Abstract

BackgroundA previously developed, specific, rapid-format immunochromatographic card test that detects immunoglobulin G4 to the recombinant Onchocerca volvulus antigen Ov-16 was modified to detect antibodies in whole blood

MethodsOv-16 card test results were assessed in 1511 subjects ⩾2 years of age in 7 West African villages with varying histories of onchocerciasis control measures

ResultsIn villages in which control measures had been implemented, anti–Ov-16 antibody prevalence rates ranged from 5.2% to 65.1%. Antibody prevalence rates were close to zero among subjects born after effective control measures had been implemented. In 2 villages without a history of control measures where onchocerciasis was endemic, microfilariae (MF) prevalence rates were 82.8% and 65.1%, and antibody prevalence rates were 73.1% and 62.1%. In these 2 villages, the sensitivity of the Ov-16 card test was 81.1% and 76.5%, the specificity was 100%, and the positive predictive value was 91.8% and 80.5%. MF and antibody prevalence rates were correlated (Spearman’s r=0.815; P<.038)

ConclusionsThe Ov-16 card test is field applicable, exhibits high sensitivity and specificity for O. volvulus infection, and has great potential as a tool for surveillance and for evaluating the success of onchocerciasis control measures

Onchocerciasis, which is caused by the parasitic nematode Onchocerca volvulus is a disabling disease that can lead to blindness and severe dermatitis. Despite ongoing control measures, onchocerciasis affects ∼18 million people, 99% of whom reside in sub-Saharan Africa [1]. Efforts have been undertaken by the Onchocerciasis Control Programme in West Africa (OCP), the African Programme for Onchocerciasis Control (APOC), and the Onchocerciasis Elimination Program for the Americas to interrupt O. volvulus transmission. These programs have significantly decreased the prevalence of infection and the risk of blindness, initially by use of vector control (the OCP) and, more recently, by use of communitywide distribution of ivermectin [2]. At last estimate, >40 million people have been protected from onchocerciasis [3]. Although the disease is no longer a public health problem in some controlled regions, additional tools for assessing the success of elimination programs and transmission-interruption strategies are needed [4]

A variety of techniques have been used to assess the impact of control measures. After treatment completion, the detection of parasites in the vector population can indicate recrudescence [5]. However, a more direct method of surveillance would be the detection of new infections in the human population. Typically, infection is identified by detecting microfilariae (MF) in small, superficial skin biopsy samples (skin snips). In areas in which onchocerciasis control programs have been relatively successful, MF are often absent in skin snips, making the detection of early infection difficult [6]. Moreover, the prepatent period (the time from the initiation of infection to the appearance of MF in the skin) is estimated to range from 9 to 15 months [7]. Obviously, the ability to identify infection during the prepatent period is important for early intervention to prevent subsequent transmission

In addition to its inability to detect infection during the prepatent period, the skin snip technique is an invasive procedure, and acceptance is decreasing among populations in areas of endemicity [8–10]. Therefore, an affordable, more reliable, and less invasive field-applicable diagnostic method was developed, one based on the recombinant antigen Ov-16, an O. volvulus–specific antigen to which antibodies develop during the prepatent period of infection [11, 12]. We previously described a rapid-format immunochromatographic card test for the detection of IgG4 antibodies to Ov-16 and demonstrated that this test is specific for O. volvulus [9]. That evaluation used banked human serum samples. The aim of the present study was to evaluate the performance of this test in field studies, primarily to assess its utility for estimating infection prevalence rates in populations in areas of endemicity, and secondarily to determine its usefulness for monitoring the effect that onchocerciasis control programs have on the transmission of O. volvulus to children

Subjects, Materials, and Methods

Study area and sample collectionThe present study was conducted during late 1999 and early 2000 in 5 villages in Burkina Faso and 2 villages in Côte d’Ivoire that had varying rates of O. volvulus infection and varying histories of control measures (table 1). Table 1 indicates when control measures were implemented in each village. Annual communitywide ivermectin distribution was begun in Badone, Koumoon, and Salimbor in 1996, after a resumption of infection in the Bougouriba basin in Burkina Faso. In Linoguin and Wayen, vector-control measures were implemented in 1979 and 1980, respectively, in which weekly larvicidal agents were sprayed over vector-breeding sites [2, 13] as part of the OCP; annual ivermectin treatment began in 3 of the study villages in 1996. No control measures had been implemented in the 2 study villages in Côte d’Ivoire at the time of the present study

