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Background: Molecular xenomonitoring (MX), the detection of parasite nucleic acid in the vector population, is recommended for onchocerciasis surveillance in elimination settings. However, the sensitivity of MX for detecting onchocerciasis-positive communities has not previously been evaluated. MX may have additional applications for control programmes but its utility is restricted by a limited understanding of the relationship between MX results and human prevalence., Methods: We conducted a systematic review of studies reporting the prevalence of Onchocerca volvulus DNA in wild-caught Simulium spp. flies (MX rate) and corresponding prevalence of microfilaria (mf) in humans. We evaluated the sensitivity of MX for detecting onchocerciasis-positive communities and describe the characteristics of studies with reduced sensitivity. We conducted a linear regression to evaluate the relationship between mf prevalence and MX rate., Results: We identified 15 relevant studies, with 13 studies comprising 34 study communities included in the quantitative analyses. Most communities were at advanced stages towards elimination and had no or extremely low human prevalence. MX detected positive flies in every study area with >1% mf prevalence, with the exception of one study conducted in the Venezuelan Amazonian focus. We identified a significant relationship between the two measurements, with mf prevalence accounting for half of the variation in MX rate (R2 0.50, p<0.001)., Conclusion: MX is sensitive to communities with ongoing onchocerciasis transmission. It has potential to predict human mf prevalence, but further data is required to understand this relationship, particularly from MX surveys conducted earlier in control programmes before transmission has been interrupted., Competing Interests: The authors have declared that no competing interests exist.

The Simulium damnosum Theobald complex transmits Onchocerca volvulus Leuckart (Spirurida: Onchocercidae), the causative agent of onchocerciasis. Recent evidence suggests that control efforts have strongly suppressed parasite populations, but vector surveillance is needed in parts of Africa where the disease remains endemic. Here, studies on biting rates and infectivity status of suspected vector species were conducted in three onchocerciasis-endemic areas, namely Iwo, Ede, and Obokun, in Osun State, Nigeria. A total of 3,035 black flies were collected between October 2014 and September 2016, and examined for parity and parasites using standard methods. A separate collection of 2,000 black flies was pool-screened for infectivity using polymerase chain reaction (PCR) amplification of the O-150 marker. Results showed that parous flies were significantly less common than nulliparous flies with overall parous rates of 8.02% in Iwo and 35.38% in Ede at the end of the study period. Obokun had a parous rate of 22.22% obtained in the first year only. None of the dissected parous flies were infected with O. volvulus and PCR assays showed no amplification of O-150 O. volvulus-specific repeats in head and body pools. However, annual biting rates exceeded the World Health Organization threshold of 1,000 bites/person/yr. Thus it appears that, with such high rates of biting, even low levels of vector infection can sustain onchocerciasis in African communities., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Onchocerciasis, or river blindness, is a dermal filariasis caused by infection with the nematode parasite Onchocerca volvulus, transmitted to humans through the bites of blackflies of the genus Simulium. Despite the decade-long West African Regional Programme for the Elimination of Onchocerciasis, involving the mass administration of ivermectin to populations in endemic areas, recrudescence has occurred. An example is in the Cascades Region of south-west Burkina Faso where the resumption of transmission had resulted in infection prevalences of up to 70% in some villages. In 2011, a strategy for community-directed distribution of ivermectin (CDTI) was set up to respond to this worrying re-emergence. Here, we report on a study of Onchocerca spp. transmission in the affected area carried out from January to December 2012. Every month, host-seeking adult females of the S. damnosum complex were collected at sites on the River Comoé near the four villages (Bodadiougou, Bolibana, Badara Karaboro and Badara Dogossè) that had recorded the highest prevalences in 2010. Collected blackflies were dissected and infective larvae were identified using the O-150 PCR method., Results: A total of 9114 S. damnosum (s.l.) adult females were collected, of which 5142 were parous (56.4%) and 78 (1.51%) were infective carrying a total of 137 infective larvae. The annual transmission potential (ATP) was calculated as 0, 30, 255 and 771 infective larvae/man/year in Badara Dogossè, Bolibana, Badara Karaboro and Bodadiougou, respectively. Transmission levels in the latter two are of particular concern as they were higher than 100 infective larvae/person/year, the designated minimum threshold required for elimination of severe pathology, including damage to vision., Conclusions: These results confirm that recrudescence of onchocerciasis has occurred, and that transmission of O. volvulus was active at sites on the Comoé River in the Cascades region in 2012. In accordance with WHO recommendations, CDTI should be continued and the situation in the Cascades region should be closely monitored if further spread of this outbreak is to be avoided.

