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Background Annual mass drug administrations (MDA) of ivermectin will strongly reduce Onchocerca volvulus microfilariae (mf) in the skin and in the onchocerciasis patients’ eyes. Ivermectin treatment will also affect the expression of immunity in patients, such that activated immune defenses may help control and contribute to clearance of mf of O. volvulus. Longitudinal surveys are a prerequisite to determining the impact of ivermectin on the status of anti-parasite immunity, notably in risk zones where parasite transmission and active O. volvulus infections persist. Methodology/Principal findings Onchocerciasis patients were treated annually with ivermectin and their Onchocerca volvulus antigen (OvAg) specific IgG and cellular responses were investigated before and at 30 years post initial ivermectin treatment (30yPT). Repeated annual ivermectin treatments eliminated persisting O. volvulus microfilariae (mf) from the skin of patients and abrogated patent infections. The OvAg-specific IgG1 and IgG4 responses were diminished at 30yPT to the levels observed in endemic controls. Prior to starting ivermectin treatment, OvAg-induced cellular productions of IL-10, IFN-γ, CCL13, CCL17 and CCL18 were low in patients, and at 30yPT, cellular cytokine and chemokine responses increased to the levels observed in endemic controls. In contrast, mitogen(PHA)- induced IL-10, IFN-γ, CCL17 and CCL18 cellular production was diminished. This divergent response profile thus revealed increased parasite antigen-specific but reduced polyclonal cellular responsiveness in patients. The transmission of O. volvulus continued at the patients’ location in the Mô river basin in central Togo 2018 and 2019 when 0.58% and 0.45%, respectively, of Simulium damnosum s.l. vector blackflies carried O. volvulus infections. Conclusions/Significance Repeated annual ivermectin treatment of onchocerciasis patients durably inhibited their patent O. volvulus infections despite ongoing low-level parasite transmission in the study area. Repeated MDA with ivermectin affects the expression of immunity in patients. O. volvulus parasite-specific antibody levels diminished to levels seen in infection-free endemic controls. With low antibody levels, antibody-dependent cellular cytotoxic responses against tissue-dwelling O. volvulus larvae will weaken. O. volvulus antigen inducible cytokine and chemokine production increased in treated mf-negative patients, while their innate responsiveness to mitogen declined. Such lower innate responsiveness in elderly patients could contribute to reduced adaptive immune responses to parasite infections and vaccines. On the other hand, increased specific cellular chemokine responses in mf-negative onchocerciasis patients could reflect effector cell activation against tissue invasive larval stages of O. volvulus. The annual Simulium damnosum s.l. biting rate observed in the Mô river basin was similar to levels prior to initiation of MDA with ivermectin, and the positive rtPCR results reported here confirm ongoing O. volvulus transmission. Author summary Onchocerciasis is a neglected tropical disease, and a major cause of debilitating skin disease and ocular damage that can lead to irreversible blindness. Annual mass drug administrations (MDA) of ivermectin strongly reduces the load of Onchocerca volvulus microfilaria (mf) in the skin and in the patients’ eyes. Evolution of onchocerciasis as a disease is prevented by MDA, but recent studies have shown that O. volvulus transmission has not been completely interrupted. Repeated MDA with ivermectin affects immune responses, such that activated immune defenses may enhance clearance of mf of O. volvulus. Longitudinal surveys are required to determine the impact of ivermectin on the status of immunity, notably in risk zones where parasite transmission and active O. volvulus infections persist. We examined the changes of O. volvulus parasite-specific antibody and cellular immune responsiveness in patients treated annually with ivermectin for 30 years. Treatment prevented patent O. volvulus infections, whilst parasite antigen-specific cytokine and chemokine responses increased but O.volvulus-specific antibody responses declined. Such decreased antibody levels could weaken antibody-dependent cellular cytotoxic responses to infective and tissue-dwelling O. volvulus larvae. Strengthened monocyte attracting and activation regulated chemokine responses could enhance effector cell migration and activation against larval stages of O.volvulus, possibly also eliciting resistance to further parasite infections.

