chemistry. 2008;16(15):7206-9. Epub 2008/07/18 34. Pincock S. Brian Oliver Lyndhurst Duke. Lancet. 2006;368(9531):196. Epub 2006/07/29 35. Preventive chemotherapy: planning, requesting medicines, and reporting. Releve epidemiologique hebdomadaire / Section d'hygiene du Secretariat de la Societe des Nations = Weekly epidemiological record / Health Section of the Secretariat of the League of Nations. 2014;89(8):61-71. Epub 2014/04/08 36. Progress towards eliminating onchocerciasis in the WHO Region of the Americas: verification by WHO of elimination of transmission in Colombia. Releve epidemiologique hebdomadaire / Section d'hygiene du Secretariat de la Societe des Nations = Weekly epidemiological record / Health Section of the Secretariat of the League of Nations. 2013;88(36):381-5. Epub 2013/09/24 37. Roberts JM, Neumann E, Gockel CW, Highton RB. Onchocerciasis in Kenya 9, 11 and 18 years after elimination of the vector. Bulletin of the World Health Organization. 1967;37(2):195-212. 38. Rodriguez-Perez MA, Dominguez-Vazquez A, Unnasch TR, Hassan HK, Arredondo-Jimenez JI, Orozco-Algarra ME, et al. Interruption of transmission of Onchocerca volvulus in the Southern Chiapas Focus, Mexico. PLoS neglected tropical diseases. 2013;7(3):e2133. Epub 2013/04/05 39. Rodriguez-Perez MA, Katholi CR, Hassan HK, Unnasch TR. Large-scale entomologic assessment of Onchocerca volvulus transmission by poolscreen PCR in Mexico. The American journal of tropical medicine and hygiene. 2006;74(6):1026-33. Epub 2006/06/09 40. Rodriguez-Perez MA, Lizarazo-Ortega C, Hassan HK, Dominguez-Vasquez A, Mendez-Galvan J, Lugo-Moreno P, et al. Evidence for suppression of Onchocerca volvulus transmission in the Oaxaca focus in Mexico. The American journal of tropical medicine and hygiene. 2008;78(1):147- 52. Epub 2008/01/12 41. Rodriguez-Perez MA, Unnasch TR, Dominguez-Vazquez A, Morales-Castro AL, Richards F, Jr., Pena-Flores GP, et al. Lack of active Onchocerca volvulus transmission in the northern Chiapas focus of Mexico. The American journal of tropical medicine and hygiene. 2010;83(1):15-20. Epub 2010/07/03 42. Rodriguez-Perez MA, Unnasch TR, Dominguez-Vazquez A, Morales-Castro AL, Pena-Flores GP, Orozco-Algarra ME, et al. Interruption of transmission of Onchocerca volvulus in the Oaxaca focus, Mexico. The American journal of tropical medicine and hygiene. 2010;83(1):21-7. Epub 2010/07/03 43. Tada I, Korenaga M, Mimori T. A comparative study of several diagnostic measures applied in Guatemalan onchocerciasis. Japanese Journal of Parasitology. 1985;34(4):261-71 44. Tada I, Korenaga M, Shiwaku K, Ogunba EO, Ufomadu GO, Nwoke BE. Specific serodiagnosis with adult Onchocerca volvulus antigen in an enzyme-linked immunosorbent assay. The American journal of tropical medicine and hygiene. 1987;36(2):383-6. Epub 1987/03/01

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45. Tang TH, Lopez-Velez R, Lanza M, Shelley AJ, Rubio JM, Luz SL. Nested PCR to detect and distinguish the sympatric filarial species Onchocerca volvulus, Mansonella ozzardi and Mansonella perstans in the Amazon Region. Memorias do Instituto Oswaldo Cruz. 2010;105(6):823-8. Epub 2010/10/15 46. Tekle AH, Elhassan E, Isiyaku S, Amazigo UV, Bush S, Noma M, et al. Impact of long-term treatment of onchocerciasis with ivermectin in Kaduna State, Nigeria: first evidence of the potential for elimination in the operational area of the African Programme for Onchocerciasis Control. Parasites & vectors. 2012;5:28. Epub 2012/02/09 47. Timmann C, Fuchs S, Thoma C, Lepping B, Brattig NW, Sievertsen J, et al. Promoter haplotypes of the interleukin-10 gene influence proliferation of peripheral blood cells in response to helminth antigen. Genes and immunity. 2004;5(4):256-60. Epub 2004/05/07 48. Umeh RE, Babalola OE, Mahmoud AO, Okoye OI, Asana UE, Umeh CN, et al. Utility and effectiveness of computerised motion sensitivity screening tests in rural onchocercal community survey. West African journal of medicine. 2010;29(6):412-6. Epub 2011/04/06 49. Uzoegwu PN, Aloh G. Comparison Of Clinical, Parasitological And Serological Diagnostic Methods For The Definitive Diagnosis Of Onchocerciasis In Nsukka Senatorial Zone. Animal Research International. 2008;1(3):181-9 50. Weil GJ, Ogunrinade AF, Chandrashekar R, Kale OO. IgG4 subclass antibody serology for onchocerciasis. The Journal of infectious diseases. 1990;161(3):549-54. Epub 1990/03/01 51. Weiss N, Karam M. Humoral immune responses in human onchocerciasis: detection of serum antibodies in early infections. Ciba Foundation symposium. 1987;127:180-8. Epub 1987/01/01 52. Yanagawa T, Kasagi F, Yoshimura T. A method for estimating incidence rates of onchocerciasis from skin-snip biopsies with consideration of false negatives. Biometrics. 1984;40(2):301-11. Epub 1984/06/01

7.2 Ineligible population(s) (n=4) 1. H.W. Ghalib MP, P. Soboslay et al. Specific and sensitive lgG4 immunodiagnosis of onchocerciasis with a recombinant 33 kD Onchocerca volvulus protein (Ov33). Tropical medicine and parasitology: official organ of Deutsche Tropenmedizinische Gesellschaft and of Deutsche Gesellschaft fuer Technische Zusammenarbeit (GTZ) 2. Katholi CR, Unnasch TR. Important experimental parameters for determining infection rates in arthropod vectors using pool screening approaches. The American journal of tropical medicine and hygiene. 2006;74(5):779-85. Epub 2006/05/12 3. Rodero M, Chivato T, Muro A, Cuéllar C. Enzyme-linked immunosorbent assay and Western blot antibody determination in sera from patients diagnosed with different helminthic infections with Anisakis simplex antigen purified by affinity chromatography. Memorias do Instituto Oswaldo Cruz.100(3):293-301 4. Shelley AJ, Maia-Herzog M, Calvao-Brito R. The specificity of an ELISA for detection of Onchocerca volvulus in Brazil in an area endemic for Mansonella ozzardi. Transactions of the Royal Society of Tropical Medicine and Hygiene. 2001;95(2):171-3. Epub 2001/05/18

7.3 Ineligible publication type (n=4) 1. Hopkins DR. Disease eradication. The New England journal of medicine. 2013;368(1):54-63. Epub 2013/01/04 2. Ito M, Kamiya M, Lujan A. Fluctuation of ELISA and skin biopsy results in individual inhabitants re-examined after several months in the endemic area of Guatemalan onchocerciasis. Annals of tropical medicine and parasitology. 1984;78(5):553-5. Epub 1984/10/01

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3. Kyelem D, Fischer PU, Brattig NW. The diagnostics and control of neglected tropical helminth diseases. Foreword. Acta tropica. 2011;120 Suppl 1:S1-3. Epub 2011/04/20 4. Progress towards eliminating onchocerciasis in the WHO Region of the Americas in 2011: interruption of transmission in Guatemala and Mexico. Releve epidemiologique hebdomadaire / Section d'hygiene du Secretariat de la Societe des Nations = Weekly epidemiological record / Health Section of the Secretariat of the League of Nations. 2012;87(33):309-14. Epub 2012/08/22

7.4 Ineligible or no diagnostic test (n=65) 1. Ambassa P. Development of an onchocercosis program. Bulletin de Liaison et de Documentation. 1988(86):77. 2. Edungbola LD. Onchocerciasis control in Nigeria. Parasitology Today. 1991;7(5):97-9 3. Anderson RI, Fazen LE, Buck AA. Onchocerciasis in Guatemala. II. Microfilariae in urine, blood, and sputum after diethylcarbamazine. American journal of tropical medicine and hygiene [Internet]. 1975; 24(1):[58-61 pp.]. Available from: http://onlinelibrary.wiley.com/o/cochrane/clcentral/articles/497/CN-00011497/frame.html 4. Awadzi K, Bell DR, Breckenridge AM, Gillies HM, Haddock DR, Orme MLE. The effect of indomethacin, prednisone and cyproheptadine in suppressing the adverse reactions following the use of diethylcarbamazine in patients with onchocerciasis [abstract]. British journal of clinical pharmacology [Internet]. 1981; 11(4):[418p p.]. Available from: http://onlinelibrary.wiley.com/o/cochrane/clcentral/articles/086/CN-00483086/frame.html 5. Ayong LS, Tume CB, Wembe FE, Simo G, Asonganyi T, Lando G, et al. Development and evaluation of an antigen detection dipstick assay for the diagnosis of human onchocerciasis. Tropical medicine & international health : TM & IH. 2005;10(3):228-33. Epub 2005/02/26 6. Blindness prevention programmes. PREVENT. 1972;1(4):62-4 7. Bradley JE, Trenholme KR, Gillespie AJ, Guderian R, Titanji V, Hong Y, et al. A sensitive serodiagnostic test for onchocerciasis using a cocktail of recombinant antigens. The American journal of tropical medicine and hygiene. 1993;48(2):198-204. Epub 1993/02/01 8. Cabrera Z, Parkhouse RM, Forsyth K, Gomez Priego A, Pabon R, Yarzabal L. Specific detection of human antibodies to Onchocerca volvulus. Tropical medicine and parasitology : official organ of Deutsche Tropenmedizinische Gesellschaft and of Deutsche Gesellschaft fur Technische Zusammenarbeit (GTZ). 1989;40(4):454-9. Epub 1989/12/01 9. Cabrera Z, Parkhouse RM. Isolation of an antigenic fraction for diagnosis of onchocerciasis. Parasite immunology. 1987;9(1):39-48. Epub 1987/01/01 10. Chandrashekar R, Ogunrinade AF, Weil GJ. Use of recombinant Onchocerca volvulus antigens for diagnosis and surveillance of human onchocerciasis. Tropical medicine & international health : TM & IH. 1996;1(5):575-80. Epub 1996/10/01 11. Duerr HP, Raddatz G, Eichner M. Control of onchocerciasis in Africa: threshold shifts, breakpoints and rules for elimination. International journal for parasitology. 2011;41(5):581-9. Epub 2011/01/25 12. Eyo JE, Onyishi GC, Ugokwe CU. Rapid epidemiological assessment of onchocerciasis in a tropical semi-urban community, Enugu State, Nigeria. Iranian journal of parasitology. 2013;8(1):145-51 13. Fischer P, Yocha J, Rubaale T, Garms R. PCR and DNA hybridization indicate the absence of animal filariae from vectors of Onchocerca volvulus in Uganda. The Journal of parasitology. 1997;83(6):1030-4. Epub 1997/12/24 14. Fischer P, Yocha J, Rubaale T, Garms R. PCR and DNA hybridization indicate the absence of animal filariae from vectors of Onchocerca volvulus in Uganda. The Journal of parasitology. 1997;83(6):1030-4. Epub 1997/12/24 15. Gilbert C, Francis V. Highlights from the 7th General Assembly, International Agency for the

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Prevention of Blindnes, Dubai 2004. Community Eye Health Journal. 2004;17(52):66 16. Guzman GE, Lavebratt C, Lujan R, Akuffo H. Diagnosis of onchocerciasis using highly specific and sensitive native proteins. Scandinavian journal of infectious diseases. 2002;34(8):583-90. Epub 2002/09/20 17. Kale OO, Bammeke AO, Ayeni O. An evaluation of skin snip techniques used in the quantitative assessment of microfilarial densities of Onchocerca volvulus. Bulletin of the World Health Organization. 1974;51(5):547-9. Epub 1974/01/01 18. Lavebratt C, Dalhammar G, Awadzi K, Akuffo HO. Field diagnosis of onchocerciasis in an area of high versus low endemicity: evaluation of the Dot Blot Assay. Scandinavian journal of infectious diseases. 1996;28(1):75-81. Epub 1996/01/01 19. Lavebratt C, Dalhammar G, Awadzi K, Akuffo HO. Field diagnosis of onchocerciasis in an area of high versus low endemicity: evaluation of the Dot Blot Assay. Scandinavian journal of infectious diseases. 1996;28(1):75-81. Epub 1996/01/01 20. Nignan A, Millot G, Le Bras J, Savel J. ELISA detection of O. volvulus antigenemia in snip-positive patients by monoclonal antibodies directed against a 56 kD glycoprotein of Ascaris suum. Acta Leidensia. 1990;59(1-2):299-301. Epub 1990/01/01 21. Park J, Dickerson TJ, Janda KD. Major sperm protein as a diagnostic antigen for onchocerciasis. Bioorganic & medicinal chemistry. 2008;16(15):7206-9. Epub 2008/07/18 22. Roffi J, Lafabrie B, Chovet M, Viale H, Digoutte JP. [Detection of onchocerciasis by enzyme immunoassay (ELISA) (author's transl)]. Medecine tropicale : revue du Corps de sante colonial. 1982;42(3):297-306. Epub 1982/05/01. Application du test ELISA au depistage de l'onchocercose 23. Klenk A, Geyer E, Zahner H. Serodiagnosis of human onchocerciasis: evaluation of sensitivity and specificity of a purified Litomosoides carinii adult worm antigen. Tropenmedizin und Parasitologie. 1984;35(2):81-4. Epub 1984/06/01 24. Schlie-Guzman MA, Rivas-Alcala AR. Antigen detection in onchocerciasis: correlation with worm burden. Tropical medicine and parasitology : official organ of Deutsche Tropenmedizinische Gesellschaft and of Deutsche Gesellschaft fur Technische Zusammenarbeit (GTZ). 1989;40(1):47- 50. Epub 1989/03/01 25. Taylor HR, Greene BM. Ocular changes with oral and transepidermal diethylcarbamazine therapy of onchocerciasis. British journal of ophthalmology [Internet]. 1981; 65(7):[494-502 pp.]. Available from: http://onlinelibrary.wiley.com/o/cochrane/clcentral/articles/628/CN- 00025628/frame.html 26. Evans D, Alphonsus K, Umaru J, et al. Transmission of onchocerciasis in central Nigeria: Ongoing transmission or disease elimination? American Journal of Tropical Medicine and Hygiene. 2013;89(5):444. 27. Higazi TB, Mohamed H, Zarroug I, et al. Interruption of onchocerciasis transmission in the abu hamed focus, northern sudan. American Journal of Tropical Medicine and Hygiene. 2012;87(5):195. 28. Katabarwa MN, Lakwo T, Habomugisha P, et al. Elimination of onchocerciasis transmission in Mt. Elgon focus of eastern Uganda has been attained. American Journal of Tropical Medicine and Hygiene. 2011;85(6):179-180. 29. Richards FO, Sauerbrey M, Flores GZ, et al. Update on the onchocerciasis elimination program for the Americas (OEPA). American Journal of Tropical Medicine and Hygiene. 2011;85(6):188. 30. Katabarwa MN, Lakwo T, Habomugisha P, et al. Has interruption of simulium neavei S.S. transmitted onchocerciasis been attained in the Kashoya-Kitomi focus? American Journal of Tropical Medicine and Hygiene. 2010;83(5):21. 31. Higazi T, Zarroug I, Ali H, et al. Onchocerciasis elimination in abu hamed focus, Northern Sudan: A 2007 entomological survey. American Journal of Tropical Medicine and Hygiene. 2010;83(5):65. 32. Richards F, Eigege A, Gonzales-Peralta C, et al. The challenges of the 'end-game' of african lf mda programs with respect to coendemicity of onchocerciasis: Must ivermectin treatment for