The Mahenge Mountains onchocerciasis focus in south eastern Tanzania was historically one of the most heavily infected areas in the country. The vectors of Onchocerca volvulus are mainly Simulium damnosum complex blackflies, but a species of the Simulium neavei group may also contribute to transmission in some areas. The only detailed studies of parasite transmission in Mahenge were conducted in the late 1960s. The taxonomy of the S. damnosum complex has since been revised and onchocerciasis control through annual community directed treatment with ivermectin (CDTI) commenced in 1997. This study aimed to provide a cytogenetic and molecular update of the S. damnosum complex cytoforms present in Mahenge, and to evaluate the current status of O. volvulus transmission by blackflies following 19 years of annual CDTI. Rivers were surveyed to identify sites of S. damnosum s.l. breeding among the eastern slopes of the mountains, and human landing collections of adult female blackflies were made close to breeding sites. Identification of S. damnosum complex cytoforms was by cytotaxonomy of late-instar larvae and ITS1 amplicon size polymorphisms of larvae and adults. Adult blackflies were pool screened for O. volvulus infection using a triplex real-time PCR. The cytoforms 'Nkusi', Simulium kilibanum and 'Turiani' were found breeding in perennial rivers. 'Nkusi' and S. kilibanum were collected on human bait at 7/7 catch sites and possessed ITS1 profiles most closely resembling the molecular forms 'Nkusi J' and S. kilibanum 'T'. Whereas 'Turiani' was present in rivers, it was not collected on human bait and appears to be zoophilic. Simulium nyasalandicum was collected in low numbers on human bait at 3/7 catch sites. In total, 12,452 S. damnosum s.l. were pool screened and O. volvulus infection was detected in 97/104 pools of bodies and 51/104 pools of heads. The estimated percentage of S. damnosum s.l. carrying infective L3 stage parasites was 0.57% (95% CI 0.43%-0.74%). Onchocerca volvulus transmission by S. damnosum s.l. is continuing in the Mahenge Mountains after 19 years of annual CDTI. Infection rates appear similar to those reported in the 1960s, but a more detailed study is required to fully understand the epidemiological significance of the ongoing transmission. These results provide further evidence that annual CDTI may be insufficient to eliminate the parasite in formerly hyperendemic foci., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The Esmeraldas focus of onchocerciasis in Ecuador expanded geographically during the 1980s and was associated with severe ocular and skin disease. Mass drug administration (MDA) with ivermectin started in 1991, initially once but later twice a year, in the principle endemic focus followed by all satellite foci. Treatment was stopped in 2009 when entomological assessments determined that transmission of Onchocerca volvulus had been interrupted., Methods: Three years after the cessation of ivermectin treatment in 2012, as defined by the WHO guidelines for onchocerciasis elimination, blackfly collections were done in four sentinel sites in former hyperendemic areas. The presence of infective larvae in local vectors, Simulium exiguum and Simulum quadrivittatum, was assessed by detection of O. volvulus DNA by PCR. Additional flies captured in four extra-sentinel sites located in former hyper- and mesoendemic dispersed isolated areas were also assessed., Results: The results from 68,310 captured blackflies, 40,114 from four sentinel villages in the previously hyperendemic areas (Corriente Grande, El Tigre, San Miguel on Río Cayapas and Naranjal on Río Canandé) and 28,197 from extra-sentinel locations, were all negative for the presence of O. volvulus. These extra-sentinel sites (Hualpí on Río Hoja Blanca, Capulí on Río Onzole, La Ceiba on Río Tululví and Medianía on Río Verde) were included to provide additional evidence of the impact of MDA on the transmission of O. volvulus in isolated endemic areas., Conclusions: Our data indicate that transmission of O. volvulus has been stopped in all endemic areas in Ecuador, including all satellite foci outside the main focus. These findings indicate that a strategy of ivermectin distribution twice a year to over 85% of the treatment-eligible population was effective in eliminating the infection from Ecuador in a focus with a highly competent primary vector, S. exiguum, and where the infection rates were equal to or greater than observed in many onchocerciasis foci in Africa.

Background: Mexico is one of the six countries formerly endemic for onchocerciasis in Latin America. Transmission has been interrupted in the three endemic foci of that country and mass drug distribution has ceased. Three years after mass drug distribution ended, post-treatment surveillance (PTS) surveys were undertaken which employed entomological indicators to check for transmission recrudescence., Methodology/principal Findings: In-depth entomologic assessments were performed in 18 communities in the three endemic foci of Mexico. None of the 108,212 Simulium ochraceum s.l. collected from the three foci were found to contain parasite DNA when tested by polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA), resulting in a maximum upper bound of the 95% confidence interval (95%-ULCI) of the infective rate in the vectors of 0.035/2,000 flies examined. This is an order of magnitude below the threshold of a 95%-ULCI of less than one infective fly per 2,000 flies tested, the current entomological criterion for interruption of transmission developed by the international community. The point estimate of seasonal transmission potential (STP) was zero, and the upper bound of the 95% confidence interval for the STP ranged from 1.2 to 1.7 L3/person/season in the different foci. This value is below all previous estimates for the minimum transmission potential required to maintain the parasite population., Conclusions/significance: The results from the in-depth entomological post treatment surveillance surveys strongly suggest that transmission has not resumed in the three foci of Mexico during the three years since the last distribution of ivermectin occurred; it was concluded that transmission remains undetectable without intervention, and Onchocerca volvulus has been eliminated from Mexico.