Mass drug administration (MDA) of ivermectin has become the main intervention to control onchocerciasis or "river blindness". In Togo, after many years of MDA, Onchocerca volvulus infection has declined dramatically, and elimination appears achievable, but in certain river basins the current situation remains unknown. We have conducted parasitological, serological, ophthalmological, and entomological assessments in northern and central Togo within the river basins of Ôti, Kéran and Mô.Examinations were completed in 1,455 participants from 11 onchocerciasis sentinel villages, and O. volvulus transmission by Simulium damnosum sensu lato (s.l.) was evaluated. In children (aged 1-10 years), the prevalence of microfilariae (Mf) was 2.3% and in adults it ranged from 5.1 to 13.3%. Positive IgG4 responses to O. volvulus adult (crude) worm antigen (OvAg) and the recombinant Ov16 antigen were in all-ages 48.7% and 34.4%, and 29.1% and 14.9% in children, respectively. In the river basin villages of Kéran, Mô and Ôti, the IgG4 seroprevalences to OvAg in children were 51.7%, 23.5% and 12.7%, respectively, and to the Ov16 antigen 33.3% (Kéran) and 5.2% (Ôti). Onchocerciasis ocular lesions (punctate keratitis, evolving iridocyclitis and chorioretinitis) were observed in children and young adults. O. volvulus-specific DNA (Ov150) was detected by poolscreen in vector samples collected from Tchitchira/Kéran(22.8%), Bouzalo/Mô(11.3%), Baghan/Mô(2.9%) and Pancerys/Ôti(4.9%); prevalences of O. volvulus infection in S. damnosum s.l. were, respectively, 1%, 0.5%, 0.1% and 0.2%.In the northern and central river basins in Togo, interruption of O. volvulus transmission has not yet been attained. Patent O. volvulus infections, positive antibody responses, progressive ocular onchocerciasis were diagnosed, and parasite transmission by S. damnosum s.l. occurred close to the survey locations. Future interventions may require approaches selectively targeted to non-complying endemic populations, to the seasonality of parasite transmission and national onchocerciasis control programs should harmonize cross-border MDA as a coordinated intervention.

Background: Annual mass drug administrations (MDA) of ivermectin will strongly reduce Onchocerca volvulus microfilariae (mf) in the skin and in the onchocerciasis patients' eyes. Ivermectin treatment will also affect the expression of immunity in patients, such that activated immune defenses may help control and contribute to clearance of mf of O. volvulus. Longitudinal surveys are a prerequisite to determining the impact of ivermectin on the status of anti-parasite immunity, notably in risk zones where parasite transmission and active O. volvulus infections persist., Methodology/principal Findings: Onchocerciasis patients were treated annually with ivermectin and their Onchocerca volvulus antigen (OvAg) specific IgG and cellular responses were investigated before and at 30 years post initial ivermectin treatment (30yPT). Repeated annual ivermectin treatments eliminated persisting O. volvulus microfilariae (mf) from the skin of patients and abrogated patent infections. The OvAg-specific IgG1 and IgG4 responses were diminished at 30yPT to the levels observed in endemic controls. Prior to starting ivermectin treatment, OvAg-induced cellular productions of IL-10, IFN-γ, CCL13, CCL17 and CCL18 were low in patients, and at 30yPT, cellular cytokine and chemokine responses increased to the levels observed in endemic controls. In contrast, mitogen(PHA)- induced IL-10, IFN-γ, CCL17 and CCL18 cellular production was diminished. This divergent response profile thus revealed increased parasite antigen-specific but reduced polyclonal cellular responsiveness in patients. The transmission of O. volvulus continued at the patients' location in the Mô river basin in central Togo 2018 and 2019 when 0.58% and 0.45%, respectively, of Simulium damnosum s.l. vector blackflies carried O. volvulus infections., Conclusions/significance: Repeated annual ivermectin treatment of onchocerciasis patients durably inhibited their patent O. volvulus infections despite ongoing low-level parasite transmission in the study area. Repeated MDA with ivermectin affects the expression of immunity in patients. O. volvulus parasite-specific antibody levels diminished to levels seen in infection-free endemic controls. With low antibody levels, antibody-dependent cellular cytotoxic responses against tissue-dwelling O. volvulus larvae will weaken. O. volvulus antigen inducible cytokine and chemokine production increased in treated mf-negative patients, while their innate responsiveness to mitogen declined. Such lower innate responsiveness in elderly patients could contribute to reduced adaptive immune responses to parasite infections and vaccines. On the other hand, increased specific cellular chemokine responses in mf-negative onchocerciasis patients could reflect effector cell activation against tissue invasive larval stages of O. volvulus. The annual Simulium damnosum s.l. biting rate observed in the Mô river basin was similar to levels prior to initiation of MDA with ivermectin, and the positive rtPCR results reported here confirm ongoing O. volvulus transmission., Competing Interests: The authors have declared that no competing interests exist.