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onchocerciasis continue when lf transmission has been interrupted? American Journal of Tropical Medicine and Hygiene. 2010;83(5):236. 33. Richards F, Lakwo T, Habomugisha P, et al. Prospects for the interruption of onchocerciasis transmission in Mount Elgon focus, Eastern Uganda. American Journal of Tropical Medicine and Hygiene. 2009;81(5):236. 34. Yaya G, Kobangue L, Kemata B, Galle D, Gresenguet G. [Elimination or control of the onchocerciasis in Africa? Case of Gami village in Central African Republic.]. Bulletin de la Societe de pathologie exotique (1990). May 10 2014. 35. Katabarwa M, Lakwo T, Habomugisha P, et al. Transmission of Onchocerca volvulus by Simulium neavei in Mount Elgon Focus of Eastern Uganda Has Been Interrupted. The American journal of tropical medicine and hygiene. Mar 31 2014. 36. Oguttu D, Byamukama E, Katholi CR, et al. Serosurveillance to monitor onchocerciasis elimination: the Ugandan experience. The American journal of tropical medicine and hygiene. Feb 2014;90(2):339-345. 37. Progress toward elimination of onchocerciasis in the Americas - 1993-2012. MMWR. Morbidity and mortality weekly report. May 24 2013;62(20):405-408. 38. Katabarwa MN, Eyamba A, Nwane P, et al. Fifteen years of annual mass treatment of onchocerciasis with ivermectin have not interrupted transmission in the west region of cameroon. Journal of parasitology research. 2013;2013:420928. 39. Higazi TB, Zarroug IM, Mohamed HA, et al. Interruption of Onchocerca volvulus transmission in the Abu Hamed focus, Sudan. The American journal of tropical medicine and hygiene. Jul 2013;89(1):51-57. 40. Rodriguez-Perez MA, Dominguez-Vazquez A, Unnasch TR, et al. Interruption of transmission of Onchocerca volvulus in the Southern Chiapas Focus, Mexico. PLoS neglected tropical diseases. 2013;7(3):e2133. 41. Lara-Ramirez EE, Rodriguez-Perez MA, Perez-Rodriguez MA, et al. Time series analysis of onchocerciasis data from Mexico: a trend towards elimination. PLoS neglected tropical diseases. 2013;7(2):e2033. 42. Lakwo TL, Garms R, Rubaale T, et al. The disappearance of onchocerciasis from the Itwara focus, western Uganda after elimination of the vector Simulium neavei and 19 years of annual ivermectin treatments. Acta tropica. Jun 2013;126(3):218-221. 43. Cruz-Ortiz N, Gonzalez RJ, Lindblade KA, et al. Elimination of Onchocerca volvulus Transmission in the Huehuetenango Focus of Guatemala. Journal of parasitology research. 2012;2012:638429. 44. Tekle AH, Elhassan E, Isiyaku S, et al. Impact of long-term treatment of onchocerciasis with ivermectin in Kaduna State, Nigeria: first evidence of the potential for elimination in the operational area of the African Programme for Onchocerciasis Control. Parasites & vectors. 2012;5:28. 45. Duerr HP, Raddatz G, Eichner M. Control of onchocerciasis in Africa: threshold shifts, breakpoints and rules for elimination. International journal for parasitology. Apr 2011;41(5):581-589. 46. Rodriguez-Perez MA, Unnasch TR, Dominguez-Vazquez A, et al. Interruption of transmission of Onchocerca volvulus in the Oaxaca focus, Mexico. The American journal of tropical medicine and hygiene. Jul 2010;83(1):21-27. 47. Rodriguez-Perez MA, Unnasch TR, Dominguez-Vazquez A, et al. Lack of active Onchocerca volvulus transmission in the northern Chiapas focus of Mexico. The American journal of tropical medicine and hygiene. Jul 2010;83(1):15-20. 48. Gonzalez RJ, Cruz-Ortiz N, Rizzo N, et al. Successful interruption of transmission of Onchocerca volvulus in the Escuintla-Guatemala focus, Guatemala. PLoS neglected tropical diseases. 2009;3(3):e404. 49. Rodriguez-Perez MA, Lutzow-Steiner MA, Segura-Cabrera A, et al. Rapid suppression of Onchocerca volvulus transmission in two communities of the Southern Chiapas focus, Mexico, achieved by quarterly treatments with Mectizan. The American journal of tropical medicine and

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hygiene. Aug 2008;79(2):239-244. 50. Cheke RA, Fiasorgbor GK, Walsh JF, Yameogo L. Elimination of the Djodji form of the blackfly Simulium sanctipauli sensu stricto as a result of larviciding by the WHO Onchocerciasis Control Programme in West Africa. Medical and veterinary entomology. Jun 2008;22(2):172-174. 51. Rodriguez-Perez MA, Lizarazo-Ortega C, Hassan HK, et al. Evidence for suppression of Onchocerca volvulus transmission in the Oaxaca focus in Mexico. The American journal of tropical medicine and hygiene. Jan 2008;78(1):147-152. 52. Lindblade KA, Arana B, Zea-Flores G, et al. Elimination of Onchocercia volvulus transmission in the Santa Rosa focus of Guatemala. The American journal of tropical medicine and hygiene. Aug 2007;77(2):334-341. 53. Vieira JC, Cooper PJ, Lovato R, et al. Impact of long-term treatment of onchocerciasis with ivermectin in Ecuador: potential for elimination of infection. BMC medicine. 2007;5:9. 54. Vieira JC, Cooper PJ, Lovato R, et al. Impact of long-term treatment of onchocerciasis with ivermectin in Ecuador: potential for elimination of infection. BMC medicine. 2007;5:9. 55. Pincock S. Brian Oliver Lyndhurst Duke. Lancet. Jul 15 2006;368(9531):196. 56. Ndyomugyenyi R, Tukesiga E, Buttner DW, Garms R. The impact of ivermectin treatment alone and when in parallel with Simulium neavei elimination on onchocerciasis in Uganda. Tropical medicine & international health : TM & IH. Aug 2004;9(8):882-886. 57. Amazigo UV, Brieger WR, Katabarwa M, et al. The challenges of community-directed treatment with ivermectin (CDTI) within the African Programme for Onchocerciasis Control (APOC). Annals of tropical medicine and parasitology. Mar 2002;96 Suppl 1:S41-58. 58. Boatin BA, Toe L, Alley ES, Dembele N, Weiss N, Dadzie KY. Diagnostics in onchocerciasis: future challenges. Annals of tropical medicine and parasitology. Apr 1998;92 Suppl 1:S41-45. 59. Buttner DW, Awadzi K, Opoku NO. Histological studies of onchocercomata from an area with interrupted transmission in Ghana. Acta Leidensia. 1990;59(1-2):49-50. 60. Rolland A. [Clinical aspects of ocular onchocerciasis in Ivory Coast nine years after the interruption of the vectorial transmission (author's transl)]. Medecine tropicale : revue du Corps de sante colonial. Sep-Oct 1981;41(5):505-510. 61. Rougemont A, Thylefors B, Ducam M, Prost A, Ranque P, Delmont J. [Treatment of onchocerciasis in hyperendemic communities in West Africa with small, gradually increasing doses of suramin. 1. Parasitological results and ophthalmological surveillance in a region where transmission has not been interrupted]. Bulletin of the World Health Organization. 1980;58(6):917-922. 62. Roberts JM, Neumann E, Gockel CW, Highton RB. Onchocerciasis in Kenya 9, 11 and 18 years after elimination of the vector. Bulletin of the World Health Organization. 1967;37(2):195-212. 63. Convit J, Schuler H, Borges R, et al. Interruption of Onchocerca volvulus transmission in Northern Venezuela. Parasites & vectors. 2013;6(1):289. 64. Katabarwa MN, Eyamba A, Nwane P, et al. Seventeen years of annual distribution of ivermectin has not interrupted onchocerciasis transmission in North Region, Cameroon. American Journal of Tropical Medicine and Hygiene. 2010;83(5):64. 65. Lovato R, Guevara A, Guderian R, et al. Interruption of infection transmission in the onchocerciasis focus of Ecuador leading to the cessation of ivermectin distribution. 20140523 2014(1935-2735 (Electronic)).

7.5 Ineligible comparisons (n=2) 1. Taylor HR, Keyvan-Larijani E, Newland HS, White AT, Greene BM. Sensitivity of skin snips in the diagnosis of onchocerciasis. Tropical medicine and parasitology : official organ of Deutsche Tropenmedizinische Gesellschaft and of Deutsche Gesellschaft fur Technische Zusammenarbeit (GTZ). 1987;38(2):145-7. Epub 1987/06/01

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2. Taylor HR, Munoz B, Keyvan-Larijani E, Greene BM. Reliability of detection of microfilariae in skin snips in the diagnosis of onchocerciasis. The American journal of tropical medicine and hygiene. 1989;41(4):467-71. Epub 1989/10/01

7.6 Study not obtainable (n=4) 1. Golden A, Yokobe L, Stevens E, Faulx D, Steel C, Peck R, et al. Development and evaluation of a rapid diagnostic test to support onchocerciasis control and elimination programs. American Journal of Tropical Medicine and Hygiene. 2012;87(5):90 2. Golden AL, Faulx D, Yokobe L, Stevens E, Peck R, Valdez M, et al. Criteria for testing development of rapid diagnostic test to support onchocerciasis control and elimination programs. American Journal of Tropical Medicine and Hygiene. 2013;89(5):82 3. Marchon-Silva V. Detecção molecular (DNA) de Onchocerca volvulus nos vetores em relação à distribuição da ivermectina na Amazônia brasileira Molecular detection (DNA) of Onchocerca volvulus in the vectors in relation to the distribution of the ivermectin in the brazilian amazonic.88 4. Yaya G, Kobangue L, Kemata B, Galle D, Gresenguet G. [Elimination or control of the onchocerciasis in Africa? Case of Gami village in Central African Republic.]. Bulletin de la Societe de pathologie exotique (1990). 2014. Epub 2014/05/13. Elimination ou controle de l'onchocercose en Afrique? Cas du village de Gami en Republique centrafricaine

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8 Appendix C: Data extraction sheets

8.1 Evidence for clinical pathway (overarching KQ1 & KQ2)

8.1.1 Traore 2012 Author: Traore et al. 20129

First data extractor: PM

Second data extractor: KT

Study design: Observational study

Continent; Country; Region; Cities/Villages: Africa; Mali, Senegal; R.Gambia focus, R. Faleme focus, R.Bakoye focus

Endemicity: Hyperendemic

Sponsor: Bill and Melinda Gates Foundation through the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR)

The authors have declared that no competing interests exist

Population:

 Epidemiological evaluation: stratified random sample of 40-44 villages (interruption of transmission) and 16-37 villages (elimination of onchocerciasis) in each of the 3 study sites; all persons above 1 year  Entomological evaluation: 4 to 6 fly-catching points per study site monitoring for a full transmission season

Number of persons/flies included and analysed:

 Epidemiological evaluation: 17,890 (interruption of transmission) to 9,365 persons (elimination of onchocerciasis)  Entomological evaluation: data not available for interruption of transmission; 237,000 black flies (elimination of onchocerciasis)

Tests:

 Epidemiological evaluation: 2 skin snips from the iliac crests; microscopically examined

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 Entomological evaluation: O-150 PCR on blackfly heads; Poolscreen with screening pools of 300 flies

Timepoint:

 Interruption of transmission: after 14-16 years of treatment  Elimination of onchocerciasis: 2-5 years after last treatment

Outcomes:

People (skin snips) Interruption of transmission Elimination of onchocerciasis Villages with Villages with Villages with Villages with n MF+ MF+ (%) 95% CI prev.of <1% prev.of <5% n MF+ MF+ (%) 95% CI prev.of <1% prev.of <5% R.Bakoye 6899 18 0.26% 0.15%-0.41% 95% 100% 3520 0 0.00% 0.00%-0.10% 100% 100% R.Gambia 5271 3 0.06% 0.01%-0.17% 95% 100% 1540 0 0.00% 0.00%-0.24% 100% 100% R.Faleme 5720 48 0.84% 0.62%-1.11% 80% 91% 4305 3 0.07% 0.01%-0.20% 95% 100% 17890 69 0.39% 9365 3 0.03% Black flies (PCR) Interruption of transmission Elimination of onchocerciasis n F3H/1000 n F3H/1000 95% CI R.Bakoye NA 0.14 56700 0 0.00-0.03 R.Gambia NA 0.02 73200 0 0.00-0.03 R.Faleme NA 0.04 107100 0.02 0.00-0.07 0.07 237000 0.01

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8.1.2 Cruz-Ortiz 2012 Author: Cruz-Ortiz et al.10

First data extractor: PM

Second data extractor: KT

Study design: Observational study

Continent, Country, Region, Cities/Villages: Americas; Guatemala; Huehuetenango focus

Endemicity: Endemic

Sponsors: Centers for Disease Control and Prevention (Atlanta, GA, USA)

Onchocerciasis Elimination Program for the Americas (Guatemala City, Guatemala); funds were provided through a grant by the Bill and Melinda Gates Foundation (Seattle, WA, USA) to The Carter Center (Atlanta, GA, USA).

Population:

 Epidemiological evaluation: 8 historically endemic municipios (similar to counties); 30.239 persons at risk of transmission (2008)  Entomological evaluation: 16 fly-collection sites in 4 coffee plantations (4 sites per plantation)

Number of people/flies included and analysed:

 Epidemiological evaluation: o Opthalmological evaluation: 365 people o Ov-16 serology: 3118 children (6 to 12 years)  Entomolgical evaluation: o 8.252 S. ochraceum out of 19.725 blackflies (2007-2008, interruption of transmission) o 4587 S. ochraceum out of 9.499 blackflies (2010-2011, elimination of onchocerciasis)

Tests:

 Epidemiological evaluation: o skin snips o slit lamp o Ov-16 serology (children 6-12y)  Entomological evaluation: 0-150 PCR Poolscreen on blackflies

Timepoint:

 Interruption of transmission: after 12 years of treatment

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 Elimination of onchocerciasis: 2-3 years after last treatment

Outcomes:

Interruption of transmission

n MFAS Seropositive 95% CI Slit lamp 365 0% 0-0,8% Ov-16 serology 3118 0% 0-0,1%

Black flies (PCR) Interruption of transmission Elimination of onchocerciasis

n Prevalence of infection 95% CI n Prevalence of infection 95% CI 0-0,02% 0-0,04% 8252 0% (≤0,4/2000 flies) 9499* 0% (≤0,8/2000 flies)

* due to low number of S. ochraceum collected, also S. metallicum were poolscreened

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8.2 Skin snip PCR (index test) vs. skin snip microscopy (reference test)

8.2.1 Fink 2010 Author: Fink et al. 201012

First data extractor: PM

Second data extractor: KT

Study design: Diagnostic study

Continent; Country; Region; Cities/Villages: US; Maryland; Bethesda

Endemicity: Non-endemic (patients have been in endemic regions, are resident in the US, which is a non-endemic region)

Sponsor: Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health

Population: Patients (immigrants from endemic regions or travelers to these same regions) referred to the Clinical Parasitology Unit for suspected filarial infections; diagnostic assays were selected based on compatible clinical symptoms and geographic exposure history.

Number of persons/flies included: 200 patients (887 assays) with suspected filarial infections

Number of persons/flies analysed: 129 patients with skin snips (136 assays) and PCR (218 assays) for suspected onchocerciasis

Index test(s): Skin snips with real-time PCR assay

Reference test(s): Skin snip microscopy

Timepoint: Patients seen between April 1999 and December 2009.

Threshold: Limits of detection: 400 fg/µl

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Outcomes:

Skin snip microscopy is reference standard (original study calculation) Disease (skin snip microscopy) 0.9% prev Prevalence + - 100.0% sens Sensitivity Index test + 2 12 14 94.4% spec Specificity (PCR) - 0 204 204 14.3% PPV Positive predictive value 2 216 218 100.0% NPV Negative predictive value NA DOR Diagnostic Odds Ratio 18.0 LR+ Positive likelihood ratio 0.0 LR- Negative likelihood ratio Skin snip PCR is reference standard (self calculation) Disease (skin snip PCR) 6.4% prev Prevalence + - 14.3% sens Sensitivity Index test + 2 0 2 100.0% spec Specificity (skin snip - 12 204 216 100.0% PPV Positive predictive value microscopy ) 14 204 218 94.4% NPV Negative predictive value NA DOR Diagnostic Odds Ratio NA LR+ Positive likelihood ratio 0.9 LR- Negative likelihood ratio

NA= not applicable (incalculable)

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8.2.2 Boatin 2002 Author: Boatin et al. 200221

First data extractor: KT

Second data extractor: PM

Study design: DTA

Continent, Country, Region, Cities/Villages: Arica; Guinea; Manikaya & Balan

Endemicity: Endemic, at time of study CMFL (Community MicroFilarial Load) <1mf/snip (February 2000; after 10 years of ivermectin treatment)

Sponsor: None reported

Population: 313 persons from 2 villages (112 from Manikaya & 201 from Balan). All individuals were invited to participate. No reporting on compliance.

Number of people/flies included: 313 persons

Number of people/flies analysed: 313 persons

Tests:

Index test(s): Skin snip O-150 PCR

Reference test(s): Skin snip microscopy (Statistical models used to calculate different reference standard values)

Timepoint: February 2000

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Outcomes: Skin snip microscopy is reference standard (self calculation) Disease (skin snip microscopy) 1,9% prev Prevalence + - 50,0% sens Sensitivity Index test + 3 19 22 93,8% spec Specificity (Skin snip - 3 288 291 13,6% PPV Positive predictive value PCR) 6 307 313 99,0% NPV Negative predictive value 15,2 DOR Diagnostic Odds Ratio 8,1 LR+ Positive likelihood ratio 0,5 LR- Negative likelihood ratio Skin snip PCR is reference standard (self calculation) Disease (skin snip PCR) 7,0% prev Prevalence + - 13,6% sens Sensitivity Index test + 3 3 6 99,0% spec Specificity (skin snip - 19 288 307 50,0% PPV Positive predictive value microscopy) 22 291 313 93,8% NPV Negative predictive value 15,2 DOR Diagnostic Odds Ratio 13,2 LR+ Positive likelihood ratio 0,9 LR- Negative likelihood ratio Composite reference standard (skin snip microscopy and DEC patch; self calculation) Disease (composite reference standard) 5,8% prev Prevalence + - 50,0% sens Sensitivity Index test + 9 13 22 95,6% spec Specificity (skin snip - 9 282 291 40,9% PPV Positive predictive value PCR) 18 295 313 96,9% NPV Negative predictive value 21,7 DOR Diagnostic Odds Ratio 11,3 LR+ Positive likelihood ratio 0,5 LR- Negative likelihood ratio Composite reference standard (skin snip microscopy, DEC patch and skin snip PCR; self calculation) Disease (composite reference standard) 9,9% prev Prevalence + - 71,0% sens Sensitivity Index test + 22 0 22 100,0% spec Specificity (skin snip - 9 282 291 100,0% PPV Positive predictive value PCR) 31 282 313 96,9% NPV Negative predictive value #DIV/0! DOR Diagnostic Odds Ratio #DIV/0! LR+ Positive likelihood ratio 0,3 LR- Negative likelihood ratio

Results from table 2 of original publication: Sensitivity of skin snip PCR (and DEC patch) in comparison with skin snip microscopy (upper and lower bounds):

 PCR (DNA) sensitivity o 9/18 (0.5; 0.27–0.73) to 22/25 (0.88; 0.72–0.98)  PCR (DNA) specificity o 282/295 (0.96; 0.93–0.98) to 100%  Skin snip microscopy sensitivity o 6/31 (0.19; 0.07–0.35) to 6/12 (0.5; 0.23–0.77)

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This means that, based on the positive results from the skin snip microscopy (and/or the DEC patch), the sensitivity of skin snip PCR varies between 50% and 88% and the specificity between 96% and 100%.