The posttreatment entomological surveillance (ES) of onchocerciasis in Latin America requires quite large numbers of flies to be examined for parasite infection to prove that the control strategies have worked and that the infection is on the path of elimination. Here, we report a high-throughput automated DNA isolation of Onchocerca volvulus for PCR using a major Latin American black fly vector of onchocerciasis. The sensitivity and relative effectiveness of silica-coated paramagnetic beads was evaluated in comparison with phenol chloroform (PC) method which is known as the gold standard of DNA extraction for ES in Latin America. The automated method was optimized in the laboratory and validated in the field to detect parasite DNA in Simulium ochraceum sensu lato flies in comparison with PC. The optimization of the automated method showed that it is sensitive to detect O. volvulus with a pool size of 100 flies as compared with PC which utilizes 50 flies pool size. The validation of the automated method in comparison with PC in an endemic community showed that 5/67 and 3/134 heads pools were positive for the two methods, respectively. There was no statistical variation (P < 0.05) in the estimation of transmission indices generated by automated method when compared with PC method. The fact that the automated method is sensitive to pool size up to 100 confers advantage over PC method and can, therefore, be employed in large-scale ES of onchocerciasis transmission in endemic areas of Latin America.

Background: Onchocerciasis is caused by Onchocerca volvulus and transmitted by Simulium species (black flies). In the Americas, the infection has been previously described in 13 discrete regional foci distributed among six countries (Brazil, Colombia, Ecuador, Guatemala, Mexico and Venezuela) where more than 370,000 people are currently considered at risk. Since 2001, disease control in Venezuela has relied on the mass drug administration to the at-risk communities. This report provides empirical evidence of interruption of Onchocerca volvulus transmission by Simulium metallicum in 510 endemic communities from two Northern foci of Venezuela, after 10-12 years of 6-monthly Mectizan (ivermectin) treatment to all the eligible residents., Methods: In-depth entomologic and epidemiologic surveys were serially conducted from 2001-2012 in selected (sentinel and extra-sentinel) communities from the North-central (NC) and North-east (NE) onchocerciasis foci of Venezuela in order to monitor the impact of ivermectin treatment., Results: From 2007-2009, entomological indicators in both foci confirmed that 0 out of 112,637 S. metallicum females examined by PCR contained L3 infection in insect heads. The upper bound of the 95% confidence intervals of the infective rate of the vector reached values below 1% by 2009 (NC) and 2012 (NE). Additionally, after 14 (NC) and 22 (NE) rounds of treatment, the seasonal transmission potential (±UL CIs) of S. metallicum was under the critical threshold of 20 L3 per person per season. Serological analysis in school children < 15 years-old demonstrated that 0 out of 6,590 individuals were harboring antibodies to Ov-16. Finally, epidemiological surveys made during 2010 (NC) and 2012 (NE) showed no evidence of microfilariae in the skin and eyes of the population., Conclusions: These results meet the WHO criteria for absence of parasite transmission and disease morbidity in these endemic areas which represent 91% of the population previously at-risk in the country. Consequently, the two Northern foci are currently under post-treatment onchocerciasis surveillance status in Venezuela.