Background: Togo has conducted annual, integrated, community-based mass drug administration (MDA) for soil-transmitted helminths (STH) and schistosomiasis since 2010. Treatment frequency and target populations are determined by disease prevalence, as measured by baseline surveys in 2007 and 2009, and WHO guidelines. Reported programmatic treatment coverage has averaged over 94%. Togo conducted a cross-sectional survey in 2015 to assess the impact of four to five years of MDA on these diseases., Methodology/principal Findings: In every sub-district in the country outside the capital, the same schools were visited as at baseline and a sample of fifteen children age 6 to 9 years old was drawn. Each child submitted urine and a stool sample. Urine samples were tested by dipstick for the presence of blood as a proxy measure of Schistosoma haematobium infection. Stool samples were analyzed by the Kato-Katz method for STH and Schistosoma mansoni. At baseline, 17,100 children were enrolled at 1,129 schools in 562 sub-districts; in 2015, 16,890 children were enrolled at the same schools. The overall prevalence of both STH and schistosomiasis declined significantly, from 31.5% to 11.6% for STH and from 23.5% to 5.0% for schistosomiasis (p<0.001 in both instances). Egg counts from both years were available only for hookworm and S. mansoni; intensity of infection decreased significantly for both infections from 2009 to 2015 (p<0.001 for both infections). In areas with high baseline prevalence, rebound of hookworm infection was noted in children who had not received albendazole in the past 6 months., Conclusions/significance: After four to five years of MDA in Togo, the prevalence and intensity of STH and schistosomiasis infection were significantly reduced compared to baseline. Data on STH indicate that stopping MDA in areas with high baseline prevalence may result in significant rebound of infection. Togo's findings may help refine treatment recommendations for these diseases., Competing Interests: The authors have declared that no competing interests exist.

Background: Buruli Ulcer (BU) is a neglected tropical skin infection caused by Mycobacterium ulcerans. Residence near aquatic areas has been identified as an important source of transmission of M. ulcerans with increased risk of contracting Buruli ulcer. However, the reservoir and the mode of transmission are not yet well known. The aim of this study was to identify the presence of M. ulcerans in the environment and its relationship with Buruli ulcer occurrence in Zio and Yoto districts of the maritime region in south Togo., Methods: A total of 219 environmental samples including soil (n = 119), water (n = 65), biofilms/plants (n = 29) and animals' feces (n = 6) were collected in 17 villages of Zio and Yoto districts of the maritime region in Togo. DNA of M. ulcerans including IS2404 and IS2606 insertions sequences and mycolactone ketoreductase-B gene (KR-B) was detected using real time PCR amplification (qPCR) technique. In parallel, clinical samples of patients were tested to establish a comparison of the genetic profile of M. ulcerans between the two types of samples. A calibration curve was generated for IS2404 from a synthetic gene of M. ulcerans Transposase pMUM001, the plasmid of virulence., Results: In the absence of inhibition of the qPCR, 6/219 (2.7%) samples were tested positive for M. ulcerans DNA containing three sequences (IS2404/IS2606/KR-B). Positive samples of M. ulcerans were consisting of biofilms/plants (3/29; 10.3%), water (1/65; 1.7%) and soil (2/119; 1.5%). Comparative analysis between DNA detected in environmental and clinical samples from BU patients showed the same genetic profile of M. ulcerans in the same environment. All these samples were collected in the environment of Haho and Zio rivers in the maritime region., Conclusion: This study confirms the presence of M. ulcerans in the environment of the Zio and Yoto districts of the maritime region of Togo. This may explain partially, the high rates of Buruli ulcer patients in this region. Also, water, plants and soil along the rivers could be possible reservoirs of the bacterium. Therefore, Haho and Zio rivers could be potential sources of infection with M. ulcerans in humans in these districts., Competing Interests: The authors have declared that no competing interests exist.