Likewise, due to the lack of gold standard, Bayesian methods were used for 5 statistical models with various scenarios (and different infection prevalence) tested. Specificity of skin snip microscopy was assumed to be 100%.

Skin snip PCR Skin snip microscopy Skin snip PCR Infection prevalence sensitivity sensitivity specificity (Bayesian 95% CI) (Bayesian 95% CI) (Bayesian 95% CI) (Bayesian 95% CI) Model 1 0.10 (0.07 to 0.14) 0.71 (0.54 to 0.86) 0.19 (0.07 to 0.35)2 Model 2 0.12 (0.09 to 0.15) 0.60 (0.46 to 0.73) 0.14 (0.09 to 0.21)1 Model 3 0.14 (0.09 to 0.021) 0.52 (0.32 to 0.72) 0.16 (0.06-0.29)2 Model 4 0.17 (0.12 to 0.24) 0.45 (0.33 to 0.50) 0.12 (0.05 to 0.21)1 Model 5 0.09 (0.05 to 0.14) 0.43 (0.27 to 0.50) 0.25 (0.11 to 0.44)1 0.958 (0.930 to 0.982) 1 single snip; 2 left + right snip

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8.2.3 Pischke 2002 Author: Pischke et al. 200215

First data extractor: KT

Second data extractor: PM

Study design: Diagnostic study

Continent; Country; Region; Cities/Villages: Africa, Western Uganda

Endemicity: Hyperendemic (MDA since 1991)

Sponsor: The scholarship programme “Infectiology” of the German “Bundesministerium für Bildung und Forschung”; upported also in part by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases.

Population: 127 persons

108 persons living in hyperendemic region (69 with low MF densities and 39 MF negative) 19 persons from another area non-endemic for Onchocerca volvulus (however infected with Mansonella perstans and/or Mansonella streptocera)

Number of persons/flies included: 127 persons

Number of persons/flies analysed: 127 persons

Index test(s): O-150 PCR (ELISA or rapid detection test strip)

Reference test(s): Skin snip microscopy

Timepoint: Samples taken 1993 or1995; PCR analysis only done on preserved samples in 2000-2001

Threshold: NA

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Outcomes:

Skin snip microscopy is reference standard (original study) Disease (skin snips, microscopy) 54.3% prev Prevalence + - 79.7% sens Sensitivity + 55 12 67 79.3% spec Specificity Index test - 14 46 60 82.1% PPV Positive predictive value (PCR with 69 58 127 76.7% NPV Negative predictive value test strip) 15.1 DOR Diagnostic Odds Ratio 3.9 LR+ Positive likelihood ratio 0.3 LR- Negative likelihood ratio Disease (skin snips, microscopy) 54.3% prev Prevalence + - 68.1% sens Sensitivity + 47 10 57 82.8% spec Specificity Index test - 22 48 70 82.5% PPV Positive predictive value (PCR with 69 58 127 68.6% NPV Negative predictive value ELISA) 10.3 DOR Diagnostic Odds Ratio 4.0 LR+ Positive likelihood ratio 0.4 LR- Negative likelihood ratio Skin snip PCR is reference standard (self calculation) Disease (PCR with test strip) 52.8% prev Prevalence + - 82.1% sens Sensitivity + 55 14 69 76.7% spec Specificity Index test - 12 46 58 79.7% PPV Positive predictive value (skin snip 67 60 127 79.3% NPV Negative predictive value microscopy) 15.1 DOR Diagnostic Odds Ratio 3.5 LR+ Positive likelihood ratio 0.2 LR- Negative likelihood ratio Disease (PCR with ELISA) 44.9% prev Prevalence + - 82.5% sens Sensitivity + 47 22 69 68.6% spec Specificity Index test - 10 48 58 68.1% PPV Positive predictive value (skin snip 57 70 127 82.8% NPV Negative predictive value microscopy) 10.3 DOR Diagnostic Odds Ratio 2.6 LR+ Positive likelihood ratio 0.3 LR- Negative likelihood ratio

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8.2.4 Vincent 2000 Author: Vincent et al. 200017

First data extractor: KT

Second data extractor: PM

Study design: Diagnostic study

Continent; Country; Region; Cities/Villages: Africa; Cameroon; South-West province; Kokaka, Kumba, Kurume, Bolo, Boa-Bukundu, Bombanda and Bomanda (near the town of Kumba)

Endemicity: Hyperendemic for the forest-zone form of onchocerciasis, no widespread use of ivermectin (mean 55.6 MF/skin snip)

Sponsor: This project was supported in part by grant AI 22488 from the National Institute of Allergy and Infectious Diseases, and the New York Blood Center Project on Onchocerciasis, which is partially supported by the Edna McConnell Clark Foundation.

Population: 38 persons (8-53 years old; 26 male, 12 female); only 29 tested with PCR of skin snips.

Similar skin snips from individuals with no history of exposure to onchocerciasis were tested in parallel in all assays.

Number of persons/flies included: 38 persons

Number of persons/flies analysed: 29 persons which got tested by skin snips

Index test(s): O-150 PCR of skin snips followed by ELISA

Reference test(s): Snip skin microscopy or clinical nodules (36 out of 38 positive MF in skin snips, 2 clinical diagnosis only)

Timepoint: NA

Threshold: NA

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Outcomes:

Skin snip microscopy or clinical nodules is reference standard (original study) Disease (skin snip microscopy or clinical nodules) 100.0% prev Prevalence + - 89.7% sens Sensitivity Index test + 26 0 26 100.0% spec Specificity (PCR with - 3 0 3 100.0% PPV Positive predictive value test strip) 29 0 29 0.0% NPV Negative predictive value NA DOR Diagnostic Odds Ratio NA LR+ Positive likelihood ratio 0.1 LR- Negative likelihood ratio Skin snip PCR is reference standard (self calculation) Disease (PCR with test strips) 89.7% prev Prevalence Index test + - 100.0% sens Sensitivity (skin snip + 26 3 29 100.0% spec Specificity microscopy - 0 0 0 89.7% PPV Positive predictive value or clinical 26 3 29 NA NPV Negative predictive value nodules) NA DOR Diagnostic Odds Ratio NA LR+ Positive likelihood ratio 0.0 LR- Negative likelihood ratio

NA= not applicable (incalculable)

Comments:

„No specimen from the nonendemic control subjects was found positive by any of these assays [including PCR of skin snips].” Number of non-endemic controls tested is not mentioned; therefore, we could not calculate specificity, but according to this, specificity would be 100%.

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8.2.5 Toe 1998 Author: Toe et al., 199818

First data extractor: KT

Second data extractor: PM

Study design: Diagnostic study

Continent; Country; Region; Cities/Villages: Africa, Ivory Coast, Gnankoradji

Endemicity: Outside Onchocerciasis Control Programme (OCP) control area; mean MF count 0.18 per skin snip

Sponsor: Onchocerciasis Control Programme in West Africa

Population: 99 children (age 2-15; 55 male, 44 female)

Number of persons/flies included: 99 children

Number of persons/flies analysed: 99 children

Index test(s): O-150 PCR of skin snips (DNA Hybridization with OVS-2)

Reference test(s): Skin snips (bilateral)

Timepoint: NA

Threshold: NA

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Outcomes:

Skin snip microscopy is reference standard (original study) Disease (skin snips, microscopy) 12.1% prev Prevalence + - 100.0% sens Sensitivity + 12 74 86 14.9% spec Specificity Index test (skin snip PCR) - 0 13 13 14.0% PPV Positive predictive value 12 87 99 100.0% NPV Negative predictive value NA DOR Diagnostic Odds Ratio 1.2 LR+ Positive likelihood ratio 0.0 LR- Negative likelihood ratio Skin snip PCR is reference standard (self calculation) Disease (skin snips, PCR) 86.9% prev Prevalence + - 14.0% sens Sensitivity Index test (skin snip + 12 0 12 100.0% spec Specificity microscopy) - 74 13 87 100.0% PPV Positive predictive value 86 13 99 14.9% NPV Negative predictive value NA DOR Diagnostic Odds Ratio NA LR+ Positive likelihood ratio 0.9 LR- Negative likelihood ratio Skin snip microscopy is reference standard (original study) Disease (skin snips, microscopy) 12.2% prev Prevalence + - 91.7% sens Sensitivity Index test (skin scratch + 11 66 77 23.3% spec Specificity PCR) - 1 20 21 14.3% PPV Positive predictive value 12 86 98 95.2% NPV Negative predictive value 3.3 DOR Diagnostic Odds Ratio 1.2 LR+ Positive likelihood ratio 0.4 LR- Negative likelihood ratio Skin snip microscopy is reference standard (original study) Population of 40 persons living in Birmingham, USA (known not to be exposed) "known negative population" 0.0% prev Prevalence + - NA sens Sensitivity Index test (skin scratch + 0 0 0 100.0% spec Specificity PCR) - 0 40 40 NA PPV Positive predictive value 0 40 40 100.0% NPV Negative predictive value NA DOR Diagnostic Odds Ratio NA LR+ Positive likelihood ratio NA LR- Negative likelihood ratio

NA= not applicable (incalculable)

Comments:

Only data for the population of Gnankoradji was extracted, as this is the primary study group comparing skin snip PCR and skin snip microscopy. Persons of other villages in Burkina Faso and US citizens (known not to be exposed; served as negative controls) were tested to evaluate skin scratches, which was not a test of our research interest.

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8.2.6 Fischer 1996 Author: Fischer et al. 199619

First data extractor: KT

Second data extractor: PM

Study design: Diagnostic study

Continent; Country; Region; Cities/Villages: Africa, Uganda, Kigoyera province (Western Uganda)

Endemicity: Hyperendemic (mean MF density in population analysed was 10.4 MF/mg in 1991 before MDA and 3.4 MF/mg in 1993)

Sponsor: None

Population: 246 persons

227 had MF in skin snips prior to ivermectin treatment and were analysed. Additionally, 19 persons without MF in skin snips before or after treatment served as control group, but was not included in this analysis

Number of persons/flies included: 246 persons

Number of persons/flies analysed: 227 persons

Index test(s): O-150 PCR of skin snips

Reference test(s): Skin snip microscopy (2 skin snips from the buttocks)

Timepoint: November-December 1993; baseline survey in 1991 (before MDA)

Threshold: NA

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Outcomes:

Skin snip microscopy is reference standard (original study) Disease (skin snip microscopy; saline incubation) 74.0% prev Prevalence + - 93.5% sens Sensitivity Index test + 157 16 173 72.9% spec Specificity (PCR with test strip) - 11 43 54 90.8% PPV Positive predictive value 168 59 227 79.6% NPV Negative predictive value 38.4 DOR Diagnostic Odds Ratio 3.4 LR+ Positive likelihood ratio 0.1 LR- Negative likelihood ratio

Disease (skin snip microscopy; 78.9% prev Prevalence collagenase digestion) 91.1% sens Sensitivity Index test + 163 10 173 79.2% spec Specificity (PCR with test strip) - 16 38 54 94.2% PPV Positive predictive value 179 48 227 70.4% NPV Negative predictive value 38.7 DOR Diagnostic Odds Ratio 4.4 LR+ Positive likelihood ratio 0.1 LR- Negative likelihood ratio Skin snip PCR is reference standard (self calculation) Disease (PCR with test strip) 76.2% prev Prevalence + - 90.8% sens Sensitivity Index test + 157 11 168 79.6% spec Specificity (skin snip microscopy; - 16 43 59 93.5% PPV Positive predictive value saline incubation) 173 54 227 72.9% NPV Negative predictive value 38.4 DOR Diagnostic Odds Ratio 4.5 LR+ Positive likelihood ratio 0.1 LR- Negative likelihood ratio

76.2% prev Prevalence Disease (PCR with test strip) 94.2% sens Sensitivity Index test + 163 16 179 70.4% spec Specificity (skin snip microscopy; - 10 38 48 91.1% PPV Positive predictive value collagenase digestion) 173 54 227 79.2% NPV Negative predictive value 38.7 DOR Diagnostic Odds Ratio 3.2 LR+ Positive likelihood ratio 0.1 LR- Negative likelihood ratio

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8.2.7 Zimmerman 1994 Author: Zimmerman et al. 199420

First data extractor: KT

Second data extractor: PM

Study design: Diagnostic study

Continent; Country; Region; Cities/Villages: Americas, Ecuador, Esmeraldas Province (north-western Ecuador)

Endemicity: Endemic

Sponsor: Edna McConnell-Clark Foundation; Fogarty International Center; National Research Council- National Institutes of Health (NIH) Research Associateship Award (P.A.Z.)

Population: 191 persons

94 from Esmeraldas focus in Ecuador 6 volunteers who had never visited an endemic area for onchocerciasis 91 patients with skin lesions from the NIH who had never visited an endemic area

Number of persons/flies included: 191 persons

Number of persons/flies analysed: 191 persons analysed (but skin snips only from the 94 persons from Esmeralas focus taken; those 97 persons who had never visited an endemic area were assumed to be MF negative)

Index test(s): O-150 PCR of skin snips

Reference test(s): Skin snip microscopy (negative controls were assumed to be MF negative)

Timepoint: NA

Threshold: NA

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Outcomes:

Skin snip microscopy or non-exposure is reference standard (original study) 1. Combined population Disease (skin snip microscopy or non-exposure) 31.4% prev Prevalence + - 100.0% sens Sensitivity + 60 13 73 90.1% spec Specificity Index test (PCR) - 0 118 118 82.2% PPV Positive predictive value 60 131 191 100.0% NPV Negative predictive value NA DOR Diagnostic Odds Ratio 10.1 LR+ Positive likelihood ratio 0.0 LR- Negative likelihood ratio 2. Non-exposed population Disease (skin snip microscopy or non-exposure) 0.0% prev Prevalence + - NA sens Sensitivity + 0 0 0 100.0% spec Specificity Index test (PCR) - 0 97 97 NA PPV Positive predictive value 0 97 97 100.0% NPV Negative predictive value NA DOR Diagnostic Odds Ratio NA LR+ Positive likelihood ratio NA LR- Negative likelihood ratio 3. Exposed population Disease (skin snip microscopy or non-exposure) 63.8% prev Prevalence + - 100.0% sens Sensitivity + 60 13 73 61.8% spec Specificity Index test (PCR) - 0 21 21 82.2% PPV Positive predictive value 60 34 94 100.0% NPV Negative predictive value NA DOR Diagnostic Odds Ratio 2.6 LR+ Positive likelihood ratio 0.0 LR- Negative likelihood ratio Skin snip PCR is reference standard (self calculation) 1. Combined population Disease (PCR) 38.2% prev Prevalence + - 82.2% sens Sensitivity Index test (skin snip + 60 0 60 100.0% spec Specificity microscopy or - 13 118 131 100.0% PPV Positive predictive value non-exposure) 73 118 191 90.1% NPV Negative predictive value NA DOR Diagnostic Odds Ratio NA LR+ Positive likelihood ratio 0.2 LR- Negative likelihood ratio 2. Non-exposed population Disease (PCR) 0.0% prev Prevalence + - NA sens Sensitivity Index test (skin snip + 0 0 0 100.0% spec Specificity microscopy or - 0 97 97 NA PPV Positive predictive value non-exposure) 0 97 97 100.0% NPV Negative predictive value NA DOR Diagnostic Odds Ratio NA LR+ Positive likelihood ratio NA LR- Negative likelihood ratio 3. Exposed population Disease (PCR) 77.7% prev Prevalence + - 82.2% sens Sensitivity Index test (skin snip + 60 0 60 100.0% spec Specificity microscopy or - 13 21 34 100.0% PPV Positive predictive value non-exposure) 73 21 94 61.8% NPV Negative predictive value NA DOR Diagnostic Odds Ratio NA LR+ Positive likelihood ratio 0.2 LR- Negative likelihood ratio

NA= not applicable (incalculable)

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8.3 Ov-16-serology (index test) vs. skin snip microscopy (reference test) [or different Ov-16-serology methods compared to each other]

8.3.1 Golden 2013 Author: Golden et al. 201311

First data extractor: PM

Second data extractor: KT

Study design: Diagnostic study

Continent; Country; Region; Cities/Villages: Study carried out in the US (specimens from Ghana, Liberia, Guatemala, Ecuador, Mali, US travellers, Cook Islands, India)

Endemicity: NA

Sponsor: Bill & Melinda Gates Foundation

Population: Samples (whole blood and sera) taken from persons with known MF+ status

Number of persons/flies included: 449 specimens of known MF+ status (sera), 100 specimens (whole blood)

Number of persons/flies analysed: 449 specimens of known MF+ status (sera), 100 specimens (whole blood)

Index test(s):

 OV-16 ELISA  OV-16 lateral-flow-based assay (rapid tool) with sera  OV-16 lateral-flow-based assay (rapid tool) with whole blood

Reference test(s): "microfilaria status" (very probably skin snips)

Timepoint: NA (but test was done at different read times (20min-70days)

Threshold: NA

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Outcomes:

Skin snip microscopy is reference standard (original study) Disease (skin snip microscopy) 50.0% prev Prevalence + - 94.0% sens Sensitivity + 47 2 49 96.0% spec Specificity - 3 48 51 95.9% PPV Positive predictive value Index test 50 50 100 94.1% NPV Negative predictive value (ELISA) 376.0 DOR Diagnostic Odds Ratio 23.5 LR+ Positive likelihood ratio 0.1 LR- Negative likelihood ratio Disease (skin snip microscopy) 50.0% prev Prevalence + - 98.0% sens Sensitivity Index test + 49 1 50 98.0% spec Specificity (Ov-16 lateral - 1 49 50 98.0% PPV Positive predictive value flow, sera) 50 50 100 98.0% NPV Negative predictive value 2401.0 DOR Diagnostic Odds Ratio 49.0 LR+ Positive likelihood ratio 0.0 LR- Negative likelihood ratio Disease (skin snip microscopy) 50.0% prev Prevalence + - 96.0% sens Sensitivity Index test + 48 2 50 96.0% spec Specificity (Ov-16 lateral - 2 48 50 96.0% PPV Positive predictive value flow, whoole 50 50 100 96.0% NPV Negative predictive value blood) 576.0 DOR Diagnostic Odds Ratio 24.0 LR+ Positive likelihood ratio 0.0 LR- Negative likelihood ratio

Sensitivity (95% CI reported in study):  OV-16 ELISA 94% (89.3-98.7)  OV-16 lateral-flow-based assay (rapid tool) with sera 98% (95.3–100.7)  OV-16 lateral-flow-based assay (rapid tool) with whole blood 96% (92.2–99.8)

Specificity (95% CI reported in study):  OV-16 ELISA 96% (92.2-99.8)  OV-16 lateral-flow-based assay (rapid tool) with sera 98% (95.3–100.7)  OV-16 lateral-flow-based assay (rapid tool) with whole blood 96% (92.2–99.8)

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8.3.2 Lipner 2006 Author: Lipner et al. 200613

First data extractor: PM

Second data extractor: KT

Study design: Diagnostic study

Continent; Country; Region; Cities/Villages: Africa; Burkina Faso; Linoguin, Wayen, Badone, Koumoon, Salimbor Africa; Ivory Coast; Maﬁa, Zakpaberi

Endemicity: Varying rates of onchocerciasis

Sponsor: Division of Intramural Research, National Institute of Allergy and Infectious Diseases (US)

Population: 1,511 persons (52% female; 54% <20years) out of the 7 villages (122-367 per village). In Badone only ≤15 years included; in the other 6 villages all persons who volunteered to participate, excluding pregnant or lactating women and children <2years of age.