Onchocerciasis in savanna zones is generally more severe than in the forest and pathologies also differ geographically, differences often ascribed to the existence of two or more strains and incompatibilities between vectors and strains. However, flies in the forest transmit more infective larvae than their savanna counterparts, even in sympatry, contradicting expectations based on the forest and savanna strains paradigm. We analysed data on the numbers of Onchocerca volvulus larvae of different stages found in 10 different taxonomic categories of the Simulium damnosum complex derived from more than 48,800 dissections of flies from Sierra Leone in the west of Africa to Uganda in the east. The samples were collected before widespread ivermectin distribution and thus provide a baseline for evaluating control measures. Savanna species contained fewer larvae per infected or per infective fly than the forest species, even when biting and parous rates were accounted for. The highest transmission indices were found in the forest-dwelling Pra form of Simulium sanctipauli (616 L3/1000 parous flies) and the lowest in the savanna-inhabiting species S. damnosum/S. sirbanum (135) and S. kilibanum (65). Frequency distributions of numbers of L1-2 and L3 larvae found in parous S. damnosum/S. sirbanum, S. kilibanum, S. squamosum, S. yahense, S. sanctipauli, S. leonense and S. soubrense all conformed to the negative binomial distribution, with the mainly savanna-dwelling species (S. damnosum/S. sirbanum) having less overdispersed distributions than the mainly forest-dwelling species. These infection patterns were maintained even when forest and savanna forms were sympatric and biting the same human population. Furthermore, for the first time, levels of blindness were positively correlated with infection intensities of the forest vector S. yahense, consistent with relations previously reported for savanna zones. Another novel result was that conversion rates of L1-2 larvae to L3s were equivalent for both forest and savanna vectors. We suggest that either a multiplicity of factors are contributing to the observed disease patterns or that many parasite strains exist within a continuum., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Entomological surveys of Simulium vectors are an important component in the criteria used to determine if Onchocerca volvulus transmission has been interrupted and if focal elimination of the parasite has been achieved. However, because infection in the vector population is quite rare in areas where control has succeeded, large numbers of flies need to be examined to certify transmission interruption. Currently, this is accomplished through PCR pool screening of large numbers of flies. The efficiency of this process is limited by the size of the pools that may be screened, which is in turn determined by the constraints imposed by the biochemistry of the assay. The current method of DNA purification from pools of vector black flies relies upon silica adsorption. This method can be applied to screen pools containing a maximum of 50 individuals (from the Latin American vectors) or 100 individuals (from the African vectors)., Methodology/principal Findings: We have evaluated an alternative method of DNA purification for pool screening of black flies which relies upon oligonucleotide capture of Onchocerca volvulus genomic DNA from homogenates prepared from pools of Latin American and African vectors. The oligonucleotide capture assay was shown to reliably detect one O. volvulus infective larva in pools containing 200 African or Latin American flies, representing a two-four fold improvement over the conventional assay. The capture assay requires an equivalent amount of technical time to conduct as the conventional assay, resulting in a two-four fold reduction in labor costs per insect assayed and reduces reagent costs to $3.81 per pool of 200 flies, or less than $0.02 per insect assayed., Conclusions/significance: The oligonucleotide capture assay represents a substantial improvement in the procedure used to detect parasite prevalence in the vector population, a major metric employed in the process of certifying the elimination of onchocerciasis.

The Itwara focus of onchocerciasis covers an area of approximately 600 km(2) in western Uganda about 20 km north of Fort Portal. The vector is Simulium neavei, whose larvae and pupae live in a phoretic association on freshwater crabs. The phoretic host in the Itwara focus is the crab Potamonautes aloysiisabaudiae. Before any onchocerciasis control, ATPs were estimated to reach between 4500 and 6500 infective larvae per person per year. S. neavei was found to be a very efficient vector with 40% of parous flies harbouring developing larvae of Onchocerca volvulus. After 4 years of community-based distribution of ivermectin transmission was still considerable and in 1995 monthly treatment of streams with the larvicide temephos commenced in the first of three sub-foci, and was gradually extended to the whole focus. Biting S. neavei disappeared from the first sub-focus (Itwara main) in June 1996, and the last infested crab was caught in November 1996. In the second sub-focus (Siisa) treatment commenced towards the end of 1995, and the last biting fly was caught in March 1997, but a deterioration in the security situation interrupted the programme (after only three treatments in the third sub-focus). Monthly treatments restarted in the second and third sub-foci (Aswa) in September 1998, and when the situation was reassessed in 2003 no biting flies were found anywhere, and the flies had not reinvaded the first sub-focus, but infected crabs were found in the second and third sub-foci. The last treatments were carried out in April-June 2003, and since then no infested crabs have been found. In summary, no S. neavei-infested crabs have been found anywhere in the focus since June 2003 and the vector is considered eliminated from that date. However, transmission had already been halted since February 2001, when the last biting flies had been collected. The parasite reservoir should die out in the human population by 2016.

To eliminate transmission of Onchocerca volvulus, semiannual mass treatment with ivermectin (Mectizan; donated by Merck & Co) has been underway in Guatemala since 2000. We applied the 2001 World Health Organization (WHO) elimination criteria in the Santa Rosa focus of onchocerciasis transmission in Guatemala (10,923 persons at risk). No evidence of parasite DNA was found in 2,221 Simulium ochraceum vectors (one-sided 95% confidence interval [CI], 0-0.086%), and no IgG4 antibody positives to recombinant antigen OV16 were found in a sample of 3,232 school children (95% CI, 0-0.009%). We also found no evidence of microfilariae in the anterior segment of the eye in 363 area residents (95% CI, 0-0.08%). Our interpretation of these data, together with historical information, suggest that transmission of O. volvulus is permanently interrupted in Santa Rosa and that ivermectin treatments there can be halted.