Background: Mass drug administration (MDA) of ivermectin has become the main intervention to control onchocerciasis or "river blindness". In Togo, after many years of MDA, Onchocerca volvulus infection has declined dramatically, and elimination appears achievable, but in certain river basins the current situation remains unknown. We have conducted parasitological, serological, ophthalmological, and entomological assessments in northern and central Togo within the river basins of Ôti, Kéran and Mô., Methodology/principal Findings: Examinations were completed in 1,455 participants from 11 onchocerciasis sentinel villages, and O. volvulus transmission by Simulium damnosum sensu lato (s.l.) was evaluated. In children (aged 1-10 years), the prevalence of microfilariae (Mf) was 2.3% and in adults it ranged from 5.1 to 13.3%. Positive IgG4 responses to O. volvulus adult (crude) worm antigen (OvAg) and the recombinant Ov16 antigen were in all-ages 48.7% and 34.4%, and 29.1% and 14.9% in children, respectively. In the river basin villages of Kéran, Mô and Ôti, the IgG4 seroprevalences to OvAg in children were 51.7%, 23.5% and 12.7%, respectively, and to the Ov16 antigen 33.3% (Kéran) and 5.2% (Ôti). Onchocerciasis ocular lesions (punctate keratitis, evolving iridocyclitis and chorioretinitis) were observed in children and young adults. O. volvulus-specific DNA (Ov150) was detected by poolscreen in vector samples collected from Tchitchira/Kéran(22.8%), Bouzalo/Mô(11.3%), Baghan/Mô(2.9%) and Pancerys/Ôti(4.9%); prevalences of O. volvulus infection in S. damnosum s.l. were, respectively, 1%, 0.5%, 0.1% and 0.2%., Conclusions/significance: In the northern and central river basins in Togo, interruption of O. volvulus transmission has not yet been attained. Patent O. volvulus infections, positive antibody responses, progressive ocular onchocerciasis were diagnosed, and parasite transmission by S. damnosum s.l. occurred close to the survey locations. Future interventions may require approaches selectively targeted to non-complying endemic populations, to the seasonality of parasite transmission and national onchocerciasis control programs should harmonize cross-border MDA as a coordinated intervention.

Background: As the major burden of Buruli ulcer disease (BUD) occurs in remote rural areas, development of point-of-care (POC) tests is considered a research priority to bring diagnostic services closer to the patients. Loop-mediated isothermal amplification (LAMP), a simple, robust and cost-effective technology, has been selected as a promising POC test candidate. Three BUD-specific LAMP assays are available to date, but various technical challenges still hamper decentralized application. To overcome the requirement of cold-chains for transport and storage of reagents, the aim of this study was to establish a dry-reagent-based LAMP assay (DRB-LAMP) employing lyophilized reagents., Methodology/principal Findings: Following the design of an IS2404 based conventional LAMP (cLAMP) assay suitable to apply lyophilized reagents, a lyophylization protocol for the DRB-LAMP format was developed. Clinical performance of cLAMP was validated through testing of 140 clinical samples from 91 suspected BUD cases by routine assays, i.e. IS2404 dry-reagent-based (DRB) PCR, conventional IS2404 PCR (cPCR), IS2404 qPCR, compared to cLAMP. Whereas qPCR rendered an additional 10% of confirmed cases and samples respectively, case confirmation and positivity rates of DRB-PCR or cPCR (64.84% and 56.43%; 100% concordant results in both assays) and cLAMP (62.64% and 52.86%) were comparable and there was no significant difference between the sensitivity of the assays (DRB PCR and cPCR, 86.76%; cLAMP, 83.82%). Likewise, sensitivity of cLAMP (95.83%) and DRB-LAMP (91.67%) were comparable as determined on a set of 24 samples tested positive in all routine assays., Conclusions/significance: Both LAMP formats constitute equivalent alternatives to conventional PCR techniques. Provided the envisaged availability of field friendly DNA extraction formats, both assays are suitable for decentralized laboratory confirmation of BUD, whereby DRB-LAMP scores with the additional advantage of not requiring cold-chains. As validation of the assays was conducted in a third-level laboratory environment, field based evaluation trials are necessary to determine the clinical performance at peripheral health care level.

