Diagnostics for onchocerciasis in the era of elimination

Diagnostics for onchocerciasis in the era of elimination Abstract In the past few years, efforts to eliminate onchocerciasis from Africa have intensified. These efforts are primarily based on the mass distribution of the anti-helminthic drug Mectizan™ (ivermectin). This program has led to the development of new guidelines by the World Health Organization for the verification that transmission has been suppressed and eventually eliminated. The requirements of diagnostic tools for this purpose differ in many ways from tests used to diagnose infection in individuals. In this review, we summarize the progress that has been made to identify diagnostics that meet the specialized requirements needed to verify onchocerciasis elimination, discuss why these tests were selected and summarize the needs that still exist to complete the arsenal of diagnostic tools that will be useful as the goal of elimination is achieved. Black fly, Diagnostics, Ivermectin, Onchocerca volvulus, River blindness Introduction A recent review has put forth the challenges faced by onchocerciasis elimination programs and how their diagnostic needs evolve as they transition from control verification of elimination.1 Here, we will focus upon the diagnostic needs to verify suppression and interruption of transmission in the context of the current WHO guidelines. Currently, onchocerciasis elimination programs rely primarily on mass drug administration (MDA) of Mectizan™ (ivermectin) to suppress and eventually eliminate transmission of Onchocerca volvulus, the causative agent of the disease. The onchocerciasis elimination program strategy is a multi-stage process.2 Initially, programs must obtain high enough treatment coverage in the eligible population to stop transmission. Once transmission is suppressed, high coverage must be maintained until all the fertile female parasites either die or become sterile. Once a suitable number of treatments have been given, transmission is believed to have been interrupted. At this point in time, the program conducts a comprehensive survey to demonstrate that transmission has been interrupted. These surveys rely upon measuring parasite exposure in children under 10, who should have remained naive to exposure had transmission been suppressed and whose test results represent a surrogate measure of exposure incidence in the population. The current WHO guidelines state that the upper bound of the 95% confidence interval (CI) for the exposure prevalence in the population of children under 10 cannot exceed 0.1%.2 In addition to the measurement of the prevalence of exposure in children, the current guidelines also call for an entomological evaluation to be conducted.2 In this evaluation, the upper bound of the 95% CI of the prevalence of flies carrying O. volvulus infective larvae must be less than 0.05%.2 If both the epidemiological and entomological criteria are met, it may be concluded that transmission has been interrupted. At this point MDA activities can be discontinued, and the program enters the surveillance phase of the elimination process. Three to five years after treatments have been discontinued, the program conducts another entomological evaluation, where once again, the upper bound of the 95% CI of the prevalence of flies carrying O. volvulus infective larvae must be less than 0.05%.2 If this criterion is met, it can be concluded that onchocerciasis has been eliminated in the evaluation area. The capability of a diagnostic test is challenged in situations where one is trying to prove that something no longer exists. A test’s sensitivity is the probability of a positive test in a population that is infected, while specificity is the probability of a negative test in an uninfected population. Adjusting test cut-offs to improve one parameter has the opposite effect on the other parameter (e.g., increasing sensitivity will result in a decrease in specificity). Two measures that reflect the accuracy of a test are the positive and negative predictive values. The positive predictive value is the proportion of true positives in the overall number of positives reported by the test, while the negative predictive value is the proportion of true negatives in the overall number of negatives reported. These measures are driven by the sensitivity and specificity of the test and by the infection prevalence in the population. In the setting of a disease elimination program, prevalence is near to or at zero. In the absence of a highly specific test, most positive results will be false positives, and the positive predictive value of the test will be very low. Because the elimination strategy requires that all eligible individuals in a targeted community regardless of infection status be treated, the results of any particular test will not affect an individual’s treatment. Thus, test sensitivity can be sacrificed to maximize specificity and minimize the number of false positive results in a situation where one is interested in population prevalence, as the number of people sampled can be increased to compensate for decreases in sensitivity.3 For example, as discussed above, the epidemiological criterion for stopping MDA in the current WHO guidelines for verifying interruption of transmission of O. volvulus requires testing enough children to conclude that the upper bound of the 95% CI of the prevalence of exposure in at-risk children less than 10 years old is less than 0.1%.2 Assuming a test with 100% sensitivity and 100% specificity, 3000 children must be tested and have negative results to meet this criterion.4 The sample size needed for a test exhibiting less than 100% sensitivity is roughly the sample size needed for an assay with 100% sensitivity divided by actual sensitivity.3 For example, for an assay with 70% sensitivity, one needs to test approximately 4285 individuals to meet the WHO criterion. In contrast to sensitivity, the specificity of an assay sets a floor of a prevalence value below which it is very difficult to measure. For example, if we employ an assay with a specificity of 99%, roughly 1% of the samples will test falsely positive in every trial. This means that the test on average will report a prevalence rate of 1%, even if the true prevalence is 0%. To detect a true prevalence of 0.1% in this case, sufficient individuals must be tested to ensure that a result reporting a 1.1% prevalence is significantly different from the 1% false positive rate. To achieve this goal would require testing over 63 000 individuals (C. R. Katholi, personal communication). Thus, ensuring a very high degree of specificity in the assay used to verify transmission elimination is paramount. Detection of parasite presence in humans Traditionally, microscopic examination of skin biopsies (snips) has been the gold standard for diagnosis and surveillance of O. volvulus infection.5 Snips generally exhibit a high degree of specificity, as O. volvulus and Mansonella streptocerca are the only filarial parasites in onchocerciasis endemic areas whose larvae inhabit the skin of an infected individual, and the larvae of these two species are readily distinguishable. However, snips are generally insensitive indicators of infection and the sensitivity of the skin snip decreases as the density of microfilaria in the skin decreases.6 This problem is exacerbated in populations that have received MDA with Mectizan™, which is a potent microfilaricide that effectively reduces microfilarial density in communities under successful MDA. A recent report suggests that the sensitivity of the conventional skin snip assay when compared with PCR in areas subject to successful MDA ranged from 76% in Uganda to 29% in Ethiopia.6 Attempts have been made to increase the sensitivity of the skin snip assay by replacing the microscopic examination of the snip with detection of amplified parasite DNA. The original DNA amplification assay for O. volvulus targeted a tandemly repeated sequence present in the O. volvulus genome with a unit length of roughly 150 bp, designated the O-150 repeat.7 This repeat family was found to be present in other species of the genus Onchocerca, but was lacking in the other human filarial parasites.7 The repeat family consisted of genus, species and strain-specific repeat units,8 permitting the development of species and strain- specific probes that could be used to classify the amplicons generated from amplification of the O-150 repeat.9 Real-time PCR6,10 and isothermal loop amplification (LAMP)11,12 assays have also been developed for the amplification of O. volvulus DNA, decreasing the limit of detection of these assays to significantly less than a single parasite and permitting rapid colorimetric detection of the amplified products.13 Most of these assays have targeted the O-150 repeat, though similar assays have been reported that target moderately repeated DNA sequences (rRNA genes,6 mitochondrial genes11) or even single copy genes.13 As might be predicted, using a DNA amplification assay to detect the presence of parasite DNA rather than using microscopy to detect the parasite itself has generally been found to increase the sensitivity of the skin snip.6,10,14 As a result, amplification of O. volvulus parasite DNA from skin snips has become the accepted standard for the diagnosis of patent O. volvulus infection in humans.2 Despite the high specificity exhibited by the skin snip assays, these are not generally applicable for demonstration of transmission interruption for several reasons. First, Mectizan™ rapidly reduces the number of microfilariae in the skin to zero or near zero,15 reducing the positive predictive value of the assay. Second, obtaining the biopsies is both painful and carries some risk of transmitting blood-borne infections, leading to community resistance.16 Together, these drawbacks led WHO to recommend against the use of skin snip-based assays as a primary diagnostic for the verification of elimination.2 An alternative method to skin snipping is the diethylcarbamazine (DEC) patch test. This test is based upon the observation that application of the anti-helminthic DEC to the skin of microfiladermic individuals infected with O. volvulus elicits a localized rash within 24–48 h.17 This is less invasive than collecting skin biopsies and thus potentially more acceptable to communities. DEC patch performance has varied widely in the different trials, with sensitivities ranging from 36 to 83% depending upon the comparator test (skin snip microscopy or PCR) used as the gold standard.18–20 Specificities were generally not high enough for the DEC patch to be used as a stand-alone test in areas with low prevalence.19 Furthermore, the sensitivity and specificity of this test was not evaluated in situations where successful MDA programs were ongoing. These issues have prevented the DEC patch test from being recommended by WHO for the verification of elimination.2 Serological tests to detect exposure to O. volvulus Preliminary studies employing low molecular weight (LMW) O. volvulus protein fractions as antigens in serological assays resulted in promising levels of sensitivity and specificity.21,22 As a result, multiple LMW antigens were produced and evaluated for the serodiagnosis of onchocerciasis (Table 1). Table 1. Candidate antigens considered for diagnosis of onchocerciasis MW (kDa)  Antigen(s)  Associated protein  Specificity (%)  Sensitivity (%)  Reference  Test used  15  OV103  MF surface associated protein  70  57  23  ELISA  Ov-MSA-1  99  89  24  IgG4 LIPS assay  16  OV16  Phosphatidyl ethanolamine binding protein  96  96  25  ELISA  17  OV10  Cysteine proteinase inhibitor  100  61  26  ELISA  OC 9.3  83  68  26  ELISA  OC 9.3  100  84  27  ELISA  OV-CPI  99  32  24  IgG4 LIPS assay  OV7  n/a  75  28  ELISA  19–20  OvMPB/10  Not determined  100  78  29  ELISA  20  OV11  Retinol binding protein  96  54  26  ELISA  OvMPB/11  99  65  29  ELISA  Ov-Far-1  100  100  24  IgG4 LIPS assay  OV20/36M  100  45  30  ELISA  OV20/OVS1  81/85  75/89  31  ELISA  20–23  OV 31  Not determined  92  68  26  ELISA  OV 31    100  30  Microplaque spot analysis  OV22/31M  100  74  30  ELISA  28  MSP-2  Major sperm protein  85  100  32  Dot blot assay  33  OC 3.6  Aspartyl protease inhibitor  n/a  93  27  ELISA  OV33-GST  96  93  33  ELISA  C27  n/a  82  34  Recombinant OV33/ELISA  C71  n/a  85  34  Recombinant OV33/ELISA  Ov-API-1  100  100  24  IgG4 LIPS assay  OV 33/5M  100  n/a  30  ELISA  MW (kDa)  Antigen(s)  Associated protein  Specificity (%)  Sensitivity (%)  Reference  Test used  15  OV103  MF surface associated protein  70  57  23  ELISA  Ov-MSA-1  99  89  24  IgG4 LIPS assay  16  OV16  Phosphatidyl ethanolamine binding protein  96  96  25  ELISA  17  OV10  Cysteine proteinase inhibitor  100  61  26  ELISA  OC 9.3  83  68  26  ELISA  OC 9.3  100  84  27  ELISA  OV-CPI  99  32  24  IgG4 LIPS assay  OV7  n/a  75  28  ELISA  19–20  OvMPB/10  Not determined  100  78  29  ELISA  20  OV11  Retinol binding protein  96  54  26  ELISA  OvMPB/11  99  65  29  ELISA  Ov-Far-1  100  100  24  IgG4 LIPS assay  OV20/36M  100  45  30  ELISA  OV20/OVS1  81/85  75/89  31  ELISA  20–23  OV 31  Not determined  92  68  26  ELISA  OV 31    100  30  Microplaque spot analysis  OV22/31M  100  74  30  ELISA  28  MSP-2  Major sperm protein  85  100  32  Dot blot assay  33  OC 3.6  Aspartyl protease inhibitor  n/a  93  27  ELISA  OV33-GST  96  93  33  ELISA  C27  n/a  82  34  Recombinant OV33/ELISA  C71  n/a  85  34  Recombinant OV33/ELISA  Ov-API-1  100  100  24  IgG4 LIPS assay  OV 33/5M  100  n/a  30  ELISA  Currently, the field has settled upon assays detecting antibodies against the Ov16 antigen for monitoring exposure to O. volvulus. This antigen is present in all lifecycle stages35 and elicits detectable antibody responses prior to the appearance of microfilaria in some chimpanzees experimentally infected with O. volvulus36 and in some children exposed to the parasite in endemic communities.37 While the initial assessment of the utility of the Ov16 antigen was assessed using detection of total IgG against Ov16, the IgG4 subtype response was the most specific.38,39 This is perhaps not surprising, as measurements of IgG isotypes in filarial infections revealed that IgG4 accounts for up to 95% of the IgG response to these infections.40 All current versions of the Ov16 assay have focused on IgG4 detection; however, the IgG4 response takes time to develop41 and thus will not immediately reflect exposure to O. volvulus. The Ov16 ELISA is now recommended by WHO guidelines for demonstrating the interruption of transmission of O. volvulus.2 Most Ov16 ELISA methods utilize dried blood spots (DBSs) as the input sample type. DBS samples are relatively stable, and can be easily collected and transported to a central facility for testing later. The cost of the reagents alone required for processing a single sample (generally run in duplicate) are approximately US $0.30, and one individual can process roughly 15 000 samples per year. However, this estimate does not include the cost of shipping to endemic laboratories. Fully burdened costs for running Ov16 ELISA are thus location-specific and should be analyzed as part of efforts to improve laboratory capacity in countries utilizing serological surveillance. The Ov16 antigen was adapted into a rapid format card test by AMRAD ICT (Australia) with a reported sensitivity of 90.6%.42 Despite promising field performance, production of this card test ceased in the year 2000. Recently, the interest in an Ov16 rapid test was revived, spurring the development of two rapid diagnostic tests (RDTs) incorporating the Ov16 antigen which are now commercially available. These consist of a single IgG4 rapid test and a combination test utilizing Ov16 and the W. bancrofti antigen Wb123.43,44 The product inserts report the sensitivity of the Ov16 single test to be 81.1% and the Ov16 test line of the biplex test to be 81.33%, respectively.45 The specificity is listed at 99.0% for the single Ov16 test and 100% for the biplex test, although it was reported to be 1–2% lower than these values in published studies using early prototypes.43,44 As part of the development of the RDT, a recombinant human IgG4 positive control antibody specific for Ov16 was also developed,46 providing a highly pure, consistent and long-term source of positive control for both the ELISA and RDT assays. The new Ov16-containing RDTs for anti-Ov16 serology are rapidly being incorporated into field studies and surveillance activities. While feasibility and acceptability of the rapid tests in surveillance have been demonstrated,47 field-based studies that include performance data using the commercial product are still pending publication. Newly released WHO guidelines recommend evaluation of the performance of these RDTs prior to their use in stop-MDA assessments;2 hopefully, data to assess the utility of the Ov16 RDT in the verification of elimination will be available soon. Entomological surveillance of O. volvulus transmission The transmission cycle of O. volvulus includes blackfly vectors of the genus Simulium. The most direct measure of the status of transmission is to measure infectivity in the black fly vector population itself. Entomological surveillance has the advantage that it eliminates the time lag inherent in the assays focusing on the human host population, where detectable patency lags infection by 12–18 months.37 The disadvantage to entomological surveillance is that large numbers of vector insects must be caught and tested. The current WHO guidelines call for testing sufficient numbers of flies to ensure that the upper bound of the 95% CI of the prevalence of flies carrying infective larvae is less than 0.05% (1/2000).2 Meeting this criterion requires testing at least 6000 flies and having all test negative to meet this criterion.4 Annual transmission potential can be used as an alternative criterion when it is not possible to capture 6000 flies due to low prevalence of flies.2 The traditional method of determining the prevalence of flies carrying infective larvae has been through field dissection of captured flies. However, this method suffers from two disadvantages. First, dissection is expensive because it requires a trained entomologist, a field microscope and a lot of time. Second, a more significant disadvantage is that Simulium damnosum sensu lato, the major vector of O. volvulus in Africa, also serves as the vector for zoonotic species of Onchocerca that do not infect humans.48 The larvae of these animal parasites are difficult or impossible to distinguish morphologically from O. volvulus; thus, dissection data can result in overestimates of the intensity of transmission. A solution to the inability of vector dissection to accurately describe transmission was to develop a specific PCR, and techniques to overcome the cost and time implications of having to test 6000 blackflies individually. Although several DNA amplification assays have been developed, the O-150 PCR has been the assay that has been used most widely.13,49 This PCR method distinguishes O. volvulus from other Onchocerca present in S. damnosum s.l., thereby improving the accuracy of the transmission estimates.9 Screening efficiency using the PCR is superior to dissection, as the PCR assay can be applied to screening pools of flies.49 However, one potential drawback to screening pools of flies is that the O-150 PCR is not quantitative and inhibitors present in the DNA preparations can reduce the efficiency of the PCR, making it impossible to get accurate estimates of the number of parasites present in a positive pool, even when the O-150 PCR is adapted to a quantitative PCR format. Thus, it is not possible to determine whether a positive pool contains a single infectious fly or multiple infectious flies. This problem was overcome by applying probability distribution estimates when it was realized that although it was not possible to determine how many positive flies were in a positive pool, it was possible to say with certainty that negative pools contain no infectious flies. If the infectious flies are randomly distributed among the collection (something easily accomplished when arranging the flies into pools with the maximum number of flies appropriate for processing), it is possible to use a probability distribution to calculate the probability estimate of the number of infected flies in a pool, given the proportion of negative and positive pools and the number of flies contained in each pool.49 The mathematics behind this observation were incorporated into a program (PoolScreen) that calculates the prevalence of infectious flies and associated confidence intervals from the proportion of positive pools, the pool size and the number of pools screened. Field studies conducted in both Africa50 and Latin America51 validated this approach. The O-150 PCR has subsequently been widely applied to collect entomological data verifying elimination of transmission of O. volvulus in Mexico,52 Guatemala53 and one focus in Sudan.54 PCR pool-screening techniques have overcome most of the operational difficulties associated with meeting the WHO guideline’s vector criterion. Screening the 6000 flies necessary to meet this requirement by screening just 60 pools of 100 flies each would take one individual less than 1 week. Pool screening also dramatically reduces the cost and time necessary to process the samples, when compared to analyzing each insect individually. The cost of reagents to process a single pool of 100 flies roughly US$6.90 per pool, or roughly US$ 0.07 per insect. A single individual can process roughly 4000 pools or 400 000 individual insects in a year. Furthermore, the collected insects can be stored in alcohol indefinitely at room temperature, minimizing the logistical difficulties encountered when shipping the collections to a central laboratory for analysis. Collecting the necessary number of flies now represents the main challenge to implementing the entomological surveys. Currently, the standard method of collecting