Detection of Onchocerca volvulus in Simulium populations is of primary importance in the assessment of the effectiveness of onchocerciasis control programs. In Brazil, the main focus of onchocerciasis is in the Amazon region, in a Yanomami reserve. The main onchocerciasis control strategy in Brazil is the semi-annually mass distribution of the microfilaricide ivermectin. In accordance with the control strategy for the disease, polymerase chain reaction (PCR) was applied in pools of simuliids from the area to detect the helminth infection in the vectors, as recommended by the Onchocerciasis Elimination Program for the Americas and the World Health Organization. Systematic sampling was performed monthly from September 1998 to October 1999, and a total of 4942 blackflies were collected from two sites (2576 from Balawaú and 2366 from Toototobi). The molecular methodology was found to be highly sensitive and specific for the detection of infected and/or infective blackflies in pools of 50 blackflies. The results from the material collected under field conditions showed that after the sixth cycle of distribution of ivermectin, the prevalence of infected blackflies with O. volvulus had decreased from 8.6 to 0.3% in Balawaú and from 4 to 0.1% in Toototobi.

To study the impact of mass Mectizan treatment on Onchocerca volvulus transmission in Mexico, entomological surveys were carried out in the endemic foci of Oaxaca, Southern Chiapas, and Northern Chiapas. Collected flies were screened by polymerase chain reaction (PCR) for O. volvulus parasites. The prevalence of infected and infective flies was estimated using the PoolScreen algorithm and with a novel probability-based method. O. volvulus infective larvae were not detected in flies from 6/13 communities. In 7/13 communities, infective flies were detected, with prevalences ranging from 1.6/10,000 to 29.0/10,000 and seasonal transmission potentials ranging from 0.4 to 3.3. Infected and infective flies were found in a community in Northern Chiapas, suggesting that, according to World Health Organization criteria, autochthonous transmission exists in this focus. These data suggest that O. volvulus transmission in Mexico has been suppressed or brought to a level that may be insufficient to sustain the parasite population.

This article analyzes the results of a one-year entomological survey conducted near the Yalala rapids in the Inga region, Democratic Republic of Congo, prior to the implementation of Community-Directed Treatment with Ivermectin (CDTI). A vector control program had been undertaken from October 1969 to December 1980, 20 kilometers upstream from the study site. The results of the present study show that during 122 collection days, 5,824 females were captured, of which 3,899 were dissected; 2,001 (51.3%) were parous while 44 (2.2%) were infected while 4 (0.2%) were infective. The crude Annual Transmission Potential (ATP) was 65 infective larvae per man and per year at the river as against 12 in Yalala village located two kilometers from the rapids. The average biting rate was 5 flies/man/day during the dry season and 90 during the rainy season. The vector capacity of similium shows that the risk of transmission of Onchocerca volvulus to the human population was not only reduced in time (2-3 months), but was also negligible all year round.

In the Lower Cross River basin in Nigeria, no pre-control entomologic profile of Onchocerca volvulus infection in the local Simulium damnosum population was available prior to the initiation of an ivermectin control program in 1995. A longitudinal entomologic study was therefore carried out over a 12-month period (January-December 2001) at the Agbokim waterfalls and Afi River, which are breeding sites of S. damnosum in the river basin. A total of 9,287 adult S. damnosum were caught on human bait; 9,048 (97.43%) were dissected, of which 313 (3.46%) were infected. Annual biting rates (ABRs) of 42,419 and 28,346 bites per persons per year were recorded at the Agbokim Waterfalls and Afi River, respectively. The annual transmission potential (ATP) was 419 infective larvae per person per year at the Agbokim Waterfalls and 427 at the Afi River. Monthly biting rate and monthly transmission potential varied significantly (P < 0.05) at the two sites. Transmission was highly seasonal from April to September, corresponding to the peak biting period of the vector. The high ATP and ABR values are a measure of the mesoendemicity of onchocerciasis in the river basin. There was a significant F(0).05 (1, 10) (P < 0.05) variation in the relative fly abundance from both sites. It was observed that human activities such as farming, fishing, timber cutting, and hunting are done in the early morning and late afternoon, which corresponds to the peak diurnal biting period of the vector. Changes in these practices and attitudes may markedly affect the disease intensity and transmission.