Table 1

Control measures implemented and prevalence rates, by study site

Table 1

Control measures implemented and prevalence rates, by study site

A total of 1511 subjects were studied in the 7 villages. In Badone, only persons ⩽15 years of age were included. In the remaining villages, all persons who volunteered to participate were selected, excluding pregnant or lactating women and children <2 years of age. Informed consent was obtained from all study participants. Historical parasitological data for each village was obtained from the records at the OCP headquarters in Ouagadougou, Burkina Faso

Capillary blood samples (collected by fingerprick) and skin snips were obtained from each subject. Of the 1511 subjects initially recruited, all had skin snips processed, and 1484 had interpretable results from the Ov-16 card tests. Inability to interpret the card-test results resulted from either poor blood flow or insufficient blood volume

Parasitological examinationTwo skin snips were obtained from the iliac crests by use of a corneoscleral punch. The skin snips were placed in saline for 1 h at ambient temperature. Emerging MF were identified by microscopic examination. MF counts were recorded as the number of MF per skin snip

Rapid-format Ov-16 card testThe immunochromatographic card test used detects IgG4 antibodies to the recombinant antigen Ov-16 and has been described in detail elsewhere [9]. The present study, however, used 100 μL of whole blood, collected by fingerprick in capillary tubes after the finger had been cleaned with isopropyl alcohol. The tests were read at 10 min

Data analysisMF prevalence was defined as the proportion of positive results among subjects from whom skin snips were obtained; antibody prevalence was defined as the proportion of positive results among subjects who had interpretable results for the Ov-16 card test. By obtaining antibody prevalence rates in villages in which control measures had not been implemented, we were able to measure the sensitivity and positive predictive value of the Ov-16 card test under field conditions, with the skin snip test as the criterion standard. Sensitivity was defined as the proportion of MF-positive subjects who tested positive by the Ov-16 card test. Positive predictive value was defined as the proportion of antibody-positive persons who were also MF positive. Test specificity was 100% (i.e., no uninfected persons give false-positive results), as has been previously demonstrated [9]. To assess the relationship between prevalence rates based on the skin snip test and those based on the Ov-16 card test, Spearman’s&amp;rank correlation coefficient was calculated. Antibody prevalence rates in persons born before and after the initiation of control measures were compared by Fisher’s exact test (2-tailed). Data analysis was performed using SAS (version 9.1; SAS Institute)

Results

Demographics and prevalence ratesThe study populations of the 7 villages ranged from 122 to 367 subjects. Of the total study population, 52% (n=790) were female, and 54% (n = 820) were <20 years of age. MF prevalence rates in the study villages ranged from 0% to 82.8%, and anti–Ov-16 antibody prevalence rates ranged from 5.2% to 73.1% (table 1). Table 1 also shows the number of subjects tested who were born after control measures had been implemented in each village. Vector control was believed to be fully effective with respect to interruption of transmission in Linoguin and Wayen; in Badone, Koumoon, and Salimbor, vector control was only partially effective, and communitywide ivermectin distribution was implemented in 1996. No control measures had been implemented in Mafia or Zakpaberi at the time of the present study

In these 2 villages without a history of either vector control or communitywide ivermectin distribution, MF prevalence rates were 82.8% (Mafia) and 65.1% (Zakpaberi), and antibody prevalence rates were 73.1% (Mafia) and 62.1% (Zakpaberi). In Mafia, the Ov-16 card test had a sensitivity of 81.1%, with a 91.8% positive predictive value. In Zakpaberi, the test sensitivity was 76.5%, and the positive predictive value was 80.5% (table 1)

Not surprisingly, MF prevalence rates were much lower in villages where control measures had been implemented than in villages where they had not (table 1). Moreover, the relative efficacy of these various control measures can be seen by comparing the MF prevalence rates observed before the implementation of control measures with those observed after (the current prevalence rates)

