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ENTOMOLOGICAL EVALUATION BY POOL SCREEN POLYMERASE CHAIN REACTION OF ONCHOCERCA VOLVULUS TRANSMISSION IN ECUADOR FOLLOWING MASS MECTIZAN^TM DISTRIBUTION

ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The prevalence of infected and infective black flies was estimated by pool screen polymerase chain reaction (PCR) amplification in the three river basins in Ecuador endemic for onchocerciasis. Mass distribution of ivermectin (Mectizan^TM) resulted in dramatic declines in the prevalence of infected and infective flies. In the Rio Santiago river basin, no infections were detected, suggesting that transmission had ceased. The ratio of infected to infective flies in Simulium exiguum was 10-fold lower than the corresponding ratio for Simulium quadrivittatum, suggesting that S. exiguum is a more-competent vector for Onchocerca volvulus than S. quadrivittatum. However, the prevalence of infective flies in the two species was not different, suggesting that S. quadrivittatum may play an important vectorial role where it is the dominant human-biting species. The data demonstrate that pool screen PCR is an efficient way to monitor transmission in areas subject to control, and to certify an area as free of O. volvulus transmission

INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Onchocerciasis, or river blindness, is the target of three large international disease control programs: the Onchocerciasis Control Program (OCP) in West Africa, the African Program for Onchocerciasis Control (APOC), and the Onchocerciasis Elimination Program for the Americas (OEPA). These programs differ in their approaches and in their progress toward their final goal of eliminating onchocerciasis as a public health problem. However, both the OCP and OEPA share a common strategic goal: to eliminate transmission of the causative agent of the disease, the filarial parasite Onchocerca volvulus.¹ The thesis underlying this goal is that if transmission can be effectively suppressed for a suitable period of time, the reproductive rate of the parasite will be brought below the minimum replacement rate, and the infection will die out. Evidence that this approach can succeed is provided by the success of the OCP, which has succeeded after almost 20 years of transmission suppression (primarily through vector control) in eliminating onchocerciasis as a public health problem².

If one is to use a strategy of suppressing transmission using chemotherapy to eliminate human onchocerciasis, monitoring the level of transmission is necessary. This is required to demonstrate that the control strategy being applied is effective and to ensure that once control activities are in place, transmission has been brought to the point where the parasite population can no longer sustain itself. O. volvulus transmission has historically been measured through dissection of vector black flies.^3,4 This is an efficient method of monitoring transmission in areas not subject to control, because the prevalence of infection in the vector population is usually high.^5,6 However, in the face of a successful control program, the prevalence of infection in the vector populations is drastically reduced. Thus, the classic dissection method to determine the prevalence of infection becomes progressively less efficient in areas where control has succeeded. Several years ago, a method based on the polymerase chain reaction (PCR) amplification of an Onchocerca-specific repeated DNA sequence (the O-150 PCR) was developed to identify O. volvulus and to distinguish the blinding and nonblinding strains of the parasite.^7–9 The O-150 PCR can detect a single infected fly in pools containing up to 100 flies.¹⁰ Furthermore, a mathematical model has been developed to allow one to calculate prevalence of infection in the black fly population based on the size of the pools screened and on the percentage of negative pools found.¹⁰ In addition, a method for the mass separation of heads and bodies of vector black flies has recently been reported.¹¹ This allows one to obtain separate estimates of the prevalence of parasites in the head capsule (representing the L3 or vertebrate infectious stage) and bodies (representing the earlier developmental stages, i.e., the L1 and L2 larvae). In field-based studies in Africa and Mexico, the O-150 pool screen PCR assay was found to produce estimates of the prevalence of infected and infective flies in the vector population that were indistinguishable from those obtained by traditional dissection.^11,12 These results suggested that the O-150 PCR pool screen assay might be a useful tool to monitor transmission in areas where successful control measures were in place and the level of infection in the vector population was low.

Ecuador is one of the six countries in the Americas endemic for onchocerciasis and therefore is a member of the regional initiative for elimination of the disease. Onchocerciasis is endemic in three river basins in Ecuador: the Rio Santiago, the Rio Cayapas, and the Rio Canande.¹³ The vectors of O. volvulus in Ecuador include Simulium exiguum, a highly efficient limitation-type vector which is comparable to the African forest species in terms of the percentage of flies developing infective stage larvae and the number of larvae per infected fly,¹⁴ and S. quadrivittatum, a species that is thought to be a less-efficient vector.^15,16 Mectizan^TM-based elimination efforts have been under way in Ecuador for the past decade, beginning in 1990 with the Rio Santiago area, where by 1997 transmission was thought to have been brought below the level necessary to maintain the infection.¹⁷

Here we report the results of a large-scale project to assess O. volvulus transmission in Ecuador, using the O-150 pool screen PCR as a monitoring tool. This assay was used to examine in excess of 100,000 flies collected at seven sentinel communities in the three endemic river basins of onchocerciasis in Ecuador during the 2000 transmission season. The data demonstrate a sustained interruption of transmission in the Rio Santiago area since 1997, significant progress toward elimination of transmission in the other river basins, and the usefulness of the O-150 pool screen PCR to efficiently monitor transmission in areas where Mectizan^TM-based elimination programs are being implemented.

MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Black fly collection. Since 1991, Ecuador has been part of the multinational, multiagency OEPA partnership, formed to eliminate onchocerciasis through semiannual treatments with Mectizan^TM. OEPA guidelines for monitoring and evaluation of Mectizan^TM programs include the designation of sentinel communities (mostly hyperendemic) within endemic areas that are subject to in-depth evaluation at about 5-year intervals.¹⁸ Parasitologic indices (skin snip prevalence, community microfilarial skin density, ocular infection) and entomologic indices (prevalence of infective stage larvae in vector populations and monthly and annual transmission potentials) are determined as part of these in-depth evaluations. To the extent that sentinel communities represent all communities within a focus, the in-depth evaluations measure program achievement toward the ultimate goal of eliminating onchocerciasis. The communities included in this study represented the designated sentinel communities for the Rio Santiago, Rio Cayapas, and Rio Canande foci in Esmeraldas Providence, Ecuador.

Flies were collected following standard procedures.¹⁹ Collections were carried out in the transmission seasons of March–June 1996 and April–July 2000. The flies collected in 2000 were separated according to species, divided into aliquots of 50 flies each, and preserved in isopropanol at room temperature. Flies collected in 1996 were not separated according to species but were handled similarly, except that they were divided into pools of 25 each.

Material preparation and O-150 amplification. Isopropanol-preserved pools were rinsed twice in 95% ethanol, air-dried briefly, and placed into 15-mL conical centrifuge tubes. The flies were then placed in liquid nitrogen overnight. In the morning, the tubes were agitated vigorously to separate the heads from the bodies of the flies, and the heads were purified from the bodies by passage through a 25-mesh sieve. DNA was prepared from the separated head and body pools as previously described¹¹ with minor modifications. In brief, the separated head and body pools were homogenized in a solution of 100 mM NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1% SDS, 100 µg/mL proteinase K and 3 µg/mL salmon sperm DNA, and incubated at 56°C for one hour. Dithiothreitol was added to a final concentration of 10 mM and the homogenate heated to 100°C for 30 minutes. The samples were subjected to two freeze-thaw cycles, followed by extraction with phenol and chloroform (1/1 v/v). DNA was purified from the aqueous layer by two cycles of adsorption to silica powder, as previously described.¹¹ The purified DNA was eluted into a final volume of 50 µL of 10mM Tris-HCl (pH 8.0) 1 mM EDTA.

PCR amplifications were carried out in a total volume of 50 µL in a solution consisting of 60 mM Tris-Hcl (pH 9.0) 15 mM (NH₄ )2SO₄, 2 mM MgCl₂, 0.2 mM each dATP, dCTP, dGTP, and dTTP, 0.5 µM of each primer, 2.5 units of Taq polymerase (Roche Diagnostics, Indianapolis, IN), and 2.5 µL of template DNA. The sequence of the primers used was as follows: 5' GATTYTTCCGRCGAANARCGC 3' and 5' B-GCNRT-RTAAATNTGNAAATTC 3', where N = A, G, C, or T; Y = C or T; R = A or G; and B = biotin. Cycling conditions consisted of five cycles of 1 minute at 94°C, 2 minutes at 37°C, and 30 seconds at 72°C, followed by 35 cycles of 30 seconds at 94°C, 30 seconds at 37°C, and 30 seconds at 72°C. The reaction was completed by incubation at 72°C for six minutes. Reactions were run in sets of 96, which included 10 negative controls and two positive controls.