vector black flies is human landing collections (HLCs), which are quite inefficient, as a team of two collectors can only collect one person-day's worth of flies per day. However, recent reports suggest that a new trap platform, known as the Esperanza Window Trap (EWT), may represent an effective alternative to HLCs for collecting vector black flies.55 Studies in Mexico demonstrated that EWTs operated by residents of the affected communities could collect sufficient numbers of flies to certify these communities were free of O. volvulus transmission.56 If the EWT platform has an equivalent performance in Africa when operated by community members, it may overcome the difficulty in obtaining sufficient numbers of vector flies to demonstrate suppression and interruption of transmission. Conclusion and future directions Although the current diagnostic tools have served well for verifying suppression and interruption of transmission of O. volvulus in most countries in Latin America and in several foci in Africa, there are several tools that could accelerate program activities targeting the elimination of onchocerciasis. One of the most pressing needs is to define the sampling schemes for verification of elimination in Africa. In Latin America, the approach that was used by the Onchocerciasis Elimination Program for the Americas was to identify sentinel hyper-endemic communities in each focus prior to the beginning of the program.57 The epidemiological and entomological indicators described previously were then used to follow the decline and eventual elimination of transmission in these sentinels. In general, the foci in Latin America were also isolated from one another, making the definition of a focus a simple matter. In contrast, sentinel communities were often not identified prior to the start of onchocerciasis control in Africa. Furthermore, the foci that exist in Africa are often not isolated, with the potential for reintroduction of the parasite either through migration of infected people or by wind-borne flies.58 It is thus necessary to gain a better understanding of what the limits to a focus are and, once this is done, how these foci should be sampled. For example, the epidemiological metrics needed for stopping MDA still need to be clarified, including how many communities should be enrolled to obtain reliable data on transmission in a focus, how many people in each community should be sampled and how the communities to be included should be chosen. Similarly, for the entomological metrics, it is not clear how many different sites should be included, how many flies need to be collected from each site, or for how long and how often the collections should be carried out. A second need is for the current seroprevalence cut-offs in the 2016 WHO guidelines to be re-evaluated in light of the recent progress in modeling the transmission of O. volvulus. For example, a recent study has suggested that the prevalence of exposure in children that would indicate the parasite population is irreversibly headed to extinction may often be higher than 0.1%, though this is dependent on the baseline endemicity.59 A similar re-evaluation of the entomological metrics is also in order, perhaps with more focus on the annual transmission potential, rather than simple prevalence of infection. Once these analyses are completed, they should be used in conjunction with methods to both more accurately determine test performance in relevant settings and calculate sample sizes that take the sensitivity and specificity of the tests to be employed into account.3 Specificity is paramount when choosing tests for verifying elimination. However, a single-antigen antibody test often cannot deliver a very high degree of specificity without suffering a dramatic loss of sensitivity. One solution is to use a confirmatory test that is independent from the primary test, and require that both the primary and confirmatory tests be positive before declaring a sample positive. Incorporating a second parasite antigen marker in an RDT, with a distinct line for each marker, could allow tailoring of the test to provide either highest sensitivity (requirement of only a single test line present to be positive) or highest specificity (both test lines must be positive). In a similar vein, while the sensitivity and specificity of the PCR assays used in black flies generally approach 100%, a major technical obstacle with these assays is the potential for amplicon contamination resulting in false positive signals. Including an independent PCR assay targeting a second genomic sequence is one way to overcome this problem. Given that there are other antigens already available that can be used to detect exposure to O. volvulus, and other PCR targets already identified for detection of parasite DNA, studies and necessary product development should be undertaken to determine which combination of tests would result in the highest combined sensitivity and specificity for verifying elimination. Finally, the available diagnostics do not directly detect potentially fertile female parasites in the human population that could restart reproduction once MDA is stopped and which may pose a risk for recrudescence. Even though not all adult worms need to be sterile or dead for transmission to be irreversibly interrupted, it would be beneficial to have a method to detect and treat people who harbor adult worm infections. The detection of adult worm infections could allow targeted treatment with medications that can permanently sterilize or kill the worms, either to accelerate program progress to achieving interruption of transmission or to further lower the probability of recrudescence of transmission in mop-up operations that occur after MDA has been stopped but transient transmission is detected. Safe treatments are available that can permanently sterilize and eventually kill the adult females,60 so the missing piece is the diagnostic test to identify people infected with fecund adult females. Some progress has been made in developing assays to detect viable adult parasites in humans. These include specific metabolites produced by female worms61,62 and detection of parasite miRNA in the blood of infected individuals.63 Assays that could detect patent infections in the face of an effective MDA program have great potential for both speeding up the process of elimination and ensuring that the infection does not recrudesce once MDA is withdrawn. Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the US Centers for Disease Control and Prevention or the World Health Organization. Author’s contributions: All authors (TRU, AG, VC and PTC) contributed to the writing of this manuscript. All authors have read and reviewed the final version of the manuscript. Funding: None Competing interests: None declared Ethical approval: Not required. References 1 Vlaminck J , Fischer PU, Weil GJ. Diagnostic tools for onchocerciasis elimination programs. Trends Parasitol  2015; 31( 11): 571– 82. Google Scholar CrossRef Search ADS PubMed  2 World Health Organization . Guidelines for stopping mass drug administration and verifying elimination of human onchocerciasis: Criteria and Procedures . Document # WHO/HTM/NTD/PCT/2016.1; Geneva: WHO Press; 2016. 3 Johnson WO , Su C-L, Gardner IAet al.  . Sample size calculations for surveys to substantiate freedom of populations from infectious agents. Biometrics  2004; 60( 1): 165– 71. Google Scholar CrossRef Search ADS PubMed  4 Basanez MG , Rodriguez-Perez MA, Reyes-Villanueva Fet al.  . Determination of sample sizes for the estimation of Onchocerca volvulus (Filarioidea: Onchocercidae) infection rates in biting populations of Simulium ochraceum s.l. (Diptera: Simuliidae) and its application to ivermectin control programs. J Med Entomol  1998; 35( 5): 745– 57. Google Scholar CrossRef Search ADS PubMed  5 Kale OO , Bammeke AO, Ayeni O. An evaluation of skin snip techniques used in the quantitative assessment of microfilarial densities of Onchocerca volvulus. Bull World Health Organ  1974; 51( 5): 547– 9. Google Scholar PubMed  6 Thiele EA , Cama VA, Lakwo Tet al.  . Detection of Onchocerca volvulus in skin snips by microscopy and real-time polymerase chain reaction: implications for monitoring and evaluation activities. Am J Trop Med Hyg  2016; 94( 4): 906– 11. Google Scholar CrossRef Search ADS PubMed  7 Meredith SEO , Lando G, Gbakima AAet al.  . Onchocerca volvulus: application of the polymerase chain reaction to identification and strain differentiation of the parasite. Exp Parasitol  1991; 73: 335– 44. Google Scholar CrossRef Search ADS PubMed  8 Zimmerman PA , Toe L, Unnasch TR. Design of Onchocerca DNA probes based upon analysis of a repeated sequence family. Mol Biochem Parasitol  1993; 58: 259– 69. Google Scholar CrossRef Search ADS PubMed  9 Toe L , Merriweather A, Unnasch TR. DNA probe based classification of Simulium damnosum s.l. borne and human derived filarial parasites in the Onchocerciasis Control Programme area. Am J Trop Med Hyg  1994; 51: 676– 83. Google Scholar CrossRef Search ADS PubMed  10 Lloyd MM , Gilbert R, Taha NTet al.  . Conventional parasitology and DNA-based diagnostic methods for onchocerciasis elimination programmes. Acta Trop  2015; 146: 114– 8. Google Scholar CrossRef Search ADS PubMed  11 Lagatie O , Merino M, Batsa Debrah Let al.  . An isothermal DNA amplification method for detection of Onchocerca volvulus infection in skin biopsies. Parasit Vectors  2016; 9( 1): 624. Google Scholar CrossRef Search ADS PubMed  12 Alhassan A , Osei-Atweneboana MY, Kyeremeh KFet al.  . Comparison of a new visual isothermal nucleic acid amplification test with PCR and skin snip analysis for diagnosis of onchocerciasis in humans. Mol Biochem Parasitol  2016; 210( 1– 2): 10– 12. Google Scholar CrossRef Search ADS PubMed  13 Poole CB , Li Z, Alhassan Aet al.  . Colorimetric tests for diagnosis of filarial infection and vector surveillance using non-instrumented nucleic acid loop-mediated isothermal amplification (NINA-LAMP). PLoS One  2017; 12( 2): e0169011. Google Scholar CrossRef Search ADS PubMed  14 Zimmerman PA , Guderian RH, Aruajo Eet al.  . Polymerase chain reaction-based diagnosis of Onchocerca volvulus infection: improved detection of patients with onchocerciasis. J Infect Dis  1994; 169( 3): 686– 689. Google Scholar CrossRef Search ADS PubMed  15 Remme J , Baker RH, G DSet al.  . A community trial of ivermectin in the onchocerciasis focus of Asubende, Ghana. I. Effect on the microfilarial reservoir and the transmission of Onchocerca volvulus. Trop Med Parasitol  1989; 40: 367– 74. Google Scholar PubMed  16 Diawara L , Traore MO, Badji Aet al.  . Feasibility of onchocerciasis elimination with ivermectin treatment in endemic foci in Africa: first evidence from studies in Mali and Senegal. PLoS Negl Trop Dis  2009; 3( 7): e497. Google Scholar CrossRef Search ADS PubMed  17 Kilian HD . The use of a topical Mazzotti test in the diagnosis of onchocerciasis. Trop Med Parasitol  1988; 39: 235– 8. Google Scholar PubMed  18 Ozoh G , Boussinesq M, Bissek ACet al.  . Evaluation of the diethylcarbamazine patch to evaluate onchocerciasis endemicity in Central Africa. Trop Med Int Health  2007; 12( 1): 123– 9. Google Scholar PubMed  19 Boatin BA , Toe L, Alley ESet al.  . Detection of Onchocerca volvulus infection in low prevalence areas: a comparison of three diagnostic methods. Parasitology  2002; 125( Pt 6): 545– 52. Google Scholar PubMed  20 Toe L , Adjami AG, Boatin BAet al.  . Topical application of diethylcarbamazine to detect onchocerciasis recrudescence in west Africa. Trans R Soc Trop Med Hyg  2000; 94( 5): 519– 25. Google Scholar CrossRef Search ADS PubMed  21 Lucius R , Buttner DW, Kirsten Cet al.  . A study on antigen recognition by onchocerciasis patients with different clinical forms of disease. Parasitology  1986; 92 ( Pt 3): 569– 80. Google Scholar CrossRef Search ADS PubMed  22 Cabrera Z , Parkhouse RM, Forsyth Ket al.  . Specific detection of human antibodies to Onchocerca volvulus. Trop Med Parasitol  1989; 40( 4): 454– 9. Google Scholar PubMed  23 Lustigman S , Brotman B, Johnson EHet al.  . Identification and characterization of an Onchocerca volvulus cDNA clone encoding a microfilarial surface-associated antigen. Mol Biochem Parasitol  1992; 50: 79– 94. Google Scholar CrossRef Search ADS PubMed  24 Burbelo PD , Leahy HP, Iadarola MJet al.  . A four-antigen mixture for rapid assessment of Onchocerca volvulus infection. PLoS Negl Trop Dis  2009; 3( 5): e438. Google Scholar CrossRef Search ADS PubMed  25 Lobos E , Altmann M, Mengod Get al.  . Identification of an Onchocerca volvulus cDNA encoding a low-molecular-weight antigen uniquely recognized by onchocerciasis patient sera. Mol Biochem Parasitol  1990; 39: 135– 46. Google Scholar CrossRef Search ADS PubMed  26 Ramachandran CP . Improved immunodiagnostic tests to monitor onchocerciasis control programmes—a multicenter effort. Parasitol Today  1993; 9( 3): 76– 79. Google Scholar CrossRef Search ADS   27 Chandrashekar R , Ogunrinade AF, Weil GJ. Use of recombinant Onchocerca volvulus antigens for diagnosis and surveillance of human onchocerciasis. Trop Med Int Health  1996; 1( 5): 575– 80. Google Scholar CrossRef Search ADS PubMed  28 Lustigman S , Brotman B, Huima Tet al.  . Characterization of an Onchocerca volvulus cDNA clone encoding a genus specific antigen present in infective larvae and adult worms. Mol Biochem Parasitol  1991; 45: 65– 76. Google Scholar CrossRef Search ADS PubMed  29 Bradley JE , Trenholme KR, Gillespie AJet al.  . A sensitive serodiagnostic test for onchocerciasis using a cocktail of recombinant antigens. Am J Trop Med Hyg  1993; 48( 2): 198– 204. Google Scholar CrossRef Search ADS PubMed  30 Bradley JE , Helm R, Lahaise Met al.  . cDNA clones of Onchocerca volvulus low molecular weight antigens provide immunologically specific diagnostic probes. Mol Biochem Parasitol  1991; 46( 2): 219– 27. Google Scholar CrossRef Search ADS PubMed  31 Mpagi JL , Buttner DW, Tischendorf FWet al.  . Use of the recombinant Onchocerca volvulus protein Ov20/OvS1 for the immunodiagnostic differentiation between onchocerciasis and mansonelliasis and for the characterization of hyperreactive onchocerciasis (Sowda). Trop Med Int Health  2000; 5( 12): 891– 7. Google Scholar CrossRef Search ADS PubMed  32 Park J , Dickerson TJ, Janda KD. Major sperm protein as a diagnostic antigen for onchocerciasis. Bioorg Med Chem  2008; 16( 15): 7206– 9. Google Scholar CrossRef Search ADS PubMed  33 Lucius R , Kern A, Seeber Fet al.  . Specific and sensitive IgG4 immunodiagnosis of onchocerciasis with a recombinant 33 kD Onchocerca volvulus protein (Ov33). Tropical Medicine & Parasitology  1992; 43( 3): 139– 45. 34 Tume CB , Ngu JL, McKerrow JLet al.  . Characterization of a recombinant Onchocerca volvulus antigen (Ov33) produced in yeast. Am J Trop Med Hyg  1997; 57( 5): 626– 33. Google Scholar PubMed  35 Lobos E , Weiss N, Karam Met al.  . An immunogenic Onchocerca volvulus antigen: a specific and early marker of infection. Science  1991; 251: 1603– 05. Google Scholar CrossRef Search ADS PubMed  36 Eberhard ML , Dickerson JW, Tsang VCet al.  . Onchocerca volvulus: parasitologic and serologic responses in experimentally infected chimpanzees and mangabey monkeys. Exp Parasitol  1995; 80( 3): 454– 62. Google Scholar CrossRef Search ADS PubMed  37 Ogunrinade AF , Chandrashekar R, Eberhard MLet al.  . Preliminary evaluation of recombinant Onchocerca volvulus antigens for serodiagnosis of onchocerciasis. J Clin Microbiol  1993; 31: 1741– 5. Google Scholar PubMed  38 Weil GJ , Ogunrinade AF, Chandrashekar Ret al.  . IgG subclass antibody serology for onchocerciasis. J Infect Dis  1990; 161: 549– 54. Google Scholar CrossRef Search ADS PubMed  39 Ogunrinade AF , Chandrashekar R, Weil GJet al.  . Use of a recombinant antigen (Oc3.6 cDNA) for the serological diagnosis of onchocerciasis in exposed Nigerian children. J Trop Pediatr  1992; 38: 103– 105. Google Scholar CrossRef Search ADS PubMed  40 Ottesen EA , Skvaril F, Tripathy SPet al.  . Prominence of IgG4 in the IgG antibody response to human filariasis. J Immunol  1985; 134( 4): 2707– 12. Google Scholar PubMed  41 Lighaam LC , Rispens T. The immunobiology of immunoglobulin G4. Semin Liver Dis  2016; 36( 3): 200– 15. Google Scholar CrossRef Search ADS PubMed  42 Weil GJ , Steel C, Liftis Fet al.  . A rapid-format antibody card test for diagnosis of onchocerciasis. J Infect Dis  2000; 182( 6): 1796– 9. Google Scholar CrossRef Search ADS PubMed  43 Golden A , Steel C, Yokobe Let 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. Google Scholar CrossRef Search ADS PubMed  44 Steel C , Golden A, Stevens Eet al.  . Rapid point-of-contact tool for mapping and integrated surveillance of Wuchereria bancrofti and Onchocerca volvulus infection. Clin Vaccine Immunol  2015; 22( 8): 896– 901. Google Scholar CrossRef Search ADS PubMed  45 PATH . Quality assurance program materials for onchocerciasis and lymphatic filariasis tests . http://sites.path.org/dx/ntd/training-and-qaqc-materials/ [accessed 27 April 2017). 46 Golden A , Stevens EJ, Yokobe Let al.  . A recombinant positive control for serology diagnostic tests supporting elimination of Onchocerca volvulus. PLoS Negl Trop Dis  2016; 10( 1): e0004292. Google Scholar CrossRef Search ADS PubMed  47 Dieye Y , Storey HL, Barrett KLet al.  . Feasibility of utilizing the SD BIOLINE Onchocerciasis IgG4 rapid test in onchocerciasis surveillance in Senegal. PLoS Negl Trop Dis  2017; 11( 10): e0005884. Google Scholar CrossRef Search ADS PubMed  48 Trees AJ , McCall PJ, Davies JB. On the possibility of bovine Onchocerca species infecting Simulium damnosum s.l. in the forest zone of Sierra Leone. I. Parasitological aspects. Ann Trop Med Parasitol  1989; 83( 6): 595– 601. Google Scholar CrossRef Search ADS PubMed  49 Katholi CR , Toe L, Merriweather Aet al.  . Determining the prevalence of Onchocerca volvulus infection in vector populations by polymerase chain reaction screening of pools of black flies. J Infect Dis  1995; 172( 5): 1414– 17. Google Scholar CrossRef Search ADS PubMed  50 Yameogo L , Toe L, Hougard JMet al.  . 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. Google Scholar CrossRef Search ADS PubMed  51 Rodríguez-Pérez MA , Danis-Lozano R, Rodríguez MHet al.  . Detection of Onchocerca volvulus infection in Simulium ochraceum sensu lato: comparison of a PCR assay and fly dissection in a Mexican hypoendemic community. Parasitology  1999; 119( 6): 613– 19. Google Scholar CrossRef Search ADS PubMed  52 Rodriguez-Perez MA , Fernandez-Santos NA, Orozco-Algarra MEet al.  . Elimination of onchocerciasis from Mexico. PLoS Negl Trop Dis  2015; 9( 7): e0003922. Google Scholar CrossRef Search ADS PubMed  53 Richards F Jr , Rizzo N, Diaz Espinoza CEet al.  . One hundred years after its discovery in Guatemala by Rodolfo Robles, Onchocerca volvulus transmission has been eliminated from the central endemic zone. Am J Trop Med Hyg  2015; 93( 6): 1295– 304. Google Scholar CrossRef Search ADS PubMed  54 Zarroug IM , Hashim K, ElMubark WAet al.  . The first confirmed elimination of an onchocerciasis focus in Africa: Abu Hamed, Sudan. Am J Trop Med Hyg  2016; 27: 1037– 40. Google Scholar CrossRef Search ADS   55 Rodríguez-Pérez MA , Adeleke MA, Burkett-Cadena NDet al.  . Development of a novel trap for the collection of black flies of the Simulium ochraceum complex. PLoS One  2013; 8( 10): e76814. Google Scholar CrossRef Search ADS PubMed  56 Rodríguez-Pérez MA , Adeleke MA, Rodríguez-Luna ICet al.  . Evaluation of a community-based trapping program to collect Simulium ochraceum sensu lato for verification of onchocerciasis elimination. PLoS Negl Trop Dis  2014; 8: e3249. Google Scholar CrossRef Search ADS PubMed  57 Sauerbrey M ; The Onchocerciasis Elimination Program for the Americas (OEPA). Ann Trop Med Parasitol  2008; 102( Suppl. 1): 25– 9. Google Scholar CrossRef Search ADS PubMed  58 Garms R , Walsh JF, Davies JB. Studies on the reinvasion of the Onchocerciasis Control Programme in the Volta River Basin by Simulium damnosum s.l. with emphasis on the south-western areas. Tropenmed Parasitol  1979; 30( 3): 345– 62. Google Scholar PubMed  59 Lont YL , Coffeng LE, de Vlas SJet al.  . Modelling anti-Ov16 IgG4 antibody prevalence as an indicator for evaluation and decision making in onchocerciasis elimination programmes. PLoS Negl Trop Dis  2017; 11( 1): e0005314. Google Scholar CrossRef Search ADS PubMed  60 Walker M , Specht S, Churcher TSet al.  . Therapeutic efficacy and macrofilaricidal activity of doxycycline for the treatment of river blindness. Clin Infect Dis  2015; 60( 8): 1199– 207. Google Scholar CrossRef Search ADS PubMed  61 Denery JR , Nunes AA, Hixon MSet al.  . Metabolomics-based discovery of diagnostic biomarkers for onchocerciasis. PLoS Negl Trop Dis  2010; 4( 10): pii: e834. Google Scholar CrossRef Search ADS   62 Globisch D , Moreno AY, Hixon MSet al.  . Onchocerca volvulus-neurotransmitter tyramine is a biomarker for river blindness. Proc Natl Acad Sci USA  2013; 110( 11): 4218– 23. Google Scholar CrossRef Search ADS PubMed  63 Tritten L , Burkman E, Moorhead Aet al.  . Detection of circulating parasite-derived microRNAs in filarial infections. PLoS Negl Trop Dis  2014; 8( 7): e2971. Google Scholar CrossRef Search ADS PubMed  © World Health Organization, 2018. All rights reserved. The World Health Organization has granted Publisher permission for the reproduction of this article. This is an Open Access article distributed under the terms of the Creative Commons Attribution 3.0 IGO License (https://creativecommons.org/licenses/by/3.0/igo/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png International Health Oxford University Press

Diagnostics for onchocerciasis in the era of elimination

Loading next page...
 
/lp/ou_press/diagnostics-for-onchocerciasis-in-the-era-of-elimination-zu6BJWJU0e
Publisher
Oxford University Press
Copyright
© World Health Organization, 2018. All rights reserved. The World Health Organization has granted Publisher permission for the reproduction of this article.