There was a correlation between MF prevalence rates and antibody prevalence rates (figure 1) in the villages studied (Spearman’s r=0.815; P<.038). When MF prevalence rates were zero, antibody prevalence rates were low; however, as MF prevalence rates increased to >10%, antibody prevalence rates tended to be uniformly high, above 60%. This suggests that antibody testing might be a useful tool for estimating onchocerciasis endemicity, especially in areas without a history of control measures. In each of the villages in which such a comparison could be made, antibody prevalence rates were much lower among persons born after control measures were implemented than among persons born before then (Wayen, 26.3% vs. 0% [P<.0001]; Koumoon, 66.5% vs. 10.5% [P<.0001]; Salimbor, 67.1% vs. 11.1% [P<.001]; and Linoguin, 13.5% vs. 2.2% [P<.02])

Figure 1

Relationship between anti–Ov-16 antibody prevalence rates and microfilariae (MF) prevalence rates in 7 villages in Burkina Faso and Côte d’Ivoire, 1999–2000

Figure 1

Relationship between anti–Ov-16 antibody prevalence rates and microfilariae (MF) prevalence rates in 7 villages in Burkina Faso and Côte d’Ivoire, 1999–2000

Utility of the Ov-16 card test after the implementation ofcontrol measuresIn the villages without a history of control measures, Mafia and Zakpaberi, antibody prevalence rates among children 3–4 years of age were 22.2% and 29.0%, respectively. Antibody prevalence rates among children 5–10 years of age (Mafia, 72.7%; Zakpaberi, 50.5%) were almost as high as those seen among adults (figure 2A). In Linoguin and Wayen, all children 5–15 years of age who were born after the implementation of effective control measures were antibody negative, apart from 2 children who had recently migrated with their families to these villages from Côte d’Ivoire. Antibodies to Ov-16 were fairly common in these villages among older people, who had been exposed to onchocerciasis before the implementation of the control measures (figure 2B). In contrast, there was no sharp increase in age-specific antibody prevalence rates in Koumoon, Salimbor, and Badone (as was seen in Linoguin and Wayen). This probably reflects the short duration (3 years) and incomplete coverage (with ivermectin) in these villages

Figure 2

Anti–Ov-16 antibody prevalence rates, by age. Each curve represents the age-specific antibody rates at the time of the study (1999–2000). The horizontal lines in panels B and C indicate the age groups for subjects born after control measures had been implemented; the arrows indicate the dividing points for those born before and those born after implementation. A No control measures implemented. B Vector control implemented in 1979 (Linoguin) and 1980 (Wayen). C Annual communitywide ivermectin distribution implemented in 1996

Figure 2

Anti–Ov-16 antibody prevalence rates, by age. Each curve represents the age-specific antibody rates at the time of the study (1999–2000). The horizontal lines in panels B and C indicate the age groups for subjects born after control measures had been implemented; the arrows indicate the dividing points for those born before and those born after implementation. A No control measures implemented. B Vector control implemented in 1979 (Linoguin) and 1980 (Wayen). C Annual communitywide ivermectin distribution implemented in 1996

Discussion

The purpose of the present study was to evaluate the performance of the rapid-format immunochromatographic Ov-16 card test in field settings of differing endemicity and to assess its utility after the implementation of control measures. We hoped to demonstrate that antibody testing is useful for monitoring the decreases in onchocerciasis endemicity and transmission that occur after the implementation of effective control measures. Antibody testing should also be a useful surveillance tool for the early detection of resurgent onchocerciasis after the suspension of control programs based on vector control or mass treatment

Our results show that the Ov-16 card test has good sensitivity and specificity relative to the standard skin snip test. It is a convenient diagnostic test for field use, because it provides rapid results and uses less-invasive procedures than does the skin snip test. The field results obtained with whole blood assayed by the Ov-16 card test were similar to those previously obtained with human serum samples assayed by either the Ov-16 card test or ELISA [9]. The sensitivity of the Ov-16 card test in MF-positive subjects was higher in villages of low endemicity in Burkina Faso, where partial or complete control had been achieved, than in the villages of high endemicity in Côte d’Ivoire. Prevalence rates (and, presumably, transmission rates) were higher in the Côte d’Ivoire villages than in the Burkina Faso villages; this may reflect the antigen-specific immunosuppression that has been reported in generalized onchocerciasis [14]