PCR product identification and data analysis. PCR amplification products were detected by PCR enzyme-linked immunosorbent assay (ELISA), essentially as previously described.²⁰ In brief, 96-well Immulon 2 plates (Dynex, Chantilly, VA) were coated overnight with 1 µg/mL strepavidin in 50 mM NaHCO₃ and 2 mM Na₂ CO₃. After coating, the plates were washed six times in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20 (TBST). A total of 10 µL of each PCR product was placed in each well and combined with 40 µL of a solution consisting of 4.8X SSPE, 1X Denhardt’s solution, 0.1% N-lauryl sarcosine, and 0.02% SDS (HB). The plates were incubated at room temperature for 30 minutes and washed six times with TBST. A total of 100 µL of 1N NaOH was added to each well, and the plate was incubated for 1 minute at room temperature and washed six times with TBST. A total of 50 µL of a solution of 50 ng/mL of the detection oligonucleotide OVS2-FL in HB was added to each well and the plate incubated at 42°C for 15 minutes and washed six times with TBST. The sequence of OVS2-FL was 5' AATCTCAAAAAACGGGTACATA-fl 3' where fl = fluorescein. A total of 100 µL of 1X SSPF and 0.1% SDS prewarmed to 42°C was added to each well, and the plate was incubated at 42°C for 10 minutes. The plate was washed six times with TBST and 50 µL of a 1/10,000 dilution of antifluorescein Fab fragment - AP conjugate (Roche Diagnostics) in 0.4M NaCl, 0.1M Tris-HCl (pH 7.5) 0.5% (w/v) bovine serum albumin was added to each well. The plate was incubated at 37°C for 15 minutes and washed six times with TBST. Bound probe was detected using the Amplify AP substrate kit (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Any samples giving an OD value above the mean plus three standard deviations of the 10 negative control wells was considered provisionally positive. Provisionally positive DNA samples were subjected to a second independent PCR amplification and PCR-ELISA assay. Provisionally positive samples that scored positive in the second PCR amplification were scored confirmed.

Statistical analysis. The prevalence of infection in the vector population and confidence intervals surrounding this estimate were calculated as previously described, using the algorithms contained in Poolscreen v1.1 1.¹⁰ Poolscreen uses a statistical model based on the observation that given the pool size and the probability of infection of an individual black fly, the probability that a pool is negative is easily calculated. Each pool represents an independent trial with outcome after testing of positive or negative. The number of positive pools observed in a set of m independent trials is well known to have a binomial distribution with parameters m and p, (the probability of a positive pool). A simple algebraic expression can be found that relates this probability to the unknown infection probability for a single fly. This expression is put into the binomial probability model, and the method of maximum likelihood estimation is used to obtain a point estimate for the infection probability for the individual fly. Using the well-known Clopper-Pearson method for constructing confidence intervals for the binomial parameter p, a suitable confidence interval for the unknown infection probability can be calculated.¹⁰ Comparisons were made among various estimated infection probabilities by calculating the individual confidence intervals at a common specified level (e.g., 95% or 99%). Estimates for which the confidence intervals did not overlap were considered to be significantly different, with a level of significance defined by the widest confidence intervals found not to overlap.

RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Flies were collected from seven sentinel communities in the three river basins endemic for O. volvulus in Esmeraldas Province, Ecuador. Esmeraldas is located in the north of Ecuador along the Pacific coast. The onchocerciasis endemic areas are located in the interior of the province. Demographic statistics of the seven sentinel communities relevant to the current study are presented in Table 1. Before the beginning of Mectizan^TM distribution, all seven communities were hyperendemic for O. volvulus infection, with infection prevalences (as estimated by microscopic examination of skin snips) ranging from 53–96% (Table 1). At the time of the in-depth evaluation conducted in 2000, all communities had received 6–14 rounds of Mectizan^TM treatment at annual or semiannual intervals.

As previously reported, two species of black fly serve as vectors for O. volvulus in Ecuador: S. exiguum and S. quadrivittatum.^15,16 Both species were found at all seven collection sites. However, the proportion of the two species varied. S. exiguum predominated at sites along the Rio Cayapas (Corriente Grande, El Tigre, and San Miguel) and the Rio Canande (Naranjal) while S. quadrivittatum represented the majority of flies collected at the sites on the Rio Santiago (Playa de Oro, Angostura, and Guayabal) (Table 2). In total, 102,200 flies were collected during 2000 (66,250 S. exiguum and 35,950 S. quadrivittatum).

As expected, the pool screen PCR data revealed a dramatic effect of Mectizan^TM distribution on transmission (Table 2). In San Miguel, the overall prevalence of infected flies dropped roughly 16-fold (from 176/10,000 flies to 10.7/10,000 flies) from 1996 to 2000. Similarly, the prevalence of infective flies dropped roughly 60-fold (from 65/10,000 flies to 1.1/10,000 flies). Large decreases in both the infected and infective rate also were noted in the flies collected at El Tigre (Table 2).

Vectorial capacity results from a combination of factors, including biting rate, host preference, the efficiency of parasite development in the insect vector (vector competence), parasite developmental time in the vector, and the average lifespan of the insect.²¹ It was therefore possible that S. exiguum and S. quadrivittatum might exhibit differences in their vectorial capacities that would be reflected in the fly collections examined here. To test this hypothesis, the prevalence of infected and infective S. exiguum and S. quadrivittatum were analyzed from the four collection points where the data presented in Table 2 suggested that O. volvulus transmission was still occurring. The results of this analysis is summarized in Table 3. The prevalence of infected flies was significantly higher in S. quadrivittatum than in S. exiguum (21.5/10,000 flies versus 6.2/10,000 flies, P = 0.01; fiducial confidence interval). In contrast, the prevalence of infective flies was higher in S. exiguum than in S. quadrivittatum, although this difference was not statistically significant (P = 0.05; fiducial confidence interval). Thus, the ratio of the prevalence of infected S. quadrivittatum flies to infective S. quadrivittatum flies was roughly 20/1 while the corresponding ratio for S. exiguum was roughly 2/1.