Number of persons/flies included: 1,511 persons had capillary blood samples (finger-prick) and skin snips

Number of persons/flies analysed: 1,484 had interpretable results from OV-16 card test. 501 persons (134 from Mafia and 367 from Zakpaberi) were analysed (DTA), because there were no control measures were implemented. Index test(s): Rapid-format Ov-16 card test (IgG4)

Reference test(s): Skin snip microscopy (2 skin snips from iliac crests)

Timepoint: late 1999 and early 2000

Threshold: NA

Outcomes: Sensitivity: Mafia: 81,1%; Zakpaberi: 76,5%

Specificity: Mafia: 100%; Zakpaberi: 100%

Positive predictive value (PPV): Mafia: 91,8%; Zakpaberi: 80,5%

Other outcomes:

MF and antibody prevalence rates were correlated (Spearman’s r=0,815; p<0,038) IF MF prevalence = 0%  antibody prevalence low If MF prevalence >10%  antibody prevalence uniformly high (above 60%)

Antibody prevalence was much lower among persons born after control measures were implemented than among persons born before then.

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8.3.3 Rodriguez-Perez 2003 Author: Rodriguez-Perez et al. 200314

First data extractor: PM

Second data extractor: KT

Study design: Diagnostic study

Continent; Country; Region; Cities/Villages: Americas; Mexico; Southern Chiapas; Las Golondrinas

Endemicity: Endemic

Sponsor: Consejo National de Cienca y Tecnologia, Mexico; Centro de Biotecnologia Genomica, Instituto Politecnico Nacional, Mexico; AMRAD (Melbourne, Australia) donated the ICT cards through OEPA

Population: 247 persons (out of a 369 census population in Las Golondrinas) accepted invitation to ELISA testing (compliance rate 67%) and 258 to ICT testing (compliance rate 70%); 11 ICT tested persons were omitted to enable 247 comparisons

Number of persons/flies included: 258 persons

Number of persons/flies analysed: 247 persons

Index test(s): ELISA (mixture of 3 recombinant proteins: OvMBP7, OvMBP11, OvMBP16)

Reference test(s): Rapid-format antibody card test (immunochromatographic test; ICT); IgG4 antibodies to recombinant Ov-16GST

Timepoint: April 2001

Threshold: NA

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Outcomes:

Ov-16 Rapid-format antibody card test is reference standard (original paper) Disease (card test) 69.2% prev Prevalence + - 96.5% sens Sensitivity + 165 29 194 61.8% spec Specificity - 6 47 53 85.1% PPV Positive predictive value Index test 171 76 247 88.7% NPV Negative predictive value (ELISA) 44.6 DOR Diagnostic Odds Ratio 2.5 LR+ Positive likelihood ratio 0.1 LR- Negative likelihood ratio Ov-16, Ov-11, Ov-7 ELISA is reference standard (self calculation) Disease (ELISA) 78.5% prev Prevalence + - 85.1% sens Sensitivity + 165 6 171 88.7% spec Specificity - 29 47 76 96.5% PPV Positive predictive value Index test 194 53 247 61.8% NPV Negative predictive value (card test) 44.6 DOR Diagnostic Odds Ratio 7.5 LR+ Positive likelihood ratio 0.2 LR- Negative likelihood ratio

Sensitivity of ELISA and ICT in comparison to total oncho-seroinfected population:  ELISA: 97% (194/200)  ICT: 85,5% (171/200)

Costs:

Cost estimation for ELISA: 0.30$ per blood spot.

Comments: No gold-standard reference test, i.e. skin snips. Both new tests, uncertainty about clinical implications of positive results (false positives?).

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8.3.4 Weil 2000 Author: Weil et al. 200016

First data extractor: PM

Second data extractor: KT

Study design: Diagnostic study

Continent; Country; Region; Cities/Villages: Africa; Ghana, Cameroon, Sierra Leone, others Americas; Ecuador, Guatemala Children (5-8 years) serum samples only from Ecuador

Endemicity: Serum samples from different regions regarding endemicity

Sponsor: National Institutes of Health; AMRAD ICT (Sydney, Australia) provided tests and was involved in study with employees, but was not involved in blinded test evaluations and had no restrictions on the data obtained

Population: Serum samples from onchocerciasis patients (positive skin snips) from authors’ serum banks.

US expatriates with onchocerciasis (positive skin snips; some had negative skin snip examinations, but had a diagnosis of onchocerciasis based on a history of residence in an endemic area, clinical symptoms consistent with onchocerciasis, and either a positive Mazzotti test or a positive skin snip PCR test. In addition, all MF-negative US onchocerciasis patients had serum IgG antibodies to ﬁlarial antigens and antibodies to ≥1 of 3 recombinant Onchocerca volvulus antigens by ELISA)

Persons with other ﬁlarial infections and other helminth infections.

Persons with autoimmune diseases, hyper IgE syndrome and nonendemic controls.

Number of persons/flies included: 228 serum samples

Number of persons/flies analysed: 228 serum samples

Index test(s): Rapid-format antibody card test (immunochromatographic test; ICT); IgG4 antibodies to recombinant Ov-16GST

Reference test(s): Skin snip microscopy; others (see population above)

Timepoint: NA

Threshold: NA

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Outcomes:

Skin snip microscopy or others is reference standard (original paper) Disease (skin snip microscopy or others) 53.9% prev Prevalence + - 87.8% sens Sensitivity Index test + 108 3 111 97.1% spec Specificity (card test) - 15 102 117 97.3% PPV Positive predictive value 123 105 228 87.2% NPV Negative predictive value 244.8 DOR Diagnostic Odds Ratio 30.7 LR+ Positive likelihood ratio 0.1 LR- Negative likelihood ratio Ov-16 Rapid-format antibody card test is reference standard (self calculation) Disease (card test) 48.7% prev Prevalence + - 97.3% sens Sensitivity Index test + 108 15 123 87.2% spec Specificity (skin snip - 3 102 105 87.8% PPV Positive predictive value microscopy 111 117 228 97.1% NPV Negative predictive value or others) 244.8 DOR Diagnostic Odds Ratio 7.6 LR+ Positive likelihood ratio 0.0 LR- Negative likelihood ratio

Other outcomes:

Effect of treatment on card test results: Paired serum samples were available for 7 onchocerciasis patients that were collected before treatment and several years after treatment, after apparent cure of their infections. All patients had positive card tests before treatment. Four of 7 serum samples from cured patients were negative by the card test. The other 3 serum samples were weakly positive.

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8.4 PCR in black flies (index test) vs. black fly dissection (reference test) [or different DNA extraction methods compared to each other]

8.4.1 Rodriguez-Perez 2013 Author: Rodriguez-Perez et al. 201322

First data extractor: PM

Second reviewer: KT

Study design: Diagnostic study

Continent; Country; Region; Cities/Villages: Americas, Mexico, Chiapas, Las Golondrinas

Endemicity: Endemic focus

Sponsor: Consejo Nacional de Ciencia y Tecnología (CONACyT) of México

Population: Flies (S. ochraceum s.l.) collected inside the community and near a coffee plantation; they were collected before they began feeding. The infected flies were allowed to feed on known infected individuals, then incubated for 9 days and then preserved.

Number of persons/flies included: 13,400 flies

Number of persons/flies analysed: 13,400 flies

Index test(s): O-150 PCR (high-throughput automated DNA isolation; silica-coated paramagnetic beads)

Reference test(s): O-150 PCR (phenol chloroform DNA extraction)

Timepoint: Flies were collected between February 1994 and April 1994

Threshold: NA

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Outcomes:

Pool size of 100 flies (index test) vs. pool size of 50 flies (reference test).

No statistically significant difference between methods.

Infected rate, infective rate and seasonal transmission potential are reported in the following table:

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8.4.2 Gopal 2012 Author: Gopal et al. 201223

First data extractor: PM

Second data extractor: KT

Study design: Diagnostic study

Continent; Country; Region; Cities/Villages: Americas; Mexico; Chiapas; Jose Maria Morelos y Pavon Africa; Burkina Faso; Region des Cascades; Bodajugu, Sakora

Endemicity: NA

Sponsor: NA (no competing interests declared by authors)

Population:

Simulium ochraceum collected in public areas (Jose Marıa Morelos y Pavon, Chiapas, Mexico) between the hours of 07:00 and 10:00. Previous studies demonstrated that majority of flies captured during this period were nulliparous, and the risk of infection was therefore minimal.

Simulium damnosum were obtained from breeding sites on public land located near the communities of Bodajugu and Sakora. These communities are located in the Region des ascades in Southwestern Burkina Faso.

Onchocerca volvulus L3 were obtained from experimentally infected Simulium damnosum flies 7 days after infection with skin microfilariae (parasite material was prepared in the Tropical Medicine Research Station, Kumba, Cameroon, and is being stored at the New York Blood Center).

Heads and bodies in different pool sizes were investigated.

Number of persons/flies included: NA

Number of persons/flies analysed: NA

Index test(s): O-150 PCR-ELISA in black flies (pool screening; DNA extraction with specific oligonucleotide based magnetic bead capture; pool size = up to 200)

Reference test(s): O-150 PCR-ELISA in black flies (pool screening; DNA extraction with silica-coated beads; pool size = 50-100)

Timepoint: NA

Threshold: NA

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Outcomes:

Costs:

Oligonucleotide capture assays are more expensive (per pool), but with increasing pool size costs are lower on a per fly basis.

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8.4.3 Rodriguez-Perez 1999 Author: Rodriguez-Perez et al. 199924

First data extractor: PM

Second data extractor: KT

Study design: Diagnostic study

Continent; Country; Region; Cities/Villages: Americas; Mexico; Chiapas; Las Golondrinas

Endemicity: Endemic focus

Sponsor: Consejo Nacional de Ciencia y Tecnología (CONACyT) of Mexico

Population: Entomological survey: S. ochraceum black flies were collected between April 1997 and February 1998 (74 days); flies showing recent blood meal were discarded; pool size was optimized

Number of persons/flies included: 21,322 (10,550 PCR + 10,772 dissected) flies

Number of persons/flies analysed: 10,772 flies

Index test(s): O-150 PCR

Reference test(s): Fly dissection

Timepoint: NA

Threshold: NA

Outcomes:

No significant differences (p=0,194; one-tailed probability) between the number of infected flies by PCR (1/10,550) and the number of infected flies by dissection (4/10,772) estimated by the 2x2χ² Fisher’s exact test

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8.4.4 Yameogo 1999 Author: Yameogo et al. 1999_ENREF_20

First data extractor: PM

Second data extractor: KT

Study design: Diagnostic study

Continent; Country; Region; Cities/Villages: Africa; Ghana (Bielikpong on the Kulpawn River), Burkina Faso (Nabere on the Bougouriba River and Leraba Pont on the Leraba River), Mali (Madina Diassa and N’zana, both on Baoule River)

Endemicity: NA (area subject to long term vector control)

Sponsor: NA

Population: S. damnosum black flies were obtained from ﬁve collection points

Number of persons/flies included: 25,548 black flies

Number of persons/flies analysed: 25,548 black flies (15,988 by dissection and 9,560 by PCR Poolscreen)

Index test(s): O-150 PCR (Poolscreen) on black flies

Reference test(s): Fly dissection

Timepoint: NA

Threshold: NA

Outcomes:

No statistically difference between the 2 test methods (p>0.5)

Costs:

Costs PCR assay: Initial expenditure: roughly $45,000 for specialized equipment Following costs: roughly US $3.00 per 100 black ﬂies assayed including supplies and labor

Costs dissection method: Initial expenditure: roughly $2,000 for a dissecting microscope Following costs: marginal cost is roughly US $15.00 per 100 black ﬂies

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Annex 3.1 Decision tables: O-150 PCR (Poolscreen) testing in black flies (KQ1)

Health system and public health evidence to recommendations framework KQ 1.1: In a human population receiving MDA (mass drug administration) against onchocerciasis, should O-150 PCR (Poolscreen) testing in black flies be used to demonstrate the interruption of transmission of Onchocerca volvulus for the purpose of stopping MDA?

Problem: Demonstrating the interruption of transmission of Onchocerca volvulus Background: Onchocerciasis was originally endemic in 38 countries: 30 in WHO's African Region, six in the Pan Options: Using O-150 PCR (Poolscreen) testing in black flies American Health Region (PAHO) and two in the Eastern Mediterranean Region; it is estimated that about 99% of the Comparison: No test global burden currently occurs in Africa. Recently, several countries have made exceptional progress in interrupting Setting: Areas that have received MDA and are being evaluated for the transmission and are ready to apply for independent assessment and acknowledgement of such achievement confirmation of interruption of transmission of Onchocerca volvulus (“verification of elimination”). The epidemiology of onchocerciasis is that of a vector-borne disease, where human beings 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 different disease patterns are associated with different variants or strains of the parasite, with differences in the vector capacity and blood-feeding characteristics of local black fly populations, with the seasonal abundance of the vector, and with differences in the human host responses to the parasite. These factors, together with those related to environment, geographical, social and demographic influences increase the complexity of the epidemiology of the disease in the different areas of its distribution. WHO is now updating the previous (2001) guidelines on the verification of elimination of human onchocerciasis.

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CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

What is the overall level of accuracy of Very accurate this evidence?

What is the No overall included Summary of findings: observational studies Historical data from Mali indicated that PCR certainty of studies Very low Low Moderate High

test could distinguish Onchocerca volvulus this evidence? X Outcome Number of Considerations Interruption of Elimination of Certainty of from O. ochengi while dissection of

BENEFITS & HARMS OF THE OPTIONS studies and of GRADE transmission onchocerciasis the black flies could not. design domains (MDA stopped) evidence (GRADE) Additional references: Infectivity rate  Lovato et al. 20141 2 of black flies 2 studies None CONFIRMED CONFIRMED ⊕⊕⊕⊕  Guevara et al. 2003 (F3H/1000; O- (505 439 flies) High 150 PCR, Poolscreen)

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CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

Summary of findings: DTA studies

Outcome Number of Considerations Summary of Evidence Certainty of studies and of GRADE findings – DTA regarding the evidence design domains characteristics interuption of (GRADE) transmisison

O-150 PCR (Poolscreen) vs dissection of black flies

Sensitivity and 4 studies Serious RoB No No direct specificity (True statistically evidence Positives, False significant Negatives, True Negatives, differences ⊕⊕⊕⊝ False Positives) between tests or DNA Moderate extraction methods

Inconclusive No evidence available in eligible DTA studies test

Complications No evidence available in eligible DTA studies

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CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

In general, accurate cost information on diagnostic tests for human O-150 PCR (Poolscreen) costs are estimated at US$ 0.19 per fly onchocerciasis Onchocerca volvulus is sparse. Three scientific studies examined (Gopal et al. 20124) provide information on costs for PCR that may be relevant for decision- making: Factors influencing costs include: No Probably Uncertain Probably Yes Varies* - Larger pools of lies for DNA are more cost efficient than smaller  Catching flies, often a time-consuming exercise Are the resources No Yes pools.4,5  Distance between fly catching points and laboratory

required small? X  Availability of a PCR machine (it could be borrowed - Capital costs for PCR test may be high, but ongoing costs are lower than dissection,6 and the test could serve as a reference from other health programmes, or if available, used for for other parasites and pathogens.5 other diseases)  Variability of costs between countries and continents. Overall, costs of programmes for small populations are higher than for RESOURCE USE larger regions/towns/populations.7

No incremental cost-effectiveness information was found. We retrieved one

Is the incremental cost No Probably Uncertain Probably Yes Varies systematic review from with searches performed up to 2010 that declared Cost of PCR is low if compared to the cost of continuing MDA small relative to the net No Yes

that “no studies on the costs of diagnostics for onchocerciasis were Judgement is “probably yes” due to lack of evidence. benefits? X

available”.3

Increased Probably Uncertain Probably Reduced Varies We know that there is some indication of differences in onchocercal skin What would be the impact The panel considered equity and concluded it is not relevant for the increased reduced disease in women and men and in different racial groups (Yoruba vs

on health inequities? 8 choice of the test. As such, no judgement was made. EQUITY Fulani), but not for any differences for tests for human onchocerciasis.