Background: Following introduction of antimycobacterial treatment of Buruli ulcer disease (BUD), several clinical studies evaluated treatment outcomes of BUD patients, in particular healing times, secondary lesions and functional limitations. Whereas recurrences were rarely observed, paradoxical reactions and functional limitations frequently occurred. Although systematic BUD control in Togo was established as early as 2007, treatment outcome has not been reviewed to date. Therefore, a pilot project on post-treatment follow-up of BUD patients in Togo aimed to evaluate treatment outcomes and to provide recommendations for optimization of treatment success., Methodology/principal Findings: Out of 199 laboratory confirmed BUD patients, 129 could be enrolled in the study. The lesions of 109 patients (84.5%) were completely healed without any complications, 5 patients (3.9%) had secondary lesions and 15 patients (11.6%) had functional limitations. Edema, category III ulcers >15 cm, healing times >180 days and a limitation of movement at time of discharge constituted the main risk factors significantly associated with BUD related functional limitations (P<0.01). Review of all BUD related documentation revealed major shortcomings, in particular concerning medical records on adjuvant surgical and physiotherapeutic treatment., Conclusions/significance: This study presents the first systematic analysis of treatment outcome of BUD patients from Togo. Median times to healing and the absence of recurrences were in line with findings reported by other investigators. The percentage of functional limitations of 11.6% was lower than in other studies, and edema, category III ulcers, healing time >180 days and limitation of movement at discharge constituted the main risk factors for functional limitations in Togolese BUD patients. Standardized treatment plans, patient assessment and follow-up, as well as improved management of medical records are recommended to allow for intensified monitoring of disease progression and healing process, to facilitate implementation of therapeutic measures and to optimize treatment success.

Background: The only available vaccine that could be potentially beneficial against mycobacterial diseases contains live attenuated bovine tuberculosis bacillus (Mycobacterium bovis) also called Bacillus Calmette-Guérin (BCG). Even though the BCG vaccine is still widely used, results on its effectiveness in preventing mycobacterial diseases are partially contradictory, especially regarding Buruli Ulcer Disease (BUD). The aim of this case-control study is to evaluate the possible protective effect of BCG vaccination on BUD., Methodology: The present study was performed in three different countries and sites where BUD is endemic: in the Democratic Republic of the Congo, Ghana, and Togo from 2010 through 2013. The large study population was comprised of 401 cases with laboratory confirmed BUD and 826 controls, mostly family members or neighbors., Principal Findings: After stratification by the three countries, two sexes and four age groups, no significant correlation was found between the presence of BCG scar and BUD status of individuals. Multivariate analysis has shown that the independent variables country (p = 0.31), sex (p = 0.24), age (p = 0.96), and presence of a BCG scar (p = 0.07) did not significantly influence the development of BUD category I or category II/III. Furthermore, the status of BCG vaccination was also not significantly related to duration of BUD or time to healing of lesions., Conclusions: In our study, we did not observe significant evidence of a protective effect of routine BCG vaccination on the risk of developing either BUD or severe forms of BUD. Since accurate data on BCG strains used in these three countries were not available, no final conclusion can be drawn on the effectiveness of BCG strain in protecting against BUD. As has been suggested for tuberculosis and leprosy, well-designed prospective studies on different existing BCG vaccine strains are needed also for BUD.

Background: In a previous study PCR analysis of clinical samples from suspected cases of Buruli ulcer disease (BUD) from Togo and external quality assurance (EQA) for local microscopy were conducted at an external reference laboratory in Germany. The relatively poor performance of local microscopy as well as effort and time associated with shipment of PCR samples necessitated the implementation of stringent EQA measures and availability of local laboratory capacity. This study describes the approach to implementation of a national BUD reference laboratory in Togo., Methodology: Large scale outreach activities accompanied by regular training programs for health care professionals were conducted in the regions "Maritime" and "Central," standard operating procedures defined all processes in participating laboratories (regional, national and external reference laboratories) as well as the interaction between laboratories and partners in the field. Microscopy was conducted at regional level and slides were subjected to EQA at national and external reference laboratories. For PCR analysis, sample pairs were collected and subjected to a dry-reagent-based IS2404-PCR (DRB-PCR) at national level and standard IS2404 PCR followed by IS2404 qPCR analysis of negative samples at the external reference laboratory., Principal Findings: The inter-laboratory concordance rates for microscopy ranged from 89% to 94%; overall, microscopy confirmed 50% of all suspected BUD cases. The inter-laboratory concordance rate for PCR was 96% with an overall PCR case confirmation rate of 78%. Compared to a previous study, the rate of BUD patients with non-ulcerative lesions increased from 37% to 50%, the mean duration of disease before clinical diagnosis decreased significantly from 182.6 to 82.1 days among patients with ulcerative lesions, and the percentage of category III lesions decreased from 30.3% to 19.2%., Conclusions: High inter-laboratory concordance rates as well as case confirmation rates of 50% (microscopy), 71% (PCR at national level), and 78% (including qPCR confirmation at external reference laboratory) suggest high standards of BUD diagnostics. The increase of non-ulcerative lesions, as well as the decrease in diagnostic delay and category III lesions, prove the effect of comprehensive EQA and training measures involving also procedures outside the laboratory.