Onchocerciasis is one of the major causes of blindness in the World, with about 17.7 million infected, particularly in West Africa. In Mexico, onchocerciasis is also present and has been subjected to control since 1923. The standard diagnosis of onchocerciasis is by the detection of microfilariae by skin biopsy and transmission is evaluated by detection of Onchocerca volvulus larvae in the vector. Classically, this was carried out by manual dissection of Simuliumn ochraceun s.l. However, with the use of ivermectin, a drug that kills microfilariae but not the adult worms, the skin biopsy is becoming no longer useful for detecting microfilariae levels and due to the reduced transmission, fly dissection is no longer viable. The subject of this paper is to present the immunological and molecular techniques developed to supersede the skin biopsy and fly dissection, and their diagnostic ability to assess the impact of multiple bi-annual mass ivermectin treatments on O. volvulus transmission in Mexico.

The influence of spatial and temporal factors on onchocerciasis transmission by Simulium exiguum s.l. and S. quadrivittatum in Ecuador was investigated to help develop sampling protocols for entomological surveillance of ivermectin programmes. Flies were collected in alternate months (November 1995-November 1996) at four sites each in the hyperendemic communities of San Miguel and El Tigre. A fixed-effects analysis of variance was used to explore the influence on vector abundance of locality, site, month and hour. Infectivity rates detected by dissection and PCR assays were compared. Simulium exiguum s.l. predominated at El Tigre (75%) whereas S. quadrivittatum prevailed at San Miguel (62%). Vector abundance was highest on river banks and outside houses. Biting and infection rates peaked from March to July. Hourly activity patterns were bimodal in S. exiguum but unimodal in S. quadrivittatum. Annual transmission potentials (ATP) for both species combined were 385 and 733 third stage larvae/person in San Miguel and El Tigre respectively, with S. exiguum accounting for 80% of the combined ATP at both localities. We recommend protocols that may maximize detection of parasite transmission. Infection rates thus obtained must be linked with vector density estimates to assess meaningfully host exposure as treatment progresses.

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) 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.

From 1976 through 1989, weekly aerial spraying operations against blackflies were carried out along the rivers of a wide savanna area of West Africa (approximately 700,000 km(2)) where onchocerciasis was hyperendemic. The level of endemicity began to decrease significantly after 4 years of vector control and became very low in 1989. This situation has been maintained without any vector control activity or chemotherapy, and no incidence of any new cases has been detected. An ophthalmological study carried out in 2000 has confirmed these good results, showing only cicatricial ocular lesions in the examined population. These results led to the conclusion that 14 years of vector control may achieve long-term elimination of onchocerciasis, even in the absence of chemotherapy, provided that the treated areas are not subjected to any contamination by exogenous parasites carried in infected humans or flies.

The status of onchocerciasis vectors in the former Ruwenzori focus in western Uganda was re-examined some 15 years after control measures against Simulium damnosum s.l. were suspended. The four cytoforms S. kilibanum, 'Sebwe', 'Nkusi' and S. pandanophilum were found. While the nonanthropophilic 'Sebwe' was still widely distributed in rivers north, east and south of the Ruwenzori, the only anthropophilic species and vector, S. kilibanum, had disappeared from most of its former habitats and was now restricted to two limited foci, where high biting densities were encountered. It was still a vector south of the Ruwenzori (Kasese focus), where 15.4% of the parous flies were infected with larval stages of Onchocerca volvulus and 34 infective larvae were found in the heads of 1000 parous flies. In the second focus along the Mahoma and Nsonge rivers, a chromosomally highly polymorphic population of S. kilibanum had replaced the former vector S. neavei, but does not act as a vector. Only 2.3% of the parous females were infected and just 1 infective larva was found in the heads of 1000 parous flies.

Detection of Onchocerca volvulus larvae in vector populations is of prime importance in the assessment of the effectiveness of onchocerciasis control programmes. Traditionally, detection of larvae is attained by the dissection of flies, but this time-consuming method cannot easily discriminate between species of Onchocerca. The genome of all Onchocerca species has a unique 150 bp repeat, which can be amplified by PCR, and O. volvulus-specific DNA probes can detect these products by Southern blot (SB). This study optimizes a PCR/SB assay, and compares it with fly dissection to estimate the prevalence (p) and intensity of infection (m) in the local vector population of a Mexican community that has become hypoendemic as a result of 7 years of treatment with ivermectin and nodulectomy. The PCR detected 1 infected fly in a pool of 99 uninfected flies, but the optimal pool size was 50 flies. At the community level, 1 out of 10,550 flies was positive (p = 0.0095%, 95% confidence intervals CI = 0.00024-0.05280%; m = 0.00027 larvae/parous fly, CI = -0.00026-0.00081) by PCR, and 4 out of 10,772 flies (p = 0.0371%, CI = 0.01012-0.09505%; m = 0.00107 larvae/parous fly, 95% CI = 0.00002-0.00212) by dissection (observed m = 0.0005). Both methods produce statistically similar estimates of the prevalence and intensity, indicating that pool screening is a viable alternative for entomological surveillance in areas where the intensity of transmission is becoming extremely low as a result of control interventions.