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CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

Factors that affect feasibility (points taken from a study on the implementation of CDI in Africa):9 - Existence of procurement and supply policy - Support from the Ministry of Health - Support from the Frontline Health Facilities No Probably Uncertain Probably Yes Varies* - Health workers attitudes, motivation and outreach The less feasible (capable of being accomplished or brought about) Is the option feasible to No Yes Factors that affect feasibility (in general for engaging communities, study in an option is, the less likely it is that it should be recommended (i.e.

implement? X Africa):9 the more barriers there are that would be difficult to overcome).

FEASIBILITY - Participatory approached to community mobilization - Community perceives value of interventions - Community perceives value of community-directed approach - Political leadership in communities

Acceptability might reflect who benefits (or is harmed) and who No Probably Uncertain Probably Yes Varies* “The value of properly informing and empowering communities, to enlist pays (or saves); and when the benefits, adverse effects, and costs Is the option acceptable? No Yes them as crucial allies in the disease control efforts, was also appreciated.”10

occur. X

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ALL SETTINGS (including different resources and varying vector efficiency)

Balance of consequences Undesirable consequences Undesirable consequences The balance between Desirable consequences Desirable consequences clearly outweigh probably outweigh desirable and undesirable probably outweigh clearly outweigh desirable consequences desirable consequences consequences undesirable consequences undesirable consequences is closely balanced or uncertain in most settings

Type of recommendation Strong Recommendation for O-150 PCR (Poolscreen) in black flies Conditional (weak) recommendation for O-150 PCR (Poolscreen) in black flies

Only in the context of rigorous research

Only with targeted monitoring and evaluation

Only in specific contexts

Strong Recommendation against O-150 PCR (Poolscreen) in black Conditional (weak) recommendation against O-150 PCR (Poolscreen) in flies black flies

Recommendation (text) WHO recommends using O-150 PCR (Poolscreen) testing in black flies to demonstrate the interruption of transmission of Onchocerca volvulus in a human population receiving mass drug administration (MDA) against onchocerciasis, for the purpose of stopping MDA.

(strong recommendation, high certainty of evidence)

Justification O-150 PCR (Poolscreen) has been validated in different epidemiological settings (African Region and Region of the Americas).

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Implementation Existence of regional laboratories serving the largest possible administrative area. considerations, including Sampling should be appropriately conducted and information on sampling sites and sampling strategies should be made available so as to standardize practices (APOC has monitoring and evaluation developed standard operating procedures that should be included as an annex to the guidelines). Laboratory quality control should be monitored.

Research priorities More investigations should be made to define appropriate and standardized protocols for fly catching.

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Definitions for ratings of the certainty of the evidence (GRADE)**

Ratings Definitions Implications

This research provides a very good indication of the likely effect. The likelihood that the effect will This evidence provides a very good basis for making a decision about whether to implement the be substantially different* is low. intervention. Impact evaluation and monitoring of the impact are unlikely to be needed if it is High implemented. This research provides a good indication of the likely effect. The likelihood that the effect will be This evidence provides a good basis for making a decision about whether to implement the substantially different* is moderate. intervention. Monitoring of the impact is likely to be needed and impact evaluation may be Moderate warranted if it is implemented.

This research provides some indication of the likely effect. However, the likelihood that it will be This evidence provides some basis for making a decision about whether to implement the Low substantially different* is high. intervention. Impact evaluation is likely to be warranted if it is implemented.

This research does not provide a reliable indication of the likely effect. The likelihood that the effect This evidence does not provide a good basis for making a decision about whether to implement the Very low will be substantially different* is very high. intervention. Impact evaluation is very likely to be warranted if it is implemented.

*Substantially different: large enough difference that it might have an effect on a decision

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**The Grading of Recommendations Assessment, Development and Evaluation (GRADE) Working Group began in the year 2000 as an informal collaboration of people with an interest in addressing the shortcomings of present grading systems in health care. The working group has developed a common, sensible and transparent approach to grading quality of evidence and strength of recommendations. Many international organizations have provided input into the development of the approach and have started using it.

1. Lovato R, Guevara A, Guderian R, Proaño R, Unnasch T, Criollo H et al. Interruption of infection transmission in the onchocerciasis focus of Ecuador leading to the cessation of ivermectin distribution. PLoS Negl Trop Dis. 2014;8(5):e2821. doi:10.1371/journal.pntd.0002821. 2. Guevara AG, Vieira JC, Lilley BG, López A, Vieira N, Rumbea J et al. Entomological evaluation by pool screen polymerase chain reaction of Onchocerca volvulus transmission in Ecuador following mass Mectizan distribution. Am J Trop Med Hyg. 2003;68(2):222–7. 3. Keating J, Yukich JO, Mollenkopf S, Tediosi F. Lymphatic filariasis and onchocerciasis prevention, treatment, and control costs across diverse settings: a systematic review. Acta Trop. 2014;135:86-95. doi:10.1016/j.actatropica.2014.03.017. 4. Gopal H, Hassan HK, Rodriguez-Perez MA, Toe LD, Lustigman S, Unnasch TR. Oligonucleotide based magnetic bead capture of Onchocerca volvulus DNA for PCR pool screening of vector black flies. PLoS Negl Trop Dis. 2012;6(6):e1712. doi:10.1371/journal.pntd.0001712. 5. Rodriguez-Perez MA, Gopal H, Adeleke MA, De Luna-Santillana EJ, Gurrola-Reyes JN, Guo X. Detection of Onchocerca volvulus in Latin American black flies for pool screening PCR using high-throughput automated DNA isolation for transmission surveillance. Parasitol Res. 2013;112(11):3925–31. doi:10.1007/s00436-013-3583-0. 6. Yamèogo L, Toe L, Hougard JM, Boatin BA, Unnasch TR. Pool screen polymerase chain reaction for estimating the prevalence of Onchocerca volvulus infection in Simulium damnosum sensu lato: results of a field trial in an area subject to successful vector control. Am J Trop Med Hyg. 1999;60(1):124–8. 7. Katabarwa MN, Habomugisha P, Richards FO, Jr. Implementing community-directed treatment with ivermectin for the control of onchocerciasis in Uganda (1997–2000): an evaluation. Ann Trop Med Parasitol. 2002;96(1):61–73. 8. Brieger WR, Ososanya OO, Kale OO, Oshiname FO, Oke GA. Gender and ethnic differences in onchocercal skin disease in Oyo State, Nigeria. Trop Med Int Health. 1997;2(6):529–34. 9. Mutalemwa P, Kisinza WN, Kisoka WJ, Kilima S, Njau J, Tenu F et al. Community directed approach beyond ivermectin in Tanzania: a promising mechanism for the delivery of complex health interventions. Tanzan J Health Res. 2009;11(3):116–125. 10. Boatin B. The Onchocerciasis Control Programme in West Africa (OCP). Ann Trop Med Parasitol. 2008;102 Suppl1:13–7. doi:10.1179/136485908X337427.

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Annex 3.2 Decision tables: Ov-16 serology (KQ1)

Health system and public health evidence to recommendations framework KQ 1.2: In a human population receiving MDA (mass drug administration) against onchocerciasis, should Ov-16 serology (ELISA) testing in children be used to demonstrate the interruption of transmission of Onchocerca volvulus for the purpose of stopping MDA?

Problem: Demonstrating the interruption of transmission of Onchocerca volvulus Background: Onchocerciasis was originally endemic in 38 countries: 30 in WHO's African Region, six in the Pan Options: Using Ov-16 serology in children American Health Region and two in the Eastern Mediterranean Region; it is estimated that about 99% of the global Comparison: No test burden currently occurs in Africa. Recently, several countries have made exceptional progress in interrupting Setting: Areas that have received MDA and are being evaluated for the transmission and are ready to apply for independent assessment and acknowledgement of such achievement confirmation of interruption of transmission of Onchocerca volvulus (“verification of elimination”). The epidemiology of onchocerciasis is that of a vector-borne disease, where human beings are the only natural vertebrate host, and infection rates and intensity are determined by the degree of

−

CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

What is the overall level of accuracy of Accurate this evidence?

volvulus (KQ1) and elimination of human onchocerciasis (KQ2). The results presented below are a summarized Limitations of the observational evidence form of those from the systematic review and from the evidence profiles presented in the supporting document. were that no comparisons between tests were performed. The included studies represent prospective case studies, and for Summary of findings: observational studies Ov-16 serology, skin snip microscopy, and infection of the eye only one case study Outcome Number of Consideration Interruption of Elimination of Certainty of publication was located. studies and s of GRADE transmission onchocerciasis the design domains (MDA stopped) evidence What is the No (GRADE) overall included certainty of studies Very low Low Moderate High Antibody NOT

this evidence? X prevalence (Ov- 1 study Serious CONFIRMED CONFIRMED ⊕⊕⊝⊝

16 serology) BENEFITS & HARMS OF THE OPTIONS

(3118 children) indirectness, Low serious RoB

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CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

Summary of findings: DTA studies

Outcome Number of Consideration Summary of Evidence Certainty of studies and s of GRADE findings – DTA regarding the evidence Limitations of the DTA evidence include that design domains characteristics interuption of (GRADE) the studies were mostly performed in transmisison populations with high onchocerciasis Ov-16 serology vs skin snip microscopy prevalence and that the reference standard used (generally skin snip microscopy) is Sensitivity & 4 studies (1425 Serious RoB, Sensitivity No direct imperfect. specificity (True patients/ serious ranges from evidence Positives, False samples) indirectness 81% to 98% Negatives, True Negatives, ⊕⊕⊝⊝ False Positives) Specificity Low ranges from 89% to 100%

Inconclusive No evidence available in eligible DTA studies test

Complications No evidence available in eligible DTA studies

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CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

In general, accurate cost information on diagnostic tests for human No Probably Uncertain Probably Yes Varies* Cost estimates: Are the resources onchocerciasis Onchocerca volvulus is sparse. No Yes Capital equipment: plate filler costs approximately US$ 2000

required small? X Overall, costs of programs for small populations are higher than for larger Consumables: US$ 0.15 per ELISA test regions/towns/populations.2

No incremental cost-effectiveness information was found. We retrieved one

Is the incremental cost No Probably Uncertain Probably Yes Varies RESOURCE USE systematic review from with searches performed up to 2010 that declared Final vote: 3 “probably yes” and 4 “yes” small relative to the net No Yes

that “no studies on the costs of diagnostics for onchocerciasis were Cost is low if compared to cost of continuing MDA benefits? X X

available”.1

on health inequities? 3 choice of the test. As such, no judgement was made. EQUITY Fulani), but not for any differences for tests for human onchocerciasis.

Factors that affect feasibility (points taken from a study on the implementation of CDI in Africa):4 - Existence of procurement and supply policy - Support from the Ministry of Health Support by non-governmental development organization (NGDOs) - Support from the Frontline Health Facilities is crucial to ensure feasibility. No Probably Uncertain Probably Yes Varies* - Health workers attitudes, motivation and outreach Is the option feasible to No Yes Factors that affect feasibility (in general for engaging communities, study in Testing for Ov-16 serology (ELISA) can be combined with testing

implement? X Africa):4 carried out in the context of TAS for lymphatic filariasis.

FEASIBILITY - Year-round geographical accessibility - Participatory approached to community mobilization No cold chain required. - Community perceives value of interventions - Community perceives value of community-directed approach - Political leadership in communities

Community participation (or “community driven intervention”) enhances sustainability.4,5 Acceptability might reflect who benefits (or is harmed) and who No Probably Uncertain Probably Yes Varies* pays (or saves); and when the benefits, adverse effects, and costs

Is the option acceptable? No Yes “The value of properly informing and empowering communities, to enlist occur.

X them as crucial allies in the disease control efforts, was also appreciated.”6

Minimally invasive test Finger-prick more acceptable in some communities.7

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ALL SETTINGS (including different resources and varying vector efficiency)

X X

Type of recommendation Strong Recommendation for Ov-16 serology Conditional (weak) recommendation for Ov-16 serology Balance of consequences Final vote: 1 vote for “desirable

X Only in the context of rigorous research consequences probably outweigh

Only with targeted monitoring and evaluation undesirable consequences”, and 6

votes for “desirable consequences Only in specific contexts clearly outweigh undesirable consequences in most settings”

Type of recommendation Final vote: 5 votes for “strong recommendation for Ov-16 serology” and 2 votes for “conditional (weak) recommendation for Ov-16 serology”. Voting results (> 2/3) are sufficient to adopt a “strong recommendation for Ov-16 serology” Strong Recommendation against Ov-16 serology Conditional (weak) recommendation against Ov-16 serology

Recommendation (text) WHO recommends using Ov-16 serology (ELISA) in children to demonstrate the interruption of transmission of Onchocerca volvulus in a human population receiving mass drug administration (MDA) against onchocerciasis, for the purpose of stopping MDA.

(strong recommendation, low certainty of evidence)

−

Justification Accurate test with relatively low cost

Implementation Quality control is important. Testing an average-size community requires approximately 2 days. considerations, including Some implementation difficulties are linked to sampling: it is often difficult to get an appropriate sample size. monitoring and evaluation Ov-16 serology (ELISA) should be regarded as a complementary test, to be implemented in conjunction with others.

Research priorities Sero-reversion of Ov-16 responses should be investigated (by age, over time). The Ov-16 Rapid Test should be validated as a possible replacement for the standard test.

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Definitions for ratings of the certainty of the evidence (GRADE)**

Ratings Definitions Implications This research provides a very good indication of the likely effect. The likelihood that the effect will This evidence provides a very good basis for making a decision about whether to implement the be substantially different* is low. intervention. Impact evaluation and monitoring of the impact are unlikely to be needed if it is High implemented. This research provides a good indication of the likely effect. The likelihood that the effect will be This evidence provides a good basis for making a decision about whether to implement the substantially different* is moderate. intervention. Monitoring of the impact is likely to be needed and impact evaluation may be Moderate warranted if it is implemented.

*Substantially different: large enough difference that it might have an effect on a decision

− working group has developed a common, sensible and transparent approach to grading quality of evidence and strength of recommendations. Many international organizations have provided input into the development of the approach and have started using it.

1. Keating J, Yukich JO, Mollenkopf S, Tediosi F. Lymphatic filariasis and onchocerciasis prevention, treatment, and control costs across diverse settings: a systematic review. Acta Trop. 2014;135:86–95. doi:10.1016/j.actatropica.2014.03.017. 2. Katabarwa MN, Habomugisha P, Richards FO, Jr. Implementing community-directed treatment with ivermectin for the control of onchocerciasis in Uganda (1997–2000): an evaluation. Ann Trop Med Parasitol. 2002;96(1):61–73. 3. Brieger WR, Ososanya OO, Kale OO, Oshiname FO, Oke GA. Gender and ethnic differences in onchocercal skin disease in Oyo State, Nigeria. Trop Med Int Health. 1997;2(6):529–34. 4. Mutalemwa P, Kisinza WN, Kisoka WJ, Kilima S, Njau J, Tenu F et al. Community directed approach beyond ivermectin in Tanzania: a promising mechanism for the delivery of complex health interventions. Tanzan J Health Res. 2009;11(3):116–125. 5. Makaula P, Bloch P, Banda HT, Bongolo Mbera G, Mangani C, de Sousa A et al. Primary health care in rural Malawi – a qualitative assessment exploring the relevance of the community-directed interventions approach. BMC Health Serv Res. 2012;12:328. doi:10.1186/1472-6963-12-328. 6. Boatin B. The Onchocerciasis Control Programme in West Africa (OCP). Ann Trop Med Parasitol. 2008;102 Suppl1:13–7. doi:10.1179/136485908X337427. 7. Rodriguez-Perez MA, Danis-Lozano R, Rodriguez MH, Bradley JE. Comparison of serological and parasitological assessments of Onchocerca volvulus transmission after 7 years of mass ivermectin treatment in Mexico. Trop Med Int Health. 1999;4(2):98–104.

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Annex 3.3 Decision tables: skin snip microscopy (KQ1)

Health system and public health evidence to recommendations framework KQ 1.4: In a human population receiving MDA (mass drug administration) against onchocerciasis, should skin snip microscopy be used to demonstrate the interruption of transmission of Onchocerca volvulus for the purpose of stopping MDA?

Problem: Demonstrating the interruption of transmission of Onchocerca volvulus Background: Onchocerciasis was originally endemic in 38 countries, 30 in WHO's African Region, six in the Pan Options: Using skin snip microscopy American Health Region and two in the Eastern Mediterranean Region; it is estimated that about 99% of the global Comparison: No test burden currently occurs in Africa. Recently, several countries have made exceptional progress in interrupting Setting: Areas that have received MDA and are being evaluated for the transmission and are ready to apply for independent assessment and acknowledgement of such achievement confirmation of interruption of transmission of Onchocerca volvulus (“verification of elimination”). The epidemiology of onchocerciasis is that of a vector-borne disease, where human beings are the only natural vertebrate host, and infection rates and intensity are determined by the degree of

−

CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

What is the Accurate: 2 votes overall level of Inaccurate: 2 votes accuracy of Very inaccurate: 3 votes this evidence?

Summary of findings: Observational studies Limitations of the observational evidence were that no comparisons between tests Outcome Number of Considerations Interruption of Elimination of Certainty of were performed. The included studies studies and of GRADE transmission on- the evidence represent prospective case studies, and for design domains (MDA stopped) chocerciasis (GRADE) three tests only one case study publication Microfilarial was located. What is the No prevalence 1 study Serious CONFIRMED CONFIRMED ⊕⊕⊝⊝ overall included (skin snip (29 753 indirectness, certainty of studies Very low Low Moderate High microscopy) Low Sensitivity is dependant on levels of

individuals) serious this evidence? X prevalence of infection, thus infleuncing imprecision

overall accuracy. BENEFITS & HARMS OF THE OPTIONS

Sensitivity: 20% Specificity: 100%

The test has a low scientifc confidence, as a large enough sample is difficult to obtain.