ISSN
1876-3413
eISSN
1876-3405
D.O.I.
10.1093/inthealth/ihx047
Publisher site
See Article on Publisher Site

Abstract

Abstract In the past few years, efforts to eliminate onchocerciasis from Africa have intensified. These efforts are primarily based on the mass distribution of the anti-helminthic drug Mectizan™ (ivermectin). This program has led to the development of new guidelines by the World Health Organization for the verification that transmission has been suppressed and eventually eliminated. The requirements of diagnostic tools for this purpose differ in many ways from tests used to diagnose infection in individuals. In this review, we summarize the progress that has been made to identify diagnostics that meet the specialized requirements needed to verify onchocerciasis elimination, discuss why these tests were selected and summarize the needs that still exist to complete the arsenal of diagnostic tools that will be useful as the goal of elimination is achieved. Black fly, Diagnostics, Ivermectin, Onchocerca volvulus, River blindness Introduction A recent review has put forth the challenges faced by onchocerciasis elimination programs and how their diagnostic needs evolve as they transition from control verification of elimination.1 Here, we will focus upon the diagnostic needs to verify suppression and interruption of transmission in the context of the current WHO guidelines. Currently, onchocerciasis elimination programs rely primarily on mass drug administration (MDA) of Mectizan™ (ivermectin) to suppress and eventually eliminate transmission of Onchocerca volvulus, the causative agent of the disease. The onchocerciasis elimination program strategy is a multi-stage process.2 Initially, programs must obtain high enough treatment coverage in the eligible population to stop transmission. Once transmission is suppressed, high coverage must be maintained until all the fertile female parasites either die or become sterile. Once a suitable number of treatments have been given, transmission is believed to have been interrupted. At this point in time, the program conducts a comprehensive survey to demonstrate that transmission has been interrupted. These surveys rely upon measuring parasite exposure in children under 10, who should have remained naive to exposure had transmission been suppressed and whose test results represent a surrogate measure of exposure incidence in the population. The current WHO guidelines state that the upper bound of the 95% confidence interval (CI) for the exposure prevalence in the population of children under 10 cannot exceed 0.1%.2 In addition to the measurement of the prevalence of exposure in children, the current guidelines also call for an entomological evaluation to be conducted.2 In this evaluation, the upper bound of the 95% CI of the prevalence of flies carrying O. volvulus infective larvae must be less than 0.05%.2 If both the epidemiological and entomological criteria are met, it may be concluded that transmission has been interrupted. At this point MDA activities can be discontinued, and the program enters the surveillance phase of the elimination process. Three to five years after treatments have been discontinued, the program conducts another entomological evaluation, where once again, the upper bound of the 95% CI of the prevalence of flies carrying O. volvulus infective larvae must be less than 0.05%.2 If this criterion is met, it can be concluded that onchocerciasis has been eliminated in the evaluation area. The capability of a diagnostic test is challenged in situations where one is trying to prove that something no longer exists. A test’s sensitivity is the probability of a positive test in a population that is infected, while specificity is the probability of a negative test in an uninfected population. Adjusting test cut-offs to improve one parameter has the opposite effect on the other parameter (e.g., increasing sensitivity will result in a decrease in specificity). Two measures that reflect the accuracy of a test are the positive and negative predictive values. The positive predictive value is the proportion of true positives in the overall number of positives reported by the test, while the negative predictive value is the proportion of true negatives in the overall number of negatives reported. These measures are driven by the sensitivity and specificity of the test and by the infection prevalence in the population. In the setting of a disease elimination program, prevalence is near to or at zero. In the absence of a highly specific test, most positive results will be false positives, and the positive predictive value of the test will be very low. Because the elimination strategy requires that all eligible individuals in a targeted community regardless of infection status be treated, the results of any particular test will not affect an individual’s treatment. Thus, test sensitivity can be sacrificed to maximize specificity and minimize the number of false positive results in a situation where one is interested in population prevalence, as the number of people sampled can be increased to compensate for decreases in sensitivity.3 For example, as discussed above, the epidemiological criterion for stopping MDA in the current WHO guidelines for verifying interruption of transmission of O. volvulus requires testing enough children to conclude that the upper bound of the 95% CI of the prevalence of exposure in at-risk children less than 10 years old is less than 0.1%.2 Assuming a test with 100% sensitivity and 100% specificity, 3000 children must be tested and have negative results to meet this criterion.4 The sample size needed for a test exhibiting less than 100% sensitivity is roughly the sample size needed for an assay with 100% sensitivity divided by actual sensitivity.3 For example, for an assay with 70% sensitivity, one needs to test approximately 4285 individuals to meet the WHO criterion. In contrast to sensitivity, the specificity of an assay sets a floor of a prevalence value below which it is very difficult to measure. For example, if we employ an assay with a specificity of 99%, roughly 1% of the samples will test falsely positive in every trial. This means that the test on average will report a prevalence rate of 1%, even if the true prevalence is 0%. To detect a true prevalence of 0.1% in this case, sufficient individuals must be tested to ensure that a result reporting a 1.1% prevalence is significantly different from the 1% false positive rate. To achieve this goal would require testing over 63 000 individuals (C. R. Katholi, personal communication). Thus, ensuring a very high degree of specificity in the assay used to verify transmission elimination is paramount. Detection of parasite presence in humans Traditionally, microscopic examination of skin biopsies (snips) has been the gold standard for diagnosis and surveillance of O. volvulus infection.5 Snips generally exhibit a high degree of specificity, as O. volvulus and Mansonella streptocerca are the only filarial parasites in onchocerciasis endemic areas whose larvae inhabit the skin of an infected individual, and the larvae of these two species are readily distinguishable. However, snips are generally insensitive indicators of infection and the sensitivity of the skin snip decreases as the density of microfilaria in the skin decreases.6 This problem is exacerbated in populations that have received MDA with Mectizan™, which is a potent microfilaricide that effectively reduces microfilarial density in communities under successful MDA. A recent report suggests that the sensitivity of the conventional skin snip assay when compared with PCR in areas subject to successful MDA ranged from 76% in Uganda to 29% in Ethiopia.6 Attempts have been made to increase the sensitivity of the skin snip assay by replacing the microscopic examination of the snip with detection of amplified parasite DNA. The original DNA amplification assay for O. volvulus targeted a tandemly repeated sequence present in the O. volvulus genome with a unit length of roughly 150 bp, designated the O-150 repeat.7 This repeat family was found to be present in other species of the genus Onchocerca, but was lacking in the other human filarial parasites.7 The repeat family consisted of genus, species and strain-specific repeat units,8 permitting the development of species and strain- specific probes that could be used to classify the amplicons generated from amplification of the O-150 repeat.9 Real-time PCR6,10 and isothermal loop amplification (LAMP)11,12 assays have also been developed for the amplification of O. volvulus DNA, decreasing the limit of detection of these assays to significantly less than a single parasite and permitting rapid colorimetric detection of the amplified products.13 Most of these assays have targeted the O-150 repeat, though similar assays have been reported that target moderately repeated DNA sequences (rRNA genes,6 mitochondrial genes11) or even single copy genes.13 As might be predicted, using a DNA amplification assay to detect the presence of parasite DNA rather than using microscopy to detect the parasite itself has generally been found to increase the sensitivity of the skin snip.6,10,14 As a result, amplification of O. volvulus parasite DNA from skin snips has become the accepted standard for the diagnosis of patent O. volvulus infection in humans.2 Despite the high specificity exhibited by the skin snip assays, these are not generally applicable for demonstration of transmission interruption for several reasons. First, Mectizan™ rapidly reduces the number of microfilariae in the skin to zero or near zero,15 reducing the positive predictive value of the assay. Second, obtaining the biopsies is both painful and carries some risk of transmitting blood-borne infections, leading to community resistance.16 Together, these drawbacks led WHO to recommend against the use of skin snip-based assays as a primary diagnostic for the verification of elimination.2 An alternative method to skin snipping is the diethylcarbamazine (DEC) patch test. This test is based upon the observation that application of the anti-helminthic DEC to the skin of microfiladermic individuals infected with O. volvulus elicits a localized rash within 24–48 h.17 This is less invasive than collecting skin biopsies and thus potentially more acceptable to communities. DEC patch performance has varied widely in the different trials, with sensitivities ranging from 36 to 83% depending upon the comparator test (skin snip microscopy or PCR) used as the gold standard.18–20 Specificities were generally not high enough for the DEC patch to be used as a stand-alone test in areas with low prevalence.19 Furthermore, the sensitivity and specificity of this test was not evaluated in situations where successful MDA programs were ongoing. These issues have prevented the DEC patch test from being recommended by WHO for the verification of elimination.2 Serological tests to detect