In areas endemic for onchocerciasis, a small number of individuals show no detectable infection with Onchocerca volvulus despite an apparently similar exposure to the transmitting blackflies. Such individuals have been termed putatively immune. Since several studies have indicated marked host differences in attractiveness for blood-seeking insects, putative immunity in O. volvulus infection may result, at least in part, from low vector attractiveness of the respective individuals. In an area hyperendemic for onchocerciasis (Guinea), where Simulium yahense is the predominant vector, we organized fly catches by putatively immune individuals and individuals with moderate-to-high worm counts. No differences were found between the 2 groups with respect to (i) the attraction of blackflies, (ii) the attraction of blackflies infected with O. volvulus, or (iii) the numbers of O. volvulus larvae carried by the attracted blackflies.

Detection of infective parasites in the vector population can be an early indicator of recrudescence in areas freed of new cases of onchocerciasis. However, dissection of vector black flies is inefficient in areas subject to effective control. Recently, a polymerase chain reaction (PCR)-based assay has been used to detect a single Onchocerca volvulus-infected black fly in pools containing large numbers of uninfected flies. This method had not been validated on wild-caught black flies in an area subject to effective vector control. Here, we report a method of restricting the pool screen PCR assay to infectious parasites and the results of a field test in an area subject to long-term vector control. The prevalence of infection determined by dissection did not differ from that determined by pool screen PCR. The results suggest that the PCR assay may be a useful tool for epidemiologic surveillance for 0. volvulus infection.

The presence of Onchocerca volvulus DNA in experimentally infected flies can now be detected by use of the PCR, so that, for example, one infected Simulium damnosum can be detected in a pool of 100 uninfected flies or one S. ochraceum can be detected in pools of 20-40. As this PCR technique is specific for O. volvulus, the results are not confounded by the presence of other, unimportant, Onchocerca species, and the technique could replace time-consuming, manual dissection of flies. In 1996 and 1997, pools of 16-21 Simulium ochraceum were tested by the PCR technique. These flies had been collected biting man, between 1992 and 1994, from two hyperendemic coffee estates (fincas) in Guatemala, and stored in commercial (95%) ethanol. Collections at finca Buena Vista (869 flies in 52 pools) were made 1-2 weeks and 46 weeks after 45% of eligible subjects had been treated with ivermectin for the first time. At finca El Brote, collections (360 flies in 18 pools) were made 13 weeks before and 7 weeks after 97% of eligible subjects had received their first treatment. DNA was easily recovered from simuliids that had been stored in ethanol for up to 4 years. Of the nine pools of flies with visible blood collected at Buena Vista, each of 20 flies, eight tested positive for O. volvulus DNA. In flies without blood, 13 of 22 pools collected at Buena Vista just after treatment tested positive, whereas there were 14 positives in 22 pools taken 46 weeks later (P > 0.05). At El Brote, nine of 10 pre-treatment pools were positive, compared with three of eight taken 7 weeks post-treatment (P = 0.04), indicating that the treatments in this finca had reduced infection in the vector, and possibly transmission, by about 60%. A sub-sample of Buena Vista flies was divided into 19 sets of three separate sub-pools containing heads, thoraces and abdomens. Three pools of heads alone were positive, and had corresponding pools of positive abdomens. Three positive pools of thoraces had negative corresponding pools of heads and abdomens. These results show that PCR can be used to determine the prevalence of O. volvulus DNA in wild-caught S. ochraceum. As the infection rates observed were higher than expected from dissections reported by other workers, PCR-determined rates may not be directly comparable with traditional parameters based on the dissection of flies to reveal O. volvulus larvae.

In order to identify Onchocerca volvulus larvae from vectors, DNA of filaria larvae from dissected blackflies was isolated, and a 150-bp long tandemly repeated DNA sequence (0-150), which occurs in many Onchocerca species, was amplified using polymerase chain reaction (PCR). Subsequently, the PCR product was blotted onto a nylon membrane and hybridized with DNA probes specific for O. volvulus or Onchocerca ochengi. Filaria larvae from 395 infected Simulium neavei were examined and 259 samples produced detectable PCR products. Among these samples, 239 (92%) reacted with an O. volvulus-specific oligonucleotide. A sample of 69 PCR products was tested using an O. ochengi DNA probe, but all failed to hybridize. Filaria larvae from 64 infected Simulium damnosum, presumably of the cytotypes "Nyamagasani" and "Nkusi" were studied and 0-150 was amplified from 38 samples. From these samples, 35 (92%) hybridized specifically with an O. volvulus probe but none with the O. ochengi-specific DNA sequence. Nonamplified samples were obtained mainly from blackflies that contained only 1 or 2 filaria larvae, and therefore, an insufficient DNA extraction was assumed. It can be concluded that few, if any, filaria species of animal origin were transmitted by S. neavei and S. damnosum s.l. in Kabarole and Kasese districts in Uganda.