It was observed that skin snipping humans is redundant if O-150 PCR (Poolscreen) testing is carried out in black flies, as the latter study is aimed at detecting if black flies have “skin snipped” humans

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CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

Summary of findings: DTA studies

Outcome Number of Consideration Summary of Evidence Certainty of studies and s of GRADE findings – DTA regarding the evidence design domains characteristics interuption of (GRADE) transmisison

O-150 PCR of skin snips vs skin snip microscopy

Sensitivity and Sensitivity specificity (True 7 studies Serious RoB, ranges from No direct Positives, False (1204 patients) serious 71% to evidence Negatives, True indirectness 100% Negatives, ⊕⊕⊝⊝

False Positives) Low Specificity ranges from 5 15% to 6 100%

Ov-16 serology vs. skin snip microscopy

Sensitivity and 4 studies (1425 Serious RoB, Sensitivity No direct specificity (True patients/ serious ranges from evidence Limitations of the DTA evidence include that Positives, False samples) indirectness 81% to 98% the studies were mostly performed in Negatives, True ⊕⊕⊝⊝ populations with high onchocerciasis Negatives, prevalence and that the reference standard False Positives) Specificity Low used (generally skin snip microscopy) is ranges from imperfect. 89% to In the DTA-studies included in this review, 100% skin snip microscopy was used as a All outcomes No comparative evidence available in eligible DTA studies reference standard; therefore only inverted calculations of values (see Appedix C of the Inconclusive No evidence available in eligible DTA studies supporting document) could be used to test estimate DTA performance of skin snip microscopy. Thus, observational evidence Complications No evidence available in eligible DTA studies should be considered rather than evidence from DTA studies.

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CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

Uncertain: 5 votes Probably yes: 1 vote Yes: 1 vote No Probably Uncertain Probably Yes Varies* In general, accurate cost information on diagnostic tests for human

Are the resources No Yes In most countries, capital equipment and expertise are already

required small? onchocerciasis Onchocerca volvulus is sparse. Overall, costs of programs X X X 2 available. for small populations are higher than for larger regions/towns/populations. Skin snip microscopy is 10 times less expensive than O-150 PCR of skin snips.

RESOURCE USE No incremental cost-effectiveness information was found. We retrieved

Is the incremental cost No Probably Uncertain Probably Yes Varies Uncertain: 5 votes one systematic review from with searches performed up to 2010 that small relative to the net No Yes Probably yes: 1 vote

declared that “no studies on the costs of diagnostics for onchocerciasis benefits? Yes: 1 vote were available”.1

What would be the Increased Probably Uncertain Probably Reduced Varies We know that there is some indication of differences in onchocercal skin

The panel considered equity and concluded it Is not relevant for Y impact increased reduced disease in women and men and in different racial groups (Yoruba vs

the choice of the test. As such, no judgement was made. EQUIT on health inequities? Fulani), but not for any differences for tests for human onchocerciasis.3

Factors that affect feasibility (points taken from a study on the implementation of CDI in Africa):4 The less feasible (capable of being accomplished or brought - Existence of procurement and supply policy about) an option is, the less likely it is that it should be - Support from the Ministry of Health

recommended (i.e. the more barriers there are that would be - Support from the Frontline Health Facilities difficult to overcome). No Probably Uncertain Probably Yes Varies* - Health workers attitudes, motivation and outreach

Is the option feasible to No Yes Factors that affect feasibility (in general for engaging communities, study in

The test is currently employed for monitoring and evaluation implement? X Africa):4 purposes in most onchocerciasis-endemic countries. FEASIBILITY - Year-round geographical accessibility

- Participatory approached to community mobilization Efforts in the fields are comparable to those necessary to - Community perceives value of interventions implement Ov-16 serology (ELISA) testing. - Community perceives value of community-directed approach - Political leadership in communities

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CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

Acceptability might reflect who benefits (or is harmed) and who pays (or saves); and when the benefits, adverse effects, and costs occur

Community participation (or “community driven intervention”) enhances No: 3 votes sustainability.4,5 Probably no: 2 votes No Probably Uncertain Probably Yes Varies* Yes: 2 votes Is the option acceptable? No Yes

“The value of properly informing and empowering communities, to enlist

them as crucial allies in the disease control efforts, was also High rate of refusal has been observed in some communities; as appreciated.”6,7 such communities are likely to refuse MDA as well, information generated is biased. On the other hand, as the test has been performed for many years, its acceptability is high in other communities.

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ALL SETTINGS (including different resources and varying vector efficiency)

X X X

Type of recommendation Strong Recommendation for skin snip microscopy Conditional (weak) recommendation for skin snip microscopy Balance of consequences Final vote: 2 votes for “Undesirable

X Only in the context of rigorous research consequences probably outweigh

Only with targeted monitoring and evaluation desirable consequences”; 3 votes for

“The balance between desirable and X Only in specific contexts undesirable consequences is closely balanced or uncertain”; 2 votes for “Desirable consequences clearly outweigh undesirable consequences in most settings Strong Recommendation against skin snip microscopy Conditional (weak) recommendation against skin snip microscopy Type of recommendation Final vote: 2 votes for “strong

X recommendation for skin snip microscopy”; 2 votes for “strong recommendation against skin snip microscopy”; 3 votes for “conditional (weak) recommendation for skin snip microscopy”. Voting results allow adopting a “conditional (weak) recommendation against skin snip microscopy”

Recommendation (text) WHO suggests not using skin snip microscopy to demonstrate the interruption of transmission of Onchocerca volvulus in a human population receiving mass drug administration (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 sample size providing adequate statistical certainty that programmatic goals have been reached 6 (conditional recommendation, low certainty of evidence)

−

Justification

Implementation Skin snip microscopy is currently used as a monitoring and evaluation tool during the treatment phase, which is an appropriate context as sensitivity is still relatively high. Its use to considerations, including decide on whether MDA should be stopped is discouraged, but it is still considered acceptable as a transitional test, while other replacement tests (notably Ov-16 serology) are monitoring and evaluation being introduced.

Reseach priorities Acceptability in low-prevalence settings should be investigated.

−

Definitions for ratings of the certainty of the evidence (GRADE)**

*Substantially different: large enough difference that it might have an effect on a decision

1. Keating J, Yukich JO, Mollenkopf S, Tediosi F. Lymphatic filariasis and onchocerciasis prevention, treatment, and control costs across diverse settings: A systematic review. Acta Trop. 2014;135:86–95. doi:10.1016/j.actatropica.2014.03.017. 2. Katabarwa MN, Habomugisha P, Richards FO, Jr. Implementing community-directed treatment with ivermectin for the control of onchocerciasis in Uganda (1997-2000): an evaluation. Ann Trop Med Parasitol. 2002;96(1):61–73. 3. Brieger WR, Ososanya OO, Kale OO, Oshiname FO, Oke GA. Gender and ethnic differences in onchocercal skin disease in Oyo State, Nigeria. Trop Med Int Health. 1997;2(6):529–34. 4. Mutalemwa P, Kisinza WN, Kisoka WJ, Kilima S, Njau J, Tenu F et al. Community directed approach beyond ivermectin in Tanzania: a promising mechanism for the delivery of complex health interventions. Tanzan J Health Res. 2009;11(3):116–25. 5. Makaula P, Bloch P, Banda HT, Bongolo Mbera G, Mangani C, de Sousa A et al. Primary health care in rural Malawi – a qualitative assessment exploring the relevance of the community-directed interventions approach. BMC Health Serv Res. 2012;12:328. doi:10.1186/1472-6963-12-328. 6. Boatin B. The Onchocerciasis Control Programme in West Africa (OCP). Ann Trop Med Parasitol. 2008;102 Suppl1:13–7. doi:10.1179/136485908X337427. 7. Rodriguez-Perez MA, Danis-Lozano R, Rodriguez MH, Bradley JE. Comparison of serological and parasitological assessments of Onchocerca volvulus transmission after 7 years of mass ivermectin treatment in Mexico. Trop Med Int Health. 1999;4(2):98–104.

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1 −

Annex 3.4 Decision tables: ocular infection (KQ1)

Health system and public health evidence to recommendations framework KQ 1.5: In a human population receiving MDA (mass drug administration) against onchocerciasis, should assessment of ocular infection (presence of microfilariae in the anterior chamber) be used to demonstrate the interruption of transmission of Onchocerca volvulus for the purpose of stopping MDA?

Problem: Demonstrating the interruption of transmission of Onchocerca volvulus Background: Onchocerciasis was originally endemic in 38 countries: 30 in WHO's African Region, six in the Pan Options: Using the following test(s): ocular infection American Health Region and two in the Eastern Mediterranean Region; it is estimated that about 99% of the global Comparison: No test burden currently occurs in Africa. Recently, several countries have made exceptional progress in interrupting Setting: Areas that have received MDA and are being evaluated for the transmission and are ready to apply for independent assessment and acknowledgement of such achievement confirmation of interruption of transmission of Onchocerca volvulus (“verification of elimination”). The epidemiology of onchocerciasis is that of a vector-borne disease, where human beings are the only natural vertebrate host, and infection rates and intensity are determined by the degree of

1 −

CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

What is the overall level of accuracy of Very inaccurate this evidence?

Summary of findings: observational studies

Outcome Number of Consideration Interruption of Elimination of Certainty of studies & s of GRADE transmission on- the evidence design domains (MDA stopped) chocerciasis (GRADE)

Opthalmological evaluation 1 study Serious NOT Limitations of the observational evidence What is the ⊕⊕⊝⊝ No (Microfilariae in (365 indirectness, CONFIRMED CONFIRMED were that no comparisons between tests overall included the anterior Low individuals) serious RoB were performed. The included studies certainty of studies Very low Low Moderate High segment)

represent prospective case studies, and for this evidence? X

three tests only one case study publication BENEFITS & HARMS OF THE OPTIONS was located. Summary of findings: DTA studies

Outcome Number of Considerations Summary of Evidence Certainty of studies and of GRADE findings – DTA regarding the evidence design domains characteristics interuption of (GRADE) transmisison Ocular infection vs other tests

All outcomes No comparative evidence available in eligible DTA studies

Inconclusive No evidence available in eligible DTA studies test

Complications No evidence available in eligible DTA studies

2 −

CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

No Probably Uncertain Probably Yes Varies* In general, accurate cost information on diagnostic tests for human

Are the resources No Yes

required small? onchocerciasis Onchocerca volvulus is sparse. Overall, costs of programs X 2

for small populations are higher than for larger regions/towns/populations.

No incremental cost-effectiveness information was found. We retrieved one

Is the incremental cost No Probably Uncertain Probably Yes Varies No Yes systematic review from with searches performed up to 2010 that declared

RESOURCE USE small relative to the net Benefits are small, while costs are high.

that “no studies on the costs of diagnostics for onchocerciasis were benefits? X

available”.1

Increased Probably Uncertain Probably Reduced Varies We know that there is some indication of differences in onchocercal skin What would be the impact The panel considered equity and concluded that it is not relevant increased reduced disease in women and men and in different racial groups (Yoruba vs

on health inequities? 3 for the choice of the test. As such, no judgement was made. EQUITY Fulani), but not for any differences for tests for human onchocerciasis.

Factors that affect feasibility (points taken from a study on the implementation of CDI in Africa):4 The less feasible (capable of being accomplished or brought about)

- Existence of procurement and supply policy an option is, the less likely it is that it should be recommended (i.e. - Support from the Ministry of Health the more barriers there are that would be difficult to overcome). No Probably Uncertain Probably Yes Varies* Is the option feasible to No Yes - Support from the Frontline Health Facilities

- Health workers attitudes, motivation and outreach Judgement is “no” as carrying out this test requires expensive implement? X

Factors that affect feasibility (in general for engaging communities, study in equipment and extensive technical expertise. Assessing ocular FEASIBILITY Africa):4 infection is also time-consuming especially in low-endemic settings, - Participatory approached to community mobilization thus making screening of large number of people impractical. - Community perceives value of interventions - Community perceives value of community-directed approach - Political leadership in communities

Acceptability might reflect who benefits (or is harmed) and who Community participation (or “community driven intervention”) enhances pays (or saves); and when the benefits, adverse effects, and costs sustainability.4,5 occur. No Probably Uncertain Probably Yes Varies*

Is the option acceptable? No Yes

“The value of properly informing and empowering communities, to enlist Communities are usually happy with this test, as it is a free medical X them as crucial allies in the disease control efforts, was also appreciated.”6, examination. Ophthalmologists and other involved professionals 7 are less happy because of its inaccuracy and low value for money.

3 −

ALL SETTINGS (including different resources and varying vector efficiency)

Type of recommendation Strong Recommendation for ocular infection Conditional (weak) recommendation for ocular infection

Only in the context of rigorous research

Only with targeted monitoring and evaluation

Only in specific contexts

Strong Recommendation against ocular infection Conditional (weak) recommendation against ocular infection

Recommendation (text) WHO recommends not using assessment of ocular infection (presence of microfilariae in the anterior chamber) to demonstrate the interruption of transmission of Onchocerca volvulus in a human population receiving mass drug administration (MDA) against onchocerciasis, for the purpose of stopping MDA.

(strong recommendation, low certainty of evidence)

Justification High inaccuracy, high cost and need for specialized personnel

Implementation N/A considerations, including monitoring and evaluation

Research priorities N/A

4 −

Definitions for ratings of the certainty of the evidence (GRADE)**

*Substantially different: large enough difference that it might have an effect on a decision

5 −

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Annex 3.5 Decision tables: O-150 PCR (Poolscreen) testing in black flies (KQ2)

Health system and public health evidence to recommendations framework KQ 2.1: In a human population, should O-150 PCR (Poolscreen) testing in black flies be used to confirm the interruption of transmission of Onchocerca volvulus?

Problem: Confirming the interruption of transmission of human onchocerciasis Background: Onchocerciasis was originally endemic in 38 countries: 30 in WHO's African Region, six in the Pan Options: Using O-150 PCR (Poolscreen) testing in black flies American Health Region and two in the Eastern Mediterranean Region; it is estimated that about 99% of the global Comparison: No test burden currently occurs in Africa. Recently, several countries have made exceptional progress in interrupting Setting: Primarily areas that have received MDA and are being evaluated for the transmission and are ready to apply for independent assessment and acknowledgement of such achievement confirmation of elimination of human onchocerciasis (“verification of elimination”). The epidemiology of onchocerciasis is that of a vector-borne disease, where human beings are the only natural vertebrate host, and infection rates and intensity are determined by the degree of

−

CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

What is the overall level of accuracy of Very accurate this evidence?

Summary of findings: Observational studies

Outcome Number of Considerations Interruption of Elimination of Certainty of Historical data from Mali indicated that PCR studies and of GRADE transmission on-chocerciasis the test could distinguish Onchocerca volvulus design domains (MDA stopped) evidence from Onchocerca ochengi while dissection of (GRADE) black flies could not What is the No Infectivity rate overall included of black flies Additional references: studies Very low Low Moderate High 2 studies None CONFIRMED CONFIRMED ⊕⊕⊕⊕ certainty of 1 (F3H/1000; O- (505 439 flies)  Lovato et al. 2014 this evidence? X 150 PCR, HIGH  Guevara et al. 20032

Poolscreen) BENEFITS & HARMS OF THE OPTIONS

Summary of findings: DTA studies 2 −

CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

Outcome Number of Considerations Summary of Evidence Certainty of studies and of GRADE findings – DTA regarding the evidence design domains characteristics interuption of (GRADE) transmisison

O-150 PCR (Poolscreen) vs dissection of black flies

Inconclusive No evidence available in eligible DTA studies test

Complications No evidence available in eligible DTA studies

3 −

CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

In general, accurate cost information on diagnostic tests for human O-150 PCR (Poolscreen) costs are estimated at US$ 0.19 cents of onchocerciasis Onchocerca volvulus is sparse. Three scientific studies a per fly examined (Gopal et al. 20124) provide information on costs for PCR that may be relevant for decision- making: Factors influencing costs include: No Probably Uncertain Probably Yes Varies* - Larger pools of lies for DNA are more cost efficient than smaller  Catching flies, often a time-consuming exercise Are the resources No Yes pools.4,5  Distance between fly catching points and laboratory

required small? X  Availability of a PCR machine (it could be borrowed - Capital costs for PCR test may be high, but ongoing costs are lower than dissection,6 and the test could serve as a reference from other health programmes, or if available, used for for other parasites and pathogens.5 other diseases)  Variability of costs between countries and continents. Overall, costs of programs for small populations are higher than for larger RESOURCE USE regions/towns/populations.7

No incremental cost-effectiveness information was found. We retrieved one

Is the incremental cost No Probably Uncertain Probably Yes Varies systematic review from with searches performed up to 2010 that declared small relative to the net No Yes Judgement is “probably yes” due to lack of evidence.

that “no studies on the costs of diagnostics for onchocerciasis were benefits? X

available”.3

on health inequities? 8 choice of the test. As such, no judgement was made. EQUITY Fulani), but not for any differences for tests for human onchocerciasis.