Antibody-based tests such as the Ov-16 card test can reflect prepatent, past, and active infections as well as (probably) heavy exposure to O. volvulus without active infection. Antibody testing cannot discriminate among these conditions. In contrast, MF detection by microscopy or polymerase chain reaction (PCR) of skin snips identifies mature infections, but these techniques are insensitive for prepatent infections. Although the Ov-16 card test cannot distinguish between active and past O. volvulus infection, our results show that it should be very useful for estimating prevalence rates, especially in areas without a history of control measures. Anti–Ov-16 antibodies develop long before the appearance of skin MF [9]. Because levels of antibody to Ov-16 slowly decrease after treatment (data not shown), the Ov-16 card test would overestimate prevalence rates in persons who were exposed to O. volvulus before the implementation of control programs. The goal for this field-applicable Ov-16 card test is to use it to screen children as an efficient means of detecting recent infection and (by inference) recent or ongoing transmission in areas where onchocerciasis used to be endemic or in areas that are subject to control efforts

Although PCR is a highly sensitive diagnostic tool for detecting O. volvulus DNA in skin snips or Simulium damnosum vectors, it requires expensive materials, equipment, training, and expertise. Skin snip testing has become less popular in many areas, and it requires that careful measures be taken to prevent person-to-person spread of bloodborne viruses [10]. Use of topical diethylcarbamazine (i.e., the DEC patch test) is noninvasive, but it can cause severe localized reactions, and reported sensitivity and specificity have been inconsistent [15, 16]. The rapid-format Ov-16 card test, on the other hand, requires only a fingerprick and limited training. This makes it a practical tool for field use for identifying areas of endemicity and for detecting infections in untreated individuals. We believe that this test has great potential as a tool for monitoring changes in onchocerciasis endemicity and transmission in areas where control measures have been implemented

Since the early 1970s, the OCP and the APOC have made tremendous efforts toward eliminating onchocerciasis, through both vector and parasite control measures [4, 17–19]. Control efforts lead to diminished transmission and should result in decreased prevalence rates among those born after the implementation of the control measures. In a longitudinal study conducted in Cameroon, Boussinesq et al. demonstrated a reduction in MF prevalence rates among children 5–7 years old over a period of 5 years [20]. Another study, conducted in Liberia, demonstrated that a decrease in transmission led to a significant reduction in onchocerciasis prevalence rates among untreated children [21]. Ivermectin has produced great benefits in terms of prevention of disease. However, because factors such as pregnancy, age <5 years, migration, sickness, refusal, and absence prevent the treatment of entire populations, ivermectin reduces but generally does not completely eliminate the reservoir of MF in African communities. S. damnosum the primary vector responsible for O. volvulus transmission in the present study region, is able to maintain transmission even when skin MF densities are low [20]. Thus, incomplete treatment coverage and the efficiency of the vector can undermine full interruption of the transmission of the parasite and the elimination of onchocerciasis

Our results suggest that the Ov-16 card test has great potential as a tool for monitoring changes in the transmission of O. volvulus and the recrudescence of onchocerciasis in the context of large control programs in which the test used targets persons born after the implementation of control measures. There was substantial variation in the MF and antibody prevalence rates among our study villages. These differences reflected varying baseline infection rates and the presence or absence of effective control measures over time [22]. Despite this variability, our results strongly suggest that the control measures implemented reduced the transmission of new infections to children. The low antibody prevalence rates observed among children born after the implementation of effective control measures suggest that these children had experienced little exposure to O. volvulus. Of course, some of these children had positive antibody tests. In some cases, antibody positivity was seen in children who had migrated from areas with ongoing transmission; in other cases, antibody positivity in children suggested incomplete interruption of transmission in the villages in which they were born

Experience in lymphatic filariasis has shown the value of rapid diagnostic tools for mapping disease and evaluating the success of control measures. The Ov-16 card test is similarly important for efforts to eliminate onchocerciasis. Unfortunately, the Ov-16 card test is no longer available commercially; subsequent to the present study, the manufacturer, AMRAD ICT, went out of business. This is a prime example of the failure of the market to provide critical diagnostic tools for neglected tropical diseases. The availability of diagnostic tests such as the rapid-format immunochromatographic Ov-16 card test will be important for any effort to eliminate onchocerciasis

Acknowledgment

We thank Nancy Shulman, for editorial assistance

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Potential conflicts of interest: none reported
Financial support: Division of Intramural Research, National Institute of Allergy and Infectious Diseases
G.J.W. and T.B.N. contributed equally to this study