View larger version (50K): [in this window] [in a new window]       FIGURE 1. Monthly fly collection and infection rates in the four sentinel communities with evidence of ongoing transmission.

DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

These data support the findings of previous pilot studies demonstrating that Mectizan^TM treatment can dramatically decrease transmission of O. volvulus. In these studies, multiple treatments with Mectizan^TM have been shown to reduce transmission of O. volvulus by anywhere from 92–95%.^22,23 In both El Tigre and San Miguel, similar decreases in the prevalence of both infected and infective flies were seen after six semiannual treatments with Mectizan^TM. This suggests that in countries with effective Mectizan^TM-based control programs, transmission can be dramatically reduced, in line with predictions from the previous field trails.

None of the 29,900 flies examined from the three collection sites in the Rio Santiago basin were found to contain O. volvulus. The upper boundary for the 95% confidence interval for the prevalence of infection in a fly population when 29,900 flies are examined and all are found to be negative (as calculated by Poolscreen) is 0.6 per 10,000 flies. In a study published in 1997, Guderian and co-workers reported that the vector infection prevalence in the Rio Santiago basin had been reduced by Mectizan^TM treatment over an 84-month period to roughly 8/10,000 flies, from a precontrol level of 110/ 10,000 flies. Furthermore, no new infections in the human population were detected from 1994 onward.¹⁷ Based on these results, the authors suggested that O. volvulus transmission in the Rio Santiago basin had been brought below the level needed to sustain new infections. The fact that we could not detect any infected flies during this study suggests that the downward trend in transmission in the Rio Santiago area noted by Guderian in 1997 had continued, and that infection of the vector population may have ceased by 2000.

Previous studies have noted that the percentage of O. volvulus microfilaria developing to infective larvae is greater in black flies lacking a cibarial armature when compared to flies that contain this structure.²⁴ This effect is believed to be due to damage to ingested microfilaria in black flies containing a cibarial armature.²⁵ Thus, black flies lacking a cibarial armature are generally more-competent vectors for O. volvulus than those that contain a ciberal armature. The data presented above support this, as the proportion of infected flies to infective flies was 2/1 in S. exiguum, which lacks a ciberal armature, while it was 20/1 in S. quadrivittatum, which contains this structure. However, vectorial capacity of a given species is a complex function that involves a plethora of factors, of which vector competence is only one. In this regard, it is interesting to note that although S. quadrivittatum appears to be a less-competent vector than S. exiguum, the prevalence of infection in S. quadrivittatum was significantly higher than in S. exiguum. Thus, although S. quadrivittatum was a less-competent host for larval development than S. exiguum, the prevalence of infective flies in the two species was not statistically different, suggesting that S. quadrivittatum is playing an important role in maintaining O. volvulus transmission in areas of Ecuador where transmission has not been completely interrupted.

Received June 5, 2002. Accepted for publication July 22, 2002.

Acknowledgments: This work was carried out as part of the established evaluation of impact within the Regional Initiative for the Elimination of Onchocerciasis in the Americas under the financial support of the Onchocerciasis Elimination Program for the Americas/ The Carter Center, Inc. and Inter-American Development Bank contract No. R.EPI.01.17.

Authors’ addresses: Angel G. Guevara, Juan C. Vieira, Andrea López, and Nadia Viera, Laboratory of Clinical Investigation, Community Services Vozandes, Villalengua 267 y 10 de Agosto, 1717691 Quito, Ecuador. Thomas R. Unnasch and Bruce G. Lilley, Division of Geographic Medicine, BBRB Box 7, University of Alabama at Birmingham, 1530 3rd Ave South, Birmingham, AL 35294. Charles Katholi, Department of Biostatistics, Ryals 327, University of Alabama at Birmingham, 1530 3rd Ave South, Birmingham, AL 35294. Richard Collins, Departments of Entomology and Veterinary Science, University of Arizona, P.O. Box 715, Sonoita, AZ 85637. Jose Rumbea, Ecuadorian National Programme for Onchocerciasis Elimination, Servicio Nacional de Erradicacion de la Malaria, Rosa Borja de Icaza No. 110 y Maracaibo, Guayaquil, Ecuador.

Reprint requests: Thomas R. Unnasch, Division of Geographic Medicine, BBRB Box 7, University of Alabama at Birmingham, 1530 3rd Ave South, Birmingham, AL 35294, Telephone: 205-975-7601, Fax: 205-934-5600, E-mail: trunnasch{at}geomed.dom.uab.edu

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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