4 −

ALL SETTINGS (including different resources and varying vector efficiency)

CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

implement? X Africa):9 the more barriers there are that would be difficult to overcome).

them as crucial allies in the disease control efforts, was also appreciated.”10 occur. X

5 −

Type of recommendation Strong Recommendation for O-150 PCR (Poolscreen) in black flies Conditional (weak) recommendation for O-150 PCR (Poolscreen) in black flies

Only in the context of rigorous research

Only with targeted monitoring and evaluation

Only in specific contexts

Strong Recommendation against O-150 PCR (Poolscreen) in black Conditional (weak) recommendation against O-150 PCR (Poolscreen) in flies black flies

Recommendation (text) WHO recommends using O-150 PCR (Poolscreen) testing in black flies to confirm the interruption of transmission of Onchocerca volvulus

(strong recommendation, high certainty of evidence)

Justification O-150 PCR (Poolscreen) has been validated in different epidemiological settings (African Region and Region of the Americas)

Implementation Existence of regional laboratories serving the largest possible administrative area considerations, including Sampling should be appropriately conducted and information on sampling sites and sampling strategies should be made available so as to standardize practices (the African monitoring and evaluation Programme for Onchocerciasis Control has developed standard operating procedures that should be included as an annex to the guidelines) Laboratory quality control should be monitored

Research priorities More investigations should be made to define appropriate and standardized protocols for fly catching

6 −

Definitions for ratings of the certainty of the evidence (GRADE)**

*Substantially different: large enough difference that it might have an effect on a decision

7 −

1. Lovato R, Guevara A, Guderian R, Proaño R, Unnasch T, Criollo H et al. Interruption of infection transmission in the onchocerciasis focus of Ecuador leading to the cessation of ivermectin distribution. PLoS Negl Trop Dis. 2014;8(5):e2821. doi:10.1371/journal.pntd.0002821. 2. Guevara AG, Vieira JC, Lilley BG, López A, Vieira N, Rumbea J et al. Entomological evaluation by pool screen polymerase chain reaction of Onchocerca volvulus transmission in Ecuador following mass Mectizan distribution. Am J Trop Med Hyg. 2003;68(2):222–7. 3. Keating J, Yukich JO, Mollenkopf S, Tediosi F. Lymphatic filariasis and onchocerciasis prevention, treatment, and control costs across diverse settings: a systematic review. Acta Trop. 2014;135:86-95. doi:10.1016/j.actatropica.2014.03.017. 4. Gopal H, Hassan HK, Rodriguez-Perez MA, Toe LD, Lustigman S, Unnasch TR. Oligonucleotide based magnetic bead capture of Onchocerca volvulus DNA for PCR pool screening of vector black flies. PLoS Negl Trop Dis. 2012;6(6):e1712. doi:10.1371/journal.pntd.0001712. 5. Rodriguez-Perez MA, Gopal H, Adeleke MA, De Luna-Santillana EJ, Gurrola-Reyes JN, Guo X. Detection of Onchocerca volvulus in Latin American black flies for pool screening PCR using high-throughput automated DNA isolation for transmission surveillance. Parasitology research. Nov 2013;112(11):3925-3931. 6. Yamèogo L, Toe L, Hougard JM, Boatin BA, Unnasch TR. Pool screen polymerase chain reaction for estimating the prevalence of Onchocerca volvulus infection in Simulium damnosum sensu lato: results of a field trial in an area subject to successful vector control. Am J Trop Med Hyg. 1999;60(1):124–8. 7. Katabarwa MN, Habomugisha P, Richards FO, Jr. Implementing community-directed treatment with ivermectin for the control of onchocerciasis in Uganda (1997-2000): an evaluation. Ann Trop Med Parasitol. 2002;96(1):61–73. 8. Brieger WR, Ososanya OO, Kale OO, Oshiname FO, Oke GA. Gender and ethnic differences in onchocercal skin disease in Oyo State, Nigeria. Trop Med Int Health. 1997;2(6):529–34. 9. Mutalemwa P, Kisinza WN, Kisoka WJ, Kilima S, Njau J, Tenu F et al. Community directed approach beyond ivermectin in Tanzania: a promising mechanism for the delivery of complex health interventions. Tanzan J Health Res. 2009;11(3):116–25. 10. Boatin B. The Onchocerciasis Control Programme in West Africa (OCP). Ann Trop Med Parasitol. 2008;102 Suppl1:13–7. doi:10.1179/136485908X337427.

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Annex 3.6 Decision tables: Ov-16 serology (KQ2)

Health system and public health evidence to recommendations framework KQ 2.2: In a human population, should Ov-16 serology (ELISA) testing in children be used to confirm the interruption of transmission of Onchocerca volvulus?

Problem: Confirming the interruption of transmission of human onchocerciasis Background: Onchocerciasis was originally endemic in 38 countries: 30 in WHO's African Region, six in the Pan Options: Using Ov-16 serology in children American Health Region and two in the Eastern Mediterranean Region; it is estimated that about 99% of the global Comparison: No test burden currently occurs in Africa. Recently, several countries have made exceptional progress in interrupting Setting: Primarily areas that have received MDA and are being evaluated for the transmission and are ready to apply for independent assessment and acknowledgement of such achievement confirmation of elimination of human onchocerciasis (“verification of elimination”). The epidemiology of onchocerciasis is that of a vector-borne disease, where human beings are the only natural vertebrate host, and infection rates and intensity are determined by the degree of

1 −

CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

What is the

overall level No Probably Uncertain Probably Yes Varies of accuracy of No Yes

this

evidence?

The systematic review conducted for the WHO panel identified two observational studies and 15 DTA studies that provide limited evidence on the comparative use of tests for confirmation of interruption of Onchocerca volvulus (KQ1) and elimination of human onchocerciasis (KQ2). The results presented below are a summarized Limitations of the observational evidence form of those from the systematic review and from the evidence profiles presented in the supporting document. were that no comparisons between tests were performed. The included studies represent prospective case studies, and for Summary of findings: Observational studies Ov-16 serology, skin snip microscopy, and infection of the eye only one case study publication was located. Outcome Number of Considerations Interruption of Elimination of Certainty of studies and of GRADE transmission onchocerciasis the What Is the No design domains (MDA stopped) evidence overall included (GRADE) certainty of studies Very low Low Moderate High

this evidence? X Antibody NOT

prevalence (Ov- 1 study Serious CONFIRMED CONFIRMED ⊕⊕⊝⊝ BENEFITS & HARMS OF THE OPTIONS

16 serology) (3118 children) indirectness, Low

serious RoB

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CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

Limitations of the DTA evidence include that Summary of findings: DTA studies the studies were mostly performed in populations with high onchocerciasis Outcome Number of Considerations Summary of Evidence Certainty of prevalence and that the reference standard studies and of GRADE findings – DTA regarding the evidence used (generally skin snip microscopy) is design domains characteristics interuption of (GRADE) imperfect. transmisison

Ov-16 serology vs skin snip microscopy

Sensitivity and 4 studies (1425 Serious RoB, Sensitivity No direct specificity (True patients/ serious ranges from evidence Positives, False samples) indirectness 81% to 98% Negatives, True Negatives, ⊕⊕⊝⊝ False Positives) Specificity Low ranges from 89% to 100%

Inconclusive No evidence available in eligible DTA studies test

Complications No evidence available in eligible DTA studies

3 −

CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

required small? X Overall, costs of programs for small populations are higher than for larger Consumables: US$ 0.15 cents per ELISA test regions/towns/populations.2

No incremental cost-effectiveness information was found. We retrieved one

that “no studies on the costs of diagnostics for onchocerciasis were Cost is low if compared to cost of continuing MDA benefits? X X

available”.1

Increased Probably Uncertain Probably Reduced Varies We know that there is some indication of differences in onchocercal skin What would be the impact The panel considered equity and concluded it Is not relevant for the increased reduced disease in women and men and in different racial groups (Yoruba vs

on health inequities? 3 choice of the test. As such, no judgement was made. EQUITY Fulani), but not for any differences for tests for human onchocerciasis.

Factors that affect feasibility (points taken from a study on the implementation of CDI in Africa):4 - Existence of procurement and supply policy Support by non-governmental development organization (NGDOs) - Support from the Ministry of Health is crucial to ensure feasibility. No Probably Uncertain Probably Yes Varies* - Support from the Frontline Health Facilities Is the option feasible to No Yes - Health workers attitudes, motivation and outreach Testing for Ov-16 serology (ELISA) can be combined with testing

implement? Factors that affect feasibility (in general for engaging communities, study in carried out in the context of TAS for lymphatic filariasis.

FEASIBILITY Africa): 4 - Year-round geographical accessibility No cold chain required. - Participatory approached to community mobilization - Community perceives value of interventions - Community perceives value of community-directed approach - Political leadership in communities

Community participation (or “community driven intervention”) enhances Acceptability might reflect who benefits (or is harmed) and who sustainability.4,5 pays (or saves); and when the benefits, adverse effects and costs No Probably Uncertain Probably Yes Varies* occur. Is the option acceptable? No Yes “The value of properly informing and empowering communities, to enlist

X them as crucial allies in the disease control efforts, was also appreciated.”6 Minimally invasive test

Finger-prick more acceptable in some communities.7

4 −

ALL SETTINGS (including different resources and varying vector efficiency)

X X

Type of recommendation Strong Recommendation for Ov-16 serology Conditional (weak) recommendation for Ov-16 serology Balance of consequences Final vote: 2 votes for “desirable

Only in the context of rigorous research consequences probably outweigh

Only with targeted monitoring and evaluation undesirable consequences”, and 5

votes for “desirable consequences X Only in specific contexts clearly outweigh undesirable consequences inmost settings”

Type of recommendation Conditional recommendation: only in specific contexts, i.e. when O- 150 PCR (Poolscreen) testing in blackflies is at or near threshold and cannot be considered fully satisfactory Strong Recommendation against Ov-16 serology Conditional (weak) recommendation against Ov-16 serology

Recommendation (text) WHO suggests using Ov-16 serology (ELISA) testing in children to confirm the interruption of transmission of O. volvulus, conditional to results of O-150 PCR (Poolscreen) testing in blackflies at or near threshold, and thus not fully satisfactory.

(conditional recommendation, low certainty of evidence)

Justification Accurate test with relatively low cost

5 −

Research priorities Sero-reversion of Ov-16 responses should be investigated (by age, over time). The Ov-16 Rapid Test should be validated as a possible replacement for the standard test.

6 −

Definitions for ratings of the certainty of the evidence (GRADE)**

*Substantially different: large enough difference that it might have an effect on a decision

7 −

1. Keating J, Yukich JO, Mollenkopf S, Tediosi F. Lymphatic filariasis and onchocerciasis prevention, treatment, and control costs across diverse settings: a systematic review. Acta Trop. 2014;135:86-95. doi:10.1016/j.actatropica.2014.03.017. 2. Katabarwa MN, Habomugisha P, Richards FO, Jr. Implementing community-directed treatment with ivermectin for the control of onchocerciasis in Uganda (1997-2000): an evaluation. Ann Trop Med Parasitol. 2002;96(1):61–73. 3. Brieger WR, Ososanya OO, Kale OO, Oshiname FO, Oke GA. Gender and ethnic differences in onchocercal skin disease in Oyo State, Nigeria. Trop Med Int Health. 1997;2(6):529–34. 4. Mutalemwa P, Kisinza WN, Kisoka WJ, Kilima S, Njau J, Tenu F et al. Community directed approach beyond ivermectin in Tanzania: a promising mechanism for the delivery of complex health interventions. Tanzan J Health Res. 2009;11(3):116–25. 5. Makaula P, Bloch P, Banda HT, Bongolo Mbera G, Mangani C, de Sousa A et al. Primary health care in rural Malawi – a qualitative assessment exploring the relevance of the community-directed interventions approach. BMC Health Serv Res. 2012;12:328. doi:10.1186/1472-6963-12-328. 6. Boatin B. The Onchocerciasis Control Programme in West Africa (OCP). Ann Trop Med Parasitol. 2008;102 Suppl1:13–7. doi:10.1179/136485908X337427. 7. Rodriguez-Perez MA, Danis-Lozano R, Rodriguez MH, Bradley JE. Comparison of serological and parasitological assessments of Onchocerca volvulus transmission after 7 years of mass ivermectin treatment in Mexico. Trop Med Int Health. 1999;4(2):98–104.

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Annex 3.7 Decision tables: ocular infection (KQ2)

Health system and public health evidence to recommendations framework KQ 2.5: In a human population, should assessment of ocular infection (presence of microfilariae in the anterior chamber) be used to confirm the interruption of transmission of Onchocerca volvulus?

Problem: Confirming the interruption of transmission of Onchocerca volvulus Background: Onchocerciasis was originally endemic in 38 countries: 30 in WHO's African Region, six in the Pan Options: Using the following test(s): ocular infection American Health Region and two in the Eastern Mediterranean Region; it is estimated that about 99% of the global Comparison: No test burden currently occurs in Africa. Recently, several countries have made exceptional progress in interrupting Setting: Primarily areas that have received MDA and are being evaluated for the transmission and are ready to apply for independent assessment and acknowledgement of such achievement confirmation of transmission of Onchocerca volvulus (“verification of elimination”). The epidemiology of onchocerciasis is that of a vector-borne disease, where human beings are the only natural vertebrate host, and infection rates and intensity are determined by the degree of

1 −

CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

What is the overall level Very inaccurate of accuracy of

this evidence?

The systematic review conducted for the WHO panel identified two observational studies and 15 DTA studies

that provide limited evidence on the comparative use of tests for confirmation of interruption of Onchocerca

volvulus (KQ1) and elimination of human onchocerciasis (KQ2). The results presented below are a summarized

form of those from the systematic review and from the evidence profiles presented in the supporting document.

Summary of findings: observational studies

Outcome Number of Consideration Interruption of Elimination of Certainty of studies and s of GRADE transmission onchocerciasis the design domains (MDA stopped) evidence (GRADE)

Ophthalmolo- gical evaluation 1 study Serious NOT ⊕⊕⊝⊝ (microfilariae in Limitations of the observational evidence What Is the No (365 indirectness, CONFIRMED CONFIRMED the anterior Low were that no comparisons between tests overall included individuals) serious RoB segment) were performed. The included studies certainty of studies Very low Low Moderate High

represent prospective case studies, and for this evidence? X three tests only one case study publication

BENEFITS & HARMS OF THE OPTIONS

was located. Summary of findings: DTA studies

Outcome Number of Considerations Summary of Evidence Certainty of studies and of GRADE findings – DTA regarding the design domains characteristics interuption of evidence transmisison (GRADE)

Occular morbidity vs other tests

All outcomes No comparative evidence available in eligible DTA studies

Inconclusive No evidence available in eligible DTA studies test

Complications No evidence available in eligible DTA studies

2 −

CRITERIA JUDGEMENTS RESEARCH EVIDENCE ADDITIONAL INFORMATION

No Probably Uncertain Probably Yes Varies* Are the resources In general, accurate cost information on diagnostic tests for human No Yes

required small? onchocerciasis Onchocerca volvulus is sparse. Overall, costs of programs X 2

for small populations are higher than for larger regions/towns/populations.

No incremental cost-effectiveness information was found. We retrieved one

Is the incremental cost No Probably Uncertain Probably Yes Varies RESOURCE USE systematic review from with searches performed up to 2010 that declared small relative to the net No Yes Benefits are small, while costs are high.

that “no studies on the costs of diagnostics for onchocerciasis were benefits? X

available”.1

Increased Probably Uncertain Probably Reduced Varies We know that there is some indication of differences in onchocercal skin What would be the impact increased reduced The panel considered equity and concluded that it is not relevant UITY disease in women and men and in different racial groups (Yoruba vs

on health inequities? 3 for the choice of the test. As such, no judgement was made. EQ Fulani), but not for any differences for tests for human onchocerciasis.

Factors that affect feasibility (points taken from a study on the implementation of CDI in Africa):4 - Existence of procurement and supply policy The less feasible (capable of being accomplished or brought about) - Support from the Ministry of Health

an option is, the less likely it is that it should be recommended (i.e. - Support from the Frontline Health Facilities the more barriers there are that would be difficult to overcome). No Probably Uncertain Probably Yes Varies* - Health workers attitudes, motivation and outreach

Is the option feasible to No Yes Factors that affect feasibility (in general for engaging communities, study in

Judgement is “no” as carrying out this test requires expensive implement? X Africa):4 equipment and extensive technical expertise. Assessing ocular FEASIBILITY - Year-round geographical accessibility infection is also time-consuming especially in low-endemic settings, - Participatory approached to community mobilization thus making screening of large number of people impractical. - Community perceives value of interventions - Community perceives value of community-directed approach - Political leadership in communities

Acceptability might reflect who benefits (or is harmed) and who Community participation (or “community driven intervention”) enhances pays (or saves); and when the benefits, adverse effects and costs sustainability.4,5 occur. No Probably Uncertain Probably Yes Varies*

Is the option acceptable? No Yes

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ALL SETTINGS (including different resources and varying vector efficiency)

Type of recommendation Strong Recommendation for ocular morbidity Conditional (weak) recommendation for ocular morbidity

Only in the context of rigorous research

Only with targeted monitoring and evaluation

Only in specific contexts

Strong Recommendation against ocular morbidity Conditional (weak) recommendation against ocular morbidity

Recommendation (text) WHO recommends not using assessment of ocular infection (presence of microfilariae in the anterior chamber) to confirm the interruption of Onchocerca volvulus.

(strong recommendation, low certainty of evidence)

Justification High inaccuracy, high cost and need for specialized personnel

Implementation N/A considerations, including monitoring and evaluation

Research priorities N/A

4 −

Definitions for ratings of the certainty of the evidence (GRADE)**

*Substantially different: large enough difference that it might have an effect on a decision

1. Keating J, Yukich JO, Mollenkopf S, Tediosi F. Lymphatic filariasis and onchocerciasis prevention, treatment, and control costs across diverse settings: A systematic review. Acta Trop. 2014;135:86–95. doi:10.1016/j.actatropica.2014.03.017. 2. Katabarwa MN, Habomugisha P, Richards FO, Jr. Implementing community-directed treatment with ivermectin for the control of onchocerciasis in Uganda (1997-2000): an evaluation. Ann Trop Med Parasitol. 2002;96(1):61–73. 3. Brieger WR, Ososanya OO, Kale OO, Oshiname FO, Oke GA. Gender and ethnic differences in onchocercal skin disease in Oyo State, Nigeria. Trop Med Int Health. 1997;2(6):529–34. 4. Mutalemwa P, Kisinza WN, Kisoka WJ, Kilima S, Njau J, Tenu F et al. Community directed approach beyond ivermectin in Tanzania: a promising mechanism for the delivery of complex health interventions. Tanzan J Health Res. 2009;11(3):116–125. 5. Makaula P, Bloch P, Banda HT, Bongolo Mbera G, Mangani C, de Sousa A et al. Primary health care in rural Malawi – a qualitative assessment exploring the relevance of the community-directed interventions approach. BMC Health Serv Res. 2012;12:328. doi:10.1186/1472-6963-12-328. 6. Boatin B. The Onchocerciasis Control Programme in West Africa (OCP). Ann Trop Med Parasitol. 2008;102 Suppl1:13–7. doi:10.1179/136485908X337427. 7. Rodriguez-Perez MA, Danis-Lozano R, Rodriguez MH, Bradley JE. Comparison of serological and parasitological assessments of Onchocerca volvulus transmission after 7 years of mass ivermectin treatment in Mexico. Trop Med Int Health. 1999;4(2):98–104.