exposure to O. volvulus Preliminary studies employing low molecular weight (LMW) O. volvulus protein fractions as antigens in serological assays resulted in promising levels of sensitivity and specificity.21,22 As a result, multiple LMW antigens were produced and evaluated for the serodiagnosis of onchocerciasis (Table 1). Table 1. Candidate antigens considered for diagnosis of onchocerciasis MW (kDa)  Antigen(s)  Associated protein  Specificity (%)  Sensitivity (%)  Reference  Test used  15  OV103  MF surface associated protein  70  57  23  ELISA  Ov-MSA-1  99  89  24  IgG4 LIPS assay  16  OV16  Phosphatidyl ethanolamine binding protein  96  96  25  ELISA  17  OV10  Cysteine proteinase inhibitor  100  61  26  ELISA  OC 9.3  83  68  26  ELISA  OC 9.3  100  84  27  ELISA  OV-CPI  99  32  24  IgG4 LIPS assay  OV7  n/a  75  28  ELISA  19–20  OvMPB/10  Not determined  100  78  29  ELISA  20  OV11  Retinol binding protein  96  54  26  ELISA  OvMPB/11  99  65  29  ELISA  Ov-Far-1  100  100  24  IgG4 LIPS assay  OV20/36M  100  45  30  ELISA  OV20/OVS1  81/85  75/89  31  ELISA  20–23  OV 31  Not determined  92  68  26  ELISA  OV 31    100  30  Microplaque spot analysis  OV22/31M  100  74  30  ELISA  28  MSP-2  Major sperm protein  85  100  32  Dot blot assay  33  OC 3.6  Aspartyl protease inhibitor  n/a  93  27  ELISA  OV33-GST  96  93  33  ELISA  C27  n/a  82  34  Recombinant OV33/ELISA  C71  n/a  85  34  Recombinant OV33/ELISA  Ov-API-1  100  100  24  IgG4 LIPS assay  OV 33/5M  100  n/a  30  ELISA  MW (kDa)  Antigen(s)  Associated protein  Specificity (%)  Sensitivity (%)  Reference  Test used  15  OV103  MF surface associated protein  70  57  23  ELISA  Ov-MSA-1  99  89  24  IgG4 LIPS assay  16  OV16  Phosphatidyl ethanolamine binding protein  96  96  25  ELISA  17  OV10  Cysteine proteinase inhibitor  100  61  26  ELISA  OC 9.3  83  68  26  ELISA  OC 9.3  100  84  27  ELISA  OV-CPI  99  32  24  IgG4 LIPS assay  OV7  n/a  75  28  ELISA  19–20  OvMPB/10  Not determined  100  78  29  ELISA  20  OV11  Retinol binding protein  96  54  26  ELISA  OvMPB/11  99  65  29  ELISA  Ov-Far-1  100  100  24  IgG4 LIPS assay  OV20/36M  100  45  30  ELISA  OV20/OVS1  81/85  75/89  31  ELISA  20–23  OV 31  Not determined  92  68  26  ELISA  OV 31    100  30  Microplaque spot analysis  OV22/31M  100  74  30  ELISA  28  MSP-2  Major sperm protein  85  100  32  Dot blot assay  33  OC 3.6  Aspartyl protease inhibitor  n/a  93  27  ELISA  OV33-GST  96  93  33  ELISA  C27  n/a  82  34  Recombinant OV33/ELISA  C71  n/a  85  34  Recombinant OV33/ELISA  Ov-API-1  100  100  24  IgG4 LIPS assay  OV 33/5M  100  n/a  30  ELISA  Currently, the field has settled upon assays detecting antibodies against the Ov16 antigen for monitoring exposure to O. volvulus. This antigen is present in all lifecycle stages35 and elicits detectable antibody responses prior to the appearance of microfilaria in some chimpanzees experimentally infected with O. volvulus36 and in some children exposed to the parasite in endemic communities.37 While the initial assessment of the utility of the Ov16 antigen was assessed using detection of total IgG against Ov16, the IgG4 subtype response was the most specific.38,39 This is perhaps not surprising, as measurements of IgG isotypes in filarial infections revealed that IgG4 accounts for up to 95% of the IgG response to these infections.40 All current versions of the Ov16 assay have focused on IgG4 detection; however, the IgG4 response takes time to develop41 and thus will not immediately reflect exposure to O. volvulus. The Ov16 ELISA is now recommended by WHO guidelines for demonstrating the interruption of transmission of O. volvulus.2 Most Ov16 ELISA methods utilize dried blood spots (DBSs) as the input sample type. DBS samples are relatively stable, and can be easily collected and transported to a central facility for testing later. The cost of the reagents alone required for processing a single sample (generally run in duplicate) are approximately US $0.30, and one individual can process roughly 15 000 samples per year. However, this estimate does not include the cost of shipping to endemic laboratories. Fully burdened costs for running Ov16 ELISA are thus location-specific and should be analyzed as part of efforts to improve laboratory capacity in countries utilizing serological surveillance. The Ov16 antigen was adapted into a rapid format card test by AMRAD ICT (Australia) with a reported sensitivity of 90.6%.42 Despite promising field performance, production of this card test ceased in the year 2000. Recently, the interest in an Ov16 rapid test was revived, spurring the development of two rapid diagnostic tests (RDTs) incorporating the Ov16 antigen which are now commercially available. These consist of a single IgG4 rapid test and a combination test utilizing Ov16 and the W. bancrofti antigen Wb123.43,44 The product inserts report the sensitivity of the Ov16 single test to be 81.1% and the Ov16 test line of the biplex test to be 81.33%, respectively.45 The specificity is listed at 99.0% for the single Ov16 test and 100% for the biplex test, although it was reported to be 1–2% lower than these values in published studies using early prototypes.43,44 As part of the development of the RDT, a recombinant human IgG4 positive control antibody specific for Ov16 was also developed,46 providing a highly pure, consistent and long-term source of positive control for both the ELISA and RDT assays. The new Ov16-containing RDTs for anti-Ov16 serology are rapidly being incorporated into field studies and surveillance activities. While feasibility and acceptability of the rapid tests in surveillance have been demonstrated,47 field-based studies that include performance data using the commercial product are still pending publication. Newly released WHO guidelines recommend evaluation of the performance of these RDTs prior to their use in stop-MDA assessments;2 hopefully, data to assess the utility of the Ov16 RDT in the verification of elimination will be available soon. Entomological surveillance of O. volvulus transmission The transmission cycle of O. volvulus includes blackfly vectors of the genus Simulium. The most direct measure of the status of transmission is to measure infectivity in the black fly vector population itself. Entomological surveillance has the advantage that it eliminates the time lag inherent in the assays focusing on the human host population, where detectable patency lags infection by 12–18 months.37 The disadvantage to entomological surveillance is that large numbers of vector insects must be caught and tested. The current WHO guidelines call for testing sufficient numbers of flies to ensure that the upper bound of the 95% CI of the prevalence of flies carrying infective larvae is less than 0.05% (1/2000).2 Meeting this criterion requires testing at least 6000 flies and having all test negative to meet this criterion.4 Annual transmission potential can be used as an alternative criterion when it is not possible to capture 6000 flies due to low prevalence of flies.2 The traditional method of determining the prevalence of flies carrying infective larvae has been through field dissection of captured flies. However, this method suffers from two disadvantages. First, dissection is expensive because it requires a trained entomologist, a field microscope and a lot of time. Second, a more significant disadvantage is that Simulium damnosum sensu lato, the major vector of O. volvulus in Africa, also serves as the vector for zoonotic species of Onchocerca that do not infect humans.48 The larvae of these animal parasites are difficult or impossible to distinguish morphologically from O. volvulus; thus, dissection data can result in overestimates of the intensity of transmission. A solution to the inability of vector dissection to accurately describe transmission was to develop a specific PCR, and techniques to overcome the cost and time implications of having to test 6000 blackflies individually. Although several DNA amplification assays have been developed, the O-150 PCR has been the assay that has been used most widely.13,49 This PCR method distinguishes O. volvulus from other Onchocerca present in S. damnosum s.l., thereby improving the accuracy of the transmission estimates.9 Screening efficiency using the PCR is superior to dissection, as the PCR assay can be applied to screening pools of flies.49 However, one potential drawback to screening pools of flies is that the O-150 PCR is not quantitative and inhibitors present in the DNA preparations can reduce the efficiency of the PCR, making it impossible to get accurate estimates of the number of parasites present in a positive pool, even when the O-150 PCR is adapted to a quantitative PCR format. Thus, it is not possible to determine whether a positive pool contains a single infectious fly or multiple infectious flies. This problem was overcome by applying probability distribution estimates when it was realized that although it was not possible to determine how many positive flies were in a positive pool, it was possible to say with certainty that negative pools contain no infectious flies. If the infectious flies are randomly distributed among the collection (something easily accomplished when arranging the flies into pools with the maximum number of flies appropriate for processing), it is possible to use a probability distribution to calculate the probability estimate of the number of infected flies in a pool, given the proportion of negative and positive pools and the number of flies contained in each pool.49 The mathematics behind this observation were incorporated into a program (PoolScreen) that calculates the prevalence of infectious flies and associated confidence intervals from the proportion of positive pools, the pool size and the number of pools screened. Field studies conducted in both Africa50 and Latin America51 validated this approach. The O-150 PCR has subsequently been widely applied to collect entomological data verifying elimination of transmission of O. volvulus in Mexico,52 Guatemala53 and one focus in Sudan.54 PCR pool-screening techniques have overcome most of the operational difficulties associated with meeting the WHO guideline’s vector criterion. Screening the 6000 flies necessary to meet this requirement by screening just 60 pools of 100 flies each would take one individual less than 1 week. Pool screening also dramatically reduces the cost and time necessary to process the samples, when compared to analyzing each insect individually. The cost of reagents to process a single pool of 100 flies roughly US$6.90 per pool, or roughly US$ 0.07 per insect. A single individual can process roughly 4000 pools or 400 000 individual insects in a year. Furthermore, the collected insects can be stored in alcohol indefinitely at room temperature, minimizing the logistical difficulties encountered when shipping the collections to a central laboratory for analysis. Collecting the