The detection of Onchocerca volvulus infected simuliids or blackflies is routinely done by dissection and microscopic examination of individual flies, but this method is tedious and time consuming. Here we describe a method of detecting single O. volvulus infected blackflies in pools of uninfected blackflies. Using a PCR with Onchocerca specific primers it is possible to reproducibly detect one heavily infected blackfly in a pool of 80 flies, or to detect one blackfly inoculated with one microfilaria in a pool of 20 flies. With the method described large numbers of blackflies can be rapidly screened for the presence of O. volvulus infected flies.

Onchocerca volvulus infected Simulium damnosum s.l. were analysed by directed heteroduplex analysis. Of 73 infected flies, 68 produced heteroduplex products identical to those previously identified. All 6 major sibling species, except S. leonense, were present in this group. In the 5 remaining flies, 2 new heteroduplex patterns were noted. Molecular phylogenetic analysis of these samples suggested that they belonged to the S. squamosum/S. yahense subcomplex. The ability to reliably genotype adult flies will permit studies of the vectorial capacity of the sibling species of S. damnosum s.l. for the blinding and non-blinding strains of O. volvulus.

The effect of semiannual ivermectin treatment along with nodulectomy on filarial transmission levels were estimated during the three dry seasons of 1991-1993 in a hyperendemic village in southern Mexico. Parasitologic and ophthalmologic examinations were carried out every six months until five drug treatments were completed. Ivermectin mass treatment with a coverage of approximately 80% had a significant impact (P < 0.05) on the prevalence of skin infection and the mean microfilarial skin density (CMFL), which were reduced 38% and 89%, respectively. A gradual and significant (P < 0.05) decrease in the mean microfilariae number in the anterior chamber of the eye and in corneal opacities was also observed as the CMFL was reduced. After three treatments, these were reduced 84% and 69%, respectively. However, after two years of continuous intervention, no significant differences (P > 0.05) were observed in either the daily mean infective biting density and the daily mean transmission potential. This was probably due to the remaining microfilarial load provided by the untreated resident population and migrant groups. On the whole, our results confirm both the efficacy of ivermectin to alleviate the clinical manifestations of the disease and its minimal impact on Onchocerca volvulus transmission, and indicate the need both to achieve higher levels of drug coverage and to incorporate other measures to stop transmission until a macrofilaricide drug is found.

Larvae of Simulium damnosum complex collected from rivers draining Tukuyu onchocerciasis focus were chromosomally examined. Of the 970 larvae analysed, 910 were identified as Kiwira form, a newly described cytotype found in all S. damnosum larval habitats in the Tukuyu area, while 51 were classified as a new subpopulation, tentatively named as Kasyabone form. The F1 larval progeny obtained from wild caught human biting S. damnosum and reared in the laboratory were chromosomally examined. 509 F1 larvae from 70 females blood fed on human volunteers were identified as Kiwira form. As the main human-biting member of the complex in the Tukuyu area, and because some of the wild caught females had developing filarial larvae indistinguishable from those of Onchocerca volvulus, Kiwira form is thus incriminated as the local vector of onchocerciasis. These observations are discussed with respect to previous literature.

In Guatemala, there is no doubt about the participation of Simulium ochraceum as vector of Onchocerciasis. However, in Mexico practically there are not studies focussed to determine the role of this species in the transmission. The objective of the present investigation was to determine which of the 3 species of Simulium founded in the Soconusco region of Chiapas, is the main, and which were secondary in the transmission of Onchocerciasis in that area. The locality of Morelos, in the Huixtla "municipio" of Chiapas, localized a 1200 meters over the sea level (mosl) were selected to carried out the present study. According to own parasitological studies, this locality is considered as highly endemic (more than 66% prevalence). From March 1979 to March, 1981, we performed captures of Simulium sp caught on human bait. Quantitative studies and of transmission potentials were also performed. The following results were obtained: a) Absolute black flies densities (nulliparous and pariparous) and infected black flies (with L1 and L2 larvae): S. ochraceum, S. metallicum and S. callidum, in that order was the distribution of densities. Infected black flies were obtained in the 3 species. However, in despite of an irregular distribution in all the year, it was possible to identify 2 peaks of maximum infection of S. ochraceum in March, 1979 and March 1981. b) Monthly bite densities and infective bites.(ABSTRACT TRUNCATED AT 250 WORDS)