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Annex 4. Summary of declared interests

Participants Declared interests Level of participation Elie Akl Personal: none Partial Institutional: none Eddie Cupp Personal: none Full Institutional: none Yankum Dadzie Personal: none Full Institutional: none Mark Eberhard Personal: none Full Institutional: research grant from Bill & Melinda Gates Foundation (ended May 2014) Patrick Lammie Personal: none Full Institutional: none Peter Personal: travel grant from Danube Partial Mahlknecht University Krems Institutional: none David Molyneux Personal: none Full Institutional: none Kylie Thaler Personal: none Partial Institutional: none Thomas Personal: consulting fees from Mectizan Full Unnasch Donation Program; chair, Uganda Onchocerciasis Elimination Expert Advisory Committee; member, Ethiopia Onchocerciasis Expert Advisory Committee. Institutional: research grant from Bill & Melinda Gates Foundation Laurent Personal: none Full Yameogo Institutional: none

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Annex 5. Preparation of the country report To initiate the verification process, a comprehensive written report (the dossier) is submitted to an independent national committee (e.g. National Review Committee) or other entity qualified to evaluate the programme. The length and detail of the dossier will vary widely from a brief document for countries with few foci or transmission zones to highly detailed documents with supporting data from countries with many foci and a large at-risk population. National programmes are strongly encouraged to publish data relevant to elimination in the peer-reviewed scientific literature. If available, such publications may be included in the dossier in order to strengthen the case for elimination.

The dossier should include historical, epidemiological and programmatic information related to the process that led the country to the elimination goal. All endemic communities should have been identified and treated, and at least 75% of the total population (80–85% of the population eligible for treatment with ivermectin) living in these communities should have been treated during each round of treatment. In addition, methods and results of in-depth epidemiological and entomological surveys should be provided.

The dossier should contain each of the following common elements and details.

1. Historical account and background information on human onchocerciasis in the affected country  How the disease was discovered and/or imported into the country or focus.  Demographic information, including population distribution by geographical region of the country and indicating the populations in areas endemic for onchocerciasis.  Ethnographic information on the populations affected by onchocerciasis.  Economic activities of the affected regions – agriculture, mining, forestry, etc.  Migration patterns (including which communities in onchocerciasis endemic areas migrate periodically) within the country and between adjacent countries, especially those where onchocerciasis is also endemic.  Information on primary and secondary vectors of Onchocerca volvulus (including their parous biting cycles) and with their distribution shown on maps.  Health-care infrastructure and primary health-care services of the endemic areas. 2. Methodology and findings of original assessments of the extent of human onchocerciasis in the entire country  Methods used and data obtained from any epidemiological, entomological, serological and parasitological surveys as well as any other investigation carried out (e.g. morbidity surveys).  Details on timing, methodology, location, outcomes and interpretation of the results should be provided.  Information should be organized so as to show compliance with elimination criteria.  Where possible, data should also be presented in clear graphic or tabular format.

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 Maps delineating the endemic regions, including maps of major river basins, and areas investigated for onchocerciasis. These maps should be topographical and locate communities by name.  Lists of communities surveyed for onchocerciasis, giving the rationale for including them as well as reasons for not surveying adjacent communities or elsewhere in the country.  Justification, rationale and criteria used to determine the geographical extent of the foci, and to ascertain no transmission occurred in areas outside the known foci. Details on migration patterns to and from the identified foci; on methods of surveillance employed; and on significance, testing and treatment of migrants should be provided.  Pre-treatment results of rapid entomological assessment and rapid epidemiological mapping of onchocerciasis used to stratify endemic communities and to select sentinel communities.  Results of in-depth pre-treatment epidemiological surveys carried out in sentinel communities. 3. Detailed overview of the national elimination programme A detailed description of intervention efforts to date should be provided, including the following:  Participating organizations (Ministry of Health, regional programmes, nongovernmental development organizations, etc.) and their responsibilities and sources of financing.  Organizational chart delineating areas of responsibilities and personnel.  National policy, recommendations and interventions implemented against onchocerciasis (including surveillance in endemic foci and outside such foci).  Programme management, whether horizontal or vertical, methods of distribution (community directed, mass target, house-to-house versus central point).  Methods used to assure maximum coverage, such as health-educational programmes, community participation, etc. 4. Data on geographical, therapeutic and programmatic coverage of ivermectin mass drug administration achieved in each treatment round The data should comprise the following:  Total number of communities and individuals in each community eligible for treatment.  Total number of communities and eligible individuals treated by treatment round.  Updated census for each treatment round.  Steps taken to assure validity of census, maps, treatment lists and reported coverage.  Measures taken to control ivermectin tablets used versus those planned for use.  Any challenges faced by the programme to fulfil the verification criteria should be presented clearly.

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5. Evaluations of treatment effects and post-elimination surveillance The following information should be provided:  Data and results of in-depth epidemiological and entomological evaluations in sentinel communities.  Description and operation of post-endemic surveillance systems and results.  Description of the mechanisms and plans in place to support and maintain post- elimination surveillance and case-detection activities.  Details on roles and responsibilities and on organizational and financial arrangements.  Details on the risk of reintroduction of the parasite and how this will be monitored. 6. Bibliography and summary of published literature  A bibliography on onchocerciasis in the affected country should be compiled by the programme and the national programme archive should be accessible for inspection by the International Verification Team.  Any raw data collections, documents and publications in possession of the national programme should be available for consultation.

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Annex 6. Process of verification of elimination and role and responsibilities of the international verification team

A6.1 The process of verification To start the process of verification of elimination the applicant country must send a letter, accompanied by the country dossier, addressed to the Director-General of WHO, to be forwarded through the relevant WHO Country Office and the Regional Office. The letter should state that the country claims to have met the criteria for elimination of human onchocerciasis and request WHO to start the process of verification.

Upon receipt of the letter and the accompanying dossier, on behalf of the Director-General of WHO, the relevant technical unit at WHO headquarters, in consultation with its counterpart in the relevant WHO Regional Office, reviews the dossier. If the technical unit deems the dossier to be adequate, an international verification team (IVT) is appointed. The IVT should be composed of members and observers, and the secretariat functions assured by WHO staff. Upon appointment, members and observers of the IVT should be given a copy of the country dossier, and a copy of the present publication meant to provide a reference standard throughout the verification process. The IVT may decide to conduct a country visit, at the end of which a report should be forwarded to the WHO Director-General through the relevant WHO Country Office and the WHO Regional Office. Through the report, the IVT will be asked to reach one of three possible conclusions:

1. The data indicate that elimination has been achieved. 2. The data indicate that elimination has not been achieved. 3. The data do not allow a decision and additional data are required to reach a conclusion.

The report must provide details of the reasons for reaching one of the above conclusions, and provide guidance and recommendations to the country in case conclusions (2) or (3) are reached.

Upon receipt of the report, and based on its conclusions, the WHO Director-General will decide whether the verification process should be considered successful or unsuccessful, and will subsequently inform the national authorities of the applicant country of the decision taken, through official correspondence.

A6.2 The international verification team Members and observers of the IVT are appointed by WHO headquarters upon consultation with the relevant WHO Regional Office as WHO temporary advisers. The composition of the IVT may vary but should include a minimum of four members and two observers. The secretariat should be represented by one staff each from WHO headquarters and the Regional Office. WHO staff in Country Offices can also be part of the secretariat.

Members of the IVT should be experts on control and elimination of onchocerciasis, one of whom as an entomologist. They should not be a citizen of the applicant country and should not have provided recent support to the country in reviewing its onchocerciasis programme or in developing its dossier.

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Members should provide independent opinions and have no conflict of interest with regard to the statements made in the dossier. They are fully entitled to participate in the decision-making process and the development of the report.

Observers should be individuals selected for their knowledge of the onchocerciasis programme of the applicant country. Their participation is meant to provide technical input and advice to the IVT and to support the secretariat in liaising with the national authorities to facilitate any possible request by the IVT. However, at no time should observers make direct contact with national authorities without prior consultation with the WHO secretariat. In some instances, for example in populous countries, more than two observers may be required and could include staff experts from the regional programmes (Onchocerciasis Elimination Program for the Americas, African Programme for Onchocerciasis Control) supporting the country’s elimination efforts.

The secretariat is responsible for providing the IVT members and observers with the necessary documentation, liaising with the national authorities to organize and arrange the country visit if one is required, and ensuring the development, finalization and submission of the IVT report.

It is highly recommended that members, observers and the secretariat are fluent in the language in which the dossier is written and the language of use in the applicant country.

A6.3 The country visit Should the IVT decide that a visit to the applicant country is required for proper assessment, such a visit should be arranged by the WHO Secretariat. It is suggested that the visit last at least 5 working days. In the end, the duration of the mission will depend on the number of foci to be visited in the country.

The purpose of such a visit is to allow members of the IVT to become acquainted with the operations and personnel of the elimination programme, to evaluate the reliability of the claim made by the country, and to formulate its conclusions and recommendations.

The country visit can be preceded by teleconferences attended by members and observers of the IVT and WHO Secretariat; the IVT members can thus express their requirements and needs with regard to the visit and the information and data to be examined.

The methodology of work to be followed during the country visit should include:

1. Examination of the dossier and any other relevant document related to the programme; 2. Interviews with the personnel of the programme and other technical staff involved in onchocerciasis control or elimination activities at central or peripheral levels; 3. Interviews with community members living in one or more endemic foci (a visit to one or more communities located in an endemic area is highly recommended); 4. It is also recommended that the first day in the country be dedicated to internal discussions among the IVT on procedures, clarifications and planning.

The chairperson is selected by the members from among the membership of the team. The chairperson structures the meeting and regulates the contribution of observers to the discussion and

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deliberations as appropriate. The chairperson should ensure that the procedures and timing of discussions are appropriate; the chairperson also speaks on behalf of the IVT vis-à-vis the national authorities, together with the WHO secretariat.

The IVT report should be finalized and signed during the visit; if not possible, its finalization should take place immediately after the visit. The original copy should be forwarded by the relevant WHO Country Office to the WHO Director-General through the relevant WHO Regional Office. Upon consultation with the other members, the chairperson can inform the observers whether they can participate in drafting and finalizing the report.

If the national authorities of the applicant country request a debriefing before leaving the country, information on the conclusions reached can be provided. However, the national authorities should be reminded that the final decision will be taken by the Director-General of WHO and not by the IVT. Press releases at this stage should be strongly discouraged.

A6.4 The report of the international verification team Although the IVT can decide on the format of the report, the following sections should be included.

1. Executive summary

2. Abbreviations

3. Introduction (details on the request for verification, dates and details of the country visit, composition of the IVT)

4. Methodology used by the IVT to ascertain the claim made by the country

5. Activities (daily account of meetings held, people met, in-country movements, etc.)

6. Critical discussion of the interventions carried out by the country to control and eliminate onchocerciasis, with a focus on the monitoring and evaluation activities

7. Conclusions, in which the IVT discusses the compliance of the data with the elimination criteria set by WHO, and expresses its opinion on whether the verification process should be considered as successful or not.

8. Recommendations to the country: if verification is successful, recommendations should be focused on post-elimination surveillance activities; if verification is unsuccessful or a conclusion cannot be reached because data and other information are incomplete, recommendations should be focused on what steps the country should take in order to meet the elimination goal in the future.

9. References. If published literature is cited in the report, references should be listed in full.

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Annex 7. Entomological evaluation of the impact of community-wide ivermectin distribution on Onchocerca volvulus transmission

A7.1 General remarks The effects of ivermectin distribution on parasite transmission can be evaluated by monitoring infection rates of vector black flies with larvae of Onchocerca volvulus. This method has several advantages over parasitological evaluation of the human population, especially when children are involved, for the following reasons:

 Infection rates in black flies are rapid and sensitive indicators of the change in community microfilarial load that results from ivermectin distribution.  Changes in vector infection rates correlate well with the percentage coverage of the human population with ivermectin.  Absence of infective-stage larvae in the vector population during the transmission season is the first indicator of having achieved interruption of parasite transmission. Conversely, the prepatent period for the appearance of nodules or skin microfilariae is 10–24 months.  Monitoring very low vector infection rates with polymerase chain reaction and DNA technology is easier and less expensive than monitoring very low levels of infection in children.  Use of O. volvulus-specific DNA probes guarantees absolute specificity and allows for processing large numbers of black flies, thus increasing the reliability of the results.  Vector collection teams working in the community can deliver health messages about ivermectin, thereby increasing coverage.  The process is completely non-invasive and is well accepted by the community.

For pre-treatment baseline data, vector infection rates are ideally measured over a complete year, or at least a complete transmission season. This provides baseline data for comparison with post- treatment evaluations.

The collection methodology can be used also to investigate areas that might be susceptible to introduction of the parasite. The systematic collections will determine if a competent vector is present, and if the biting density is sufficiently high to support a transmission cycle. The methods used to collect black flies were originally described for Africa (1) and the Americas (2) and have been standardized and operationalized by the respective programmes (see below).

PCR technology versus dissection of black flies. To monitor the effects of larviciding or ivermectin in trials in both Africa and the Americas the annual transmission potential (ATP) was determined by individual dissection of hundreds of thousands of black flies. This effort is technically and financially challenging and clearly beyond the capacity of an operational programme. Moreover, repetitive treatment further reduces the microfilarial load in the community and infection rates in flies drop accordingly; as a result, an increased number of black flies must be examined to produce statistically 1

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reliable data. For this reason, the use of PCR technology is recommended rather than dissections, and programmes have universally accepted and switched to PCR technology to evaluate flies. Fly collections are grouped in pools and tested for O. volvulus larvae. Maximum pool sizes vary by species and by the method used to purify DNA from the pools. To ensure that the upper bound of the 95% confidence interval for prevalence of infective larvae is below an acceptable level (0.05%) at least 6000 flies must be screened per focus-relevant catching site. If the vector density is insufficient to permit the collection of this number of flies, sufficient collections must be carried out to ensure that the upper bound of the 95% confidence interval for the ATP is less than 20 (see discussion on ATP in section 1.4.1, and see below). Simulations using Poolscreen 3.0 have demonstrated that in areas with low annual biting rates, if collections are carried out 2 days per week during the transmission season and all collected flies are tested and found to be negative, the upper bound of the 95% confidence interval for the ATP will be within acceptable limits. The data are analysed by a statistical software program (Poolscreen) made available by the University of Alabama (Birmingham, USA) and the University of South Florida (Florida, USA) (3).

A7.2 Methodology The methodology used to conduct the entomological evaluations, including use of selected (or sentinel) communities, fly collection teams, collection sites and collection schedules, has been well operationalized. Although somewhat different methodologies exist among programmes, the ultimate data and threshold values are equivalent across them. For the specific details of fly collections and processing, interested readers should refer to the documents published by the Onchocerciasis Elimination Program for the Americas [OEPA] (4) and the African Programme for Onchocerciasis Control [APOC] (5).

A.7.2.1 Determining the annual biting rate Data analysis requires a biting rate as well as an infection rate. The biting rate is calculated using the models provided by the Poolscreen v.3.0 software package. The biting rate and the infection rate are also required in order to estimate the basic reproductive rate (Ro) for that focus. Therefore, when the flies are processed for PCR testing, the number of flies collected during each collection period must be counted and recorded. The location of the collections of these flies is also an important piece of information that must be recorded.

A.7.2.2 Processing of flies for PCR In the laboratory, flies from each collection period are examined under a stereo dissecting microscope and identified as to species; any flies containing ingested blood are discarded. Flies are then counted into “pools” by species. The heads are separated from the bodies and the heads and bodies are tested separately, because most infective stage larvae are found in the head. If resources are available, testing the bodies as well as the heads is acceptable. For example, if elimination has been documented for a focus and the purpose of testing flies is surveillance for renewed transmission, then it is important to test the bodies because a positive indicates previous contact with a microfilaria-positive person.

To assess interruption of transmission, the algorithm used in the OEPA is to test bodies first and, if a positive pool is detected, to immediately stop and switch to testing heads. If no positive body pools

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are detected, there is no need to test heads. The most important point is that if any evidence of contact between the parasite and the vector is detected, all heads must be tested to estimate the prevalence of L3 in the flies. Other programmes have chosen to test only heads, the rationale being that if positive bodies are detected, the heads will need to be tested. Both approaches work and have been operationalized in the field. More information about parasite–vector contact, and infection levels in the focus, is obtained from testing of fly bodies, but for convenience and cost reasons, only testing the heads can be a practical approach.

A7.3 Data reporting and analysis Three statistics can be reported:

1. Infectivity rate 2. Biting rate, and 3. Annual transmission potential.

The minimum requirements are the infectivity rate (that is, the proportion of flies carrying infective larvae based on heads alone) and the biting rate. The proportion of flies infected based on the flies’ bodies can be calculated if the bodies are processed. The proportion of infected flies is calculated from fly bodies only because if pools of heads and bodies both test positive, it is impossible to know whether the positives were caused by infective or immature parasite stages. Having infective stage larvae in the head with first- and second-stage larvae in the bodies simultaneously (asynchronous parasite development) is more frequent in vectors with a poorly developed cibarial armature, for example in Simulium metallicum, S. exiguum or S. damnosum. The Poolscreen programs v.2.0 or 3.0 calculate infection rate, biting rate and annual transmission potential with an associated 95% confidence interval