necessary number of flies now represents the main challenge to implementing the entomological surveys. Currently, the standard method of collecting vector black flies is human landing collections (HLCs), which are quite inefficient, as a team of two collectors can only collect one person-day's worth of flies per day. However, recent reports suggest that a new trap platform, known as the Esperanza Window Trap (EWT), may represent an effective alternative to HLCs for collecting vector black flies.55 Studies in Mexico demonstrated that EWTs operated by residents of the affected communities could collect sufficient numbers of flies to certify these communities were free of O. volvulus transmission.56 If the EWT platform has an equivalent performance in Africa when operated by community members, it may overcome the difficulty in obtaining sufficient numbers of vector flies to demonstrate suppression and interruption of transmission. Conclusion and future directions Although the current diagnostic tools have served well for verifying suppression and interruption of transmission of O. volvulus in most countries in Latin America and in several foci in Africa, there are several tools that could accelerate program activities targeting the elimination of onchocerciasis. One of the most pressing needs is to define the sampling schemes for verification of elimination in Africa. In Latin America, the approach that was used by the Onchocerciasis Elimination Program for the Americas was to identify sentinel hyper-endemic communities in each focus prior to the beginning of the program.57 The epidemiological and entomological indicators described previously were then used to follow the decline and eventual elimination of transmission in these sentinels. In general, the foci in Latin America were also isolated from one another, making the definition of a focus a simple matter. In contrast, sentinel communities were often not identified prior to the start of onchocerciasis control in Africa. Furthermore, the foci that exist in Africa are often not isolated, with the potential for reintroduction of the parasite either through migration of infected people or by wind-borne flies.58 It is thus necessary to gain a better understanding of what the limits to a focus are and, once this is done, how these foci should be sampled. For example, the epidemiological metrics needed for stopping MDA still need to be clarified, including how many communities should be enrolled to obtain reliable data on transmission in a focus, how many people in each community should be sampled and how the communities to be included should be chosen. Similarly, for the entomological metrics, it is not clear how many different sites should be included, how many flies need to be collected from each site, or for how long and how often the collections should be carried out. A second need is for the current seroprevalence cut-offs in the 2016 WHO guidelines to be re-evaluated in light of the recent progress in modeling the transmission of O. volvulus. For example, a recent study has suggested that the prevalence of exposure in children that would indicate the parasite population is irreversibly headed to extinction may often be higher than 0.1%, though this is dependent on the baseline endemicity.59 A similar re-evaluation of the entomological metrics is also in order, perhaps with more focus on the annual transmission potential, rather than simple prevalence of infection. Once these analyses are completed, they should be used in conjunction with methods to both more accurately determine test performance in relevant settings and calculate sample sizes that take the sensitivity and specificity of the tests to be employed into account.3 Specificity is paramount when choosing tests for verifying elimination. However, a single-antigen antibody test often cannot deliver a very high degree of specificity without suffering a dramatic loss of sensitivity. One solution is to use a confirmatory test that is independent from the primary test, and require that both the primary and confirmatory tests be positive before declaring a sample positive. Incorporating a second parasite antigen marker in an RDT, with a distinct line for each marker, could allow tailoring of the test to provide either highest sensitivity (requirement of only a single test line present to be positive) or highest specificity (both test lines must be positive). In a similar vein, while the sensitivity and specificity of the PCR assays used in black flies generally approach 100%, a major technical obstacle with these assays is the potential for amplicon contamination resulting in false positive signals. Including an independent PCR assay targeting a second genomic sequence is one way to overcome this problem. Given that there are other antigens already available that can be used to detect exposure to O. volvulus, and other PCR targets already identified for detection of parasite DNA, studies and necessary product development should be undertaken to determine which combination of tests would result in the highest combined sensitivity and specificity for verifying elimination. Finally, the available diagnostics do not directly detect potentially fertile female parasites in the human population that could restart reproduction once MDA is stopped and which may pose a risk for recrudescence. Even though not all adult worms need to be sterile or dead for transmission to be irreversibly interrupted, it would be beneficial to have a method to detect and treat people who harbor adult worm infections. The detection of adult worm infections could allow targeted treatment with medications that can permanently sterilize or kill the worms, either to accelerate program progress to achieving interruption of transmission or to further lower the probability of recrudescence of transmission in mop-up operations that occur after MDA has been stopped but transient transmission is detected. Safe treatments are available that can permanently sterilize and eventually kill the adult females,60 so the missing piece is the diagnostic test to identify people infected with fecund adult females. Some progress has been made in developing assays to detect viable adult parasites in humans. These include specific metabolites produced by female worms61,62 and detection of parasite miRNA in the blood of infected individuals.63 Assays that could detect patent infections in the face of an effective MDA program have great potential for both speeding up the process of elimination and ensuring that the infection does not recrudesce once MDA is withdrawn. Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the US Centers for Disease Control and Prevention or the World Health Organization. Author’s contributions: All authors (TRU, AG, VC and PTC) contributed to the writing of this manuscript. All authors have read and reviewed the final version of the manuscript. Funding: None Competing interests: None declared Ethical approval: Not required. References 1 Vlaminck J , Fischer PU, Weil GJ. Diagnostic tools for onchocerciasis elimination programs. Trends Parasitol  2015; 31( 11): 571– 82. Google Scholar CrossRef Search ADS PubMed  2 World Health Organization . Guidelines for stopping mass drug administration and verifying elimination of human onchocerciasis: Criteria and Procedures . Document # WHO/HTM/NTD/PCT/2016.1; Geneva: WHO Press; 2016. 3 Johnson WO , Su C-L, Gardner IAet al.  . Sample size calculations for surveys to substantiate freedom of populations from infectious agents. Biometrics  2004; 60( 1): 165– 71. Google Scholar CrossRef Search ADS PubMed  4 Basanez MG , Rodriguez-Perez MA, Reyes-Villanueva Fet al.  . Determination of sample sizes for the estimation of Onchocerca volvulus (Filarioidea: Onchocercidae) infection rates in biting populations of Simulium ochraceum s.l. (Diptera: Simuliidae) and its application to ivermectin control programs. J Med Entomol  1998; 35( 5): 745– 57. Google Scholar CrossRef Search ADS PubMed  5 Kale OO , Bammeke AO, Ayeni O. An evaluation of skin snip techniques used in the quantitative assessment of microfilarial densities of Onchocerca volvulus. Bull World Health Organ  1974; 51( 5): 547– 9. Google Scholar PubMed  6 Thiele EA , Cama VA, Lakwo Tet al.  . Detection of Onchocerca volvulus in skin snips by microscopy and real-time polymerase chain reaction: implications for monitoring and evaluation activities. Am J Trop Med Hyg  2016; 94( 4): 906– 11. Google Scholar CrossRef Search ADS PubMed  7 Meredith SEO , Lando G, Gbakima AAet al.  . Onchocerca volvulus: application of the polymerase chain reaction to identification and strain differentiation of the parasite. Exp Parasitol  1991; 73: 335– 44. Google Scholar CrossRef Search ADS PubMed  8 Zimmerman PA , Toe L, Unnasch TR. Design of Onchocerca DNA probes based upon analysis of a repeated sequence family. Mol Biochem Parasitol  1993; 58: 259– 69. Google Scholar CrossRef Search ADS PubMed  9 Toe L , Merriweather A, Unnasch TR. DNA probe based classification of Simulium damnosum s.l. borne and human derived filarial parasites in the Onchocerciasis Control Programme area. Am J Trop Med Hyg  1994; 51: 676– 83. Google Scholar CrossRef Search ADS PubMed  10 Lloyd MM , Gilbert R, Taha NTet al.  . Conventional parasitology and DNA-based diagnostic methods for onchocerciasis elimination programmes. Acta Trop  2015; 146: 114– 8. Google Scholar CrossRef Search ADS PubMed  11 Lagatie O , Merino M, Batsa Debrah Let al.  . An isothermal DNA amplification method for detection of Onchocerca volvulus infection in skin biopsies. Parasit Vectors  2016; 9( 1): 624. Google Scholar CrossRef Search ADS PubMed  12 Alhassan A , Osei-Atweneboana MY, Kyeremeh KFet al.  . Comparison of a new visual isothermal nucleic acid amplification test with PCR and skin snip analysis for diagnosis of onchocerciasis in humans. Mol Biochem Parasitol  2016; 210( 1– 2): 10– 12. Google Scholar CrossRef Search ADS PubMed  13 Poole CB , Li Z, Alhassan Aet al.  . Colorimetric tests for diagnosis of filarial infection and vector surveillance using non-instrumented nucleic acid loop-mediated isothermal amplification (NINA-LAMP). PLoS One  2017; 12( 2): e0169011. Google Scholar CrossRef Search ADS PubMed  14 Zimmerman PA , Guderian RH, Aruajo Eet al.  . Polymerase chain reaction-based diagnosis of Onchocerca volvulus infection: improved detection of patients with onchocerciasis. J Infect Dis  1994; 169( 3): 686– 689. Google Scholar CrossRef Search ADS PubMed  15 Remme J , Baker RH, G DSet al.  . A community trial of ivermectin in the onchocerciasis focus of Asubende, Ghana. I. Effect on the microfilarial reservoir and the transmission of Onchocerca volvulus. Trop Med Parasitol  1989; 40: 367– 74. Google Scholar PubMed  16 Diawara L , Traore MO, Badji Aet al.  . Feasibility of onchocerciasis elimination with ivermectin treatment in endemic foci in Africa: first evidence from studies in Mali and Senegal. PLoS Negl Trop Dis  2009; 3( 7): e497. Google Scholar CrossRef Search ADS PubMed  17 Kilian HD . The use of a topical Mazzotti test in the diagnosis of onchocerciasis. Trop Med Parasitol  1988; 39: 235– 8. Google Scholar PubMed  18 Ozoh G , Boussinesq M, Bissek ACet al.  . Evaluation of the diethylcarbamazine patch to evaluate onchocerciasis endemicity in Central Africa. Trop Med Int Health  2007; 12( 1): 123– 9. Google Scholar PubMed  19 Boatin BA , Toe L, Alley ESet al.  . Detection of Onchocerca volvulus infection in low prevalence areas: a comparison of three diagnostic methods. Parasitology  2002; 125( Pt 6): 545– 52. Google Scholar PubMed  20 Toe L , Adjami AG, Boatin BAet al.  . Topical application of diethylcarbamazine to detect onchocerciasis recrudescence in west Africa. Trans R Soc Trop Med Hyg  2000; 94( 5): 519– 25. Google Scholar CrossRef Search ADS PubMed  21 Lucius R , Buttner DW, Kirsten Cet al.  . A study on antigen recognition by onchocerciasis patients with different clinical forms of disease. Parasitology  1986; 92 ( Pt 3): 569– 80. Google Scholar CrossRef Search ADS PubMed  22 Cabrera Z , Parkhouse RM, Forsyth Ket al.  . Specific detection of human antibodies to Onchocerca volvulus. Trop Med Parasitol  1989; 40( 4): 454– 9. Google Scholar PubMed  23 Lustigman S , Brotman B, Johnson EHet al.  . Identification and characterization of an Onchocerca volvulus cDNA clone encoding a microfilarial surface-associated antigen. Mol Biochem Parasitol  1992; 50: 79– 94. Google Scholar CrossRef Search ADS PubMed  24 Burbelo PD , Leahy HP, Iadarola MJet al.  . A four-antigen mixture for rapid assessment of Onchocerca volvulus infection. PLoS Negl Trop Dis  2009; 3( 5): e438. Google Scholar CrossRef Search ADS PubMed  25 Lobos E , Altmann M, Mengod Get al.  . Identification of an Onchocerca volvulus cDNA encoding a low-molecular-weight antigen uniquely recognized by onchocerciasis patient sera. Mol Biochem Parasitol  1990; 39: 135– 46. Google Scholar CrossRef Search ADS PubMed  26 Ramachandran CP . Improved immunodiagnostic tests to monitor onchocerciasis control programmes—a multicenter effort. Parasitol Today  1993; 9( 3): 76– 79. Google Scholar CrossRef Search ADS   27 Chandrashekar R , Ogunrinade AF, Weil GJ. Use of recombinant Onchocerca volvulus antigens for diagnosis and surveillance of human onchocerciasis. Trop Med Int Health  1996; 1( 5): 575– 80. Google Scholar CrossRef Search ADS PubMed  28 Lustigman S , Brotman B, Huima Tet al.  . Characterization of an Onchocerca volvulus cDNA clone encoding a genus specific antigen present in infective larvae and adult worms. Mol Biochem Parasitol  1991; 45: 65– 76. Google Scholar CrossRef Search ADS PubMed  29 Bradley JE , Trenholme KR, Gillespie AJet al.  . A sensitive serodiagnostic test for onchocerciasis using a cocktail of recombinant antigens. Am J Trop Med Hyg  1993; 48( 2): 198– 204. Google Scholar CrossRef Search ADS PubMed  30 Bradley JE , Helm R, Lahaise Met al.  . cDNA clones of Onchocerca volvulus low molecular weight antigens provide immunologically specific diagnostic probes. Mol Biochem Parasitol  1991; 46( 2): 219– 27. Google Scholar CrossRef Search ADS PubMed  31 Mpagi JL , Buttner DW, Tischendorf FWet al.  . Use of the recombinant Onchocerca volvulus protein Ov20/OvS1 for the immunodiagnostic differentiation between onchocerciasis and mansonelliasis and for the characterization of hyperreactive onchocerciasis (Sowda). Trop Med Int Health  2000; 5( 12): 891– 7. Google Scholar CrossRef Search ADS PubMed  32 Park J , Dickerson TJ, Janda KD. Major sperm protein as a diagnostic antigen for onchocerciasis. Bioorg Med Chem  2008; 16( 15): 7206– 9. Google Scholar CrossRef Search ADS PubMed  33 Lucius R , Kern A, Seeber Fet al.  . Specific and sensitive IgG4 immunodiagnosis of onchocerciasis with a recombinant 33 kD Onchocerca volvulus protein (Ov33). Tropical Medicine & Parasitology  1992; 43( 3): 139– 45. 34 Tume CB , Ngu JL, McKerrow JLet al.  . Characterization of a recombinant Onchocerca volvulus antigen (Ov33) produced in yeast. Am J Trop Med Hyg  1997; 57( 5): 626– 33. Google Scholar PubMed  35 Lobos E , Weiss N, Karam Met al.  . An immunogenic Onchocerca volvulus antigen: a specific and early marker of infection. Science  1991; 251: 1603– 05. Google Scholar CrossRef Search ADS PubMed  36 Eberhard ML , Dickerson JW, Tsang VCet al.  . Onchocerca volvulus: parasitologic and serologic responses in experimentally infected chimpanzees and mangabey monkeys. Exp Parasitol  1995; 80( 3): 454– 62. Google Scholar CrossRef Search ADS PubMed  37 Ogunrinade AF , Chandrashekar R, Eberhard MLet al.  . Preliminary evaluation of recombinant Onchocerca volvulus antigens for serodiagnosis of onchocerciasis. J Clin Microbiol  1993; 31: 1741– 5. Google Scholar PubMed  38 Weil GJ , Ogunrinade AF, Chandrashekar Ret al.  . IgG subclass antibody serology for onchocerciasis. J Infect Dis  1990; 161: 549– 54. Google Scholar CrossRef Search ADS PubMed  39 Ogunrinade AF , Chandrashekar R, Weil GJet al.  . Use of a recombinant antigen (Oc3.6 cDNA) for the serological diagnosis of onchocerciasis in exposed Nigerian children. J Trop Pediatr  1992; 38: 103– 105. Google Scholar CrossRef Search ADS PubMed  40 Ottesen EA , Skvaril F, Tripathy SPet al.  . Prominence of IgG4 in the IgG antibody response to human filariasis. J Immunol  1985; 134( 4): 2707– 12. Google Scholar PubMed  41 Lighaam LC , Rispens T. The immunobiology of immunoglobulin G4. Semin Liver Dis  2016; 36( 3): 200– 15. Google Scholar CrossRef Search ADS PubMed  42 Weil GJ , Steel C, Liftis Fet al.  . A rapid-format antibody card test for diagnosis of onchocerciasis. J Infect Dis  2000; 182( 6): 1796– 9. Google Scholar CrossRef Search ADS PubMed  43 Golden A , Steel C, Yokobe Let 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. Google Scholar CrossRef Search ADS PubMed  44 Steel C , Golden A, Stevens Eet al.  . Rapid point-of-contact tool for mapping and integrated surveillance of Wuchereria bancrofti and Onchocerca volvulus infection. Clin Vaccine Immunol  2015; 22( 8): 896– 901. Google Scholar CrossRef Search ADS PubMed  45 PATH . Quality assurance program materials for onchocerciasis and lymphatic filariasis tests . http://sites.path.org/dx/ntd/training-and-qaqc-materials/ [accessed 27 April 2017). 46 Golden A , Stevens EJ, Yokobe Let al.  . A recombinant positive control for serology diagnostic tests supporting elimination of Onchocerca volvulus. PLoS Negl Trop Dis  2016; 10( 1): e0004292. Google Scholar CrossRef Search ADS PubMed  47 Dieye Y , Storey HL, Barrett KLet al.  . Feasibility of utilizing the SD BIOLINE Onchocerciasis IgG4 rapid test in onchocerciasis surveillance in Senegal. PLoS Negl Trop Dis  2017; 11( 10): e0005884. Google Scholar CrossRef Search ADS PubMed  48 Trees AJ , McCall PJ, Davies JB. On the possibility of bovine Onchocerca species infecting Simulium damnosum s.l. in the forest zone of Sierra Leone. I. Parasitological aspects. Ann Trop Med Parasitol  1989; 83( 6): 595– 601. Google Scholar CrossRef Search ADS PubMed  49 Katholi CR , Toe L, Merriweather Aet al.  . Determining the prevalence of Onchocerca volvulus infection in vector populations by polymerase chain reaction screening of pools of black flies. J Infect Dis  1995; 172( 5): 1414– 17. Google Scholar CrossRef Search ADS PubMed  50 Yameogo L , Toe L, Hougard JMet al.  . 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. Google Scholar CrossRef Search ADS PubMed  51 Rodríguez-Pérez MA , Danis-Lozano R, Rodríguez MHet al.  . Detection of Onchocerca volvulus infection in Simulium ochraceum sensu lato: comparison of a PCR assay and fly dissection in a Mexican hypoendemic community. Parasitology  1999; 119( 6): 613– 19. Google Scholar CrossRef Search ADS PubMed  52 Rodriguez-Perez MA , Fernandez-Santos NA, Orozco-Algarra MEet al.  . Elimination of onchocerciasis from Mexico. PLoS Negl Trop Dis  2015; 9( 7): e0003922. Google Scholar CrossRef Search ADS PubMed  53 Richards F Jr , Rizzo N, Diaz Espinoza CEet al.  . One hundred years after its discovery in Guatemala by Rodolfo Robles, Onchocerca volvulus transmission has been eliminated from the central endemic zone. Am J Trop Med Hyg  2015; 93( 6): 1295– 304. Google Scholar CrossRef Search ADS PubMed  54 Zarroug IM , Hashim K, ElMubark WAet al.  . The first confirmed elimination of an onchocerciasis focus in Africa: Abu Hamed, Sudan. Am J Trop Med Hyg  2016; 27: 1037– 40. Google Scholar CrossRef Search ADS   55 Rodríguez-Pérez MA , Adeleke MA, Burkett-Cadena NDet al.  . Development of a novel trap for the collection of black flies of the Simulium ochraceum complex. PLoS One  2013; 8( 10): e76814. Google Scholar CrossRef Search ADS PubMed  56 Rodríguez-Pérez MA , Adeleke MA, Rodríguez-Luna ICet al.  . Evaluation of a community-based trapping program to collect Simulium ochraceum sensu lato for verification of onchocerciasis elimination. PLoS Negl Trop Dis  2014; 8: e3249. Google Scholar CrossRef Search ADS PubMed  57 Sauerbrey M ; The Onchocerciasis Elimination Program for the Americas (OEPA). Ann Trop Med Parasitol  2008; 102( Suppl. 1): 25– 9. Google Scholar CrossRef Search ADS PubMed  58 Garms R , Walsh JF, Davies JB. Studies on the reinvasion of the Onchocerciasis Control Programme in the Volta River Basin by Simulium damnosum s.l. with emphasis on the south-western areas. Tropenmed Parasitol  1979; 30( 3): 345– 62. Google Scholar PubMed  59 Lont YL , Coffeng LE, de Vlas SJet al.  . Modelling anti-Ov16 IgG4 antibody prevalence as an indicator for evaluation and decision making in onchocerciasis elimination programmes. PLoS Negl Trop Dis  2017; 11( 1): e0005314. Google Scholar CrossRef Search ADS PubMed  60 Walker M , Specht S, Churcher TSet al.  . Therapeutic efficacy and macrofilaricidal activity of doxycycline for the treatment of river blindness. Clin Infect Dis  2015; 60( 8): 1199– 207. Google Scholar CrossRef Search ADS PubMed  61 Denery JR , Nunes AA, Hixon MSet al.  . Metabolomics-based discovery of diagnostic biomarkers for onchocerciasis. PLoS Negl Trop Dis  2010; 4( 10): pii: e834. Google Scholar CrossRef Search ADS   62 Globisch D , Moreno AY, Hixon MSet al.  . Onchocerca volvulus-neurotransmitter tyramine is a biomarker for river blindness. Proc Natl Acad Sci USA  2013; 110( 11): 4218– 23. Google Scholar CrossRef Search ADS PubMed  63 Tritten L , Burkman E, Moorhead Aet al.  . Detection of circulating parasite-derived microRNAs in filarial infections. PLoS Negl Trop Dis  2014; 8( 7): e2971. Google Scholar CrossRef Search ADS PubMed  © World Health Organization, 2018. All rights reserved. The World Health Organization has granted Publisher permission for the reproduction of this article. This is an Open Access article distributed under the terms of the Creative Commons Attribution 3.0 IGO License (https://creativecommons.org/licenses/by/3.0/igo/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Journal

International HealthOxford University Press

Published: Mar 1, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off