Evaluation of different DNA extraction methods and loop-mediated isothermal amplification primers for the detection of Mycobacterium ulcerans in clinical specimens

Background: Early diagnosis and treatment of Buruli ulcer is critical in order to avoid the debilitating effects of the disease. In this regard, the development of new diagnostic and point of care tools is encouraged. The loop-mediated isothermal amplification for the detection of Mycobacterium ulcerans represents one of the new tools with a good potential of being developed into a point of care test. There is however the need to standardize the assays, reduce sample preparation times, improve the detection/visualization system and optimize them for high-throughput screening, adaptable to low resourced laboratories. Methods: In this study, we assessed two DNA extraction protocols (modified Boom and EasyNAT methods), three previously published LAMP primer sets (BURULI, MU 2404 and BU-LAMP), and compared the sensitivity and specificity of LAMP assays on three DNA amplification platforms. Results: Our results show that Buruli ulcer diagnosis using primers targeting IS2404 for the LAMP method is sensitive (73.75-91.49%), depending on the DNA extraction method used. Even though the modified Boom DNA extraction method provided the best results, its instrumentation requirement prevent it from being field applicable. The EasyNAT method on the other hand is simpler and may represent the best method for DNA extraction in less resourced settings. Conclusions: For further work on the development and use of LAMP tests for Buruli diagnosis, it is recommended that the BURULI sets of primers be used, as these yielded the best results in terms of sensitivity (87.50-91.49%) and specificity (89.23-100%), depending on the DNA extraction methods used.This study was supported by the Swiss Agency for Development and Cooperation and the UBS Optimus Foundation through FIND, Geneva-Switzerland. The funders had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.S

Assessing the presence of Wuchereria bancrofti in vector and human populations from urban communities in Conakry, Guinea

The Global Programme to Eliminate Lymphatic Filariasis was launched in 2000 with the goal of interrupting transmission of lymphatic filariasis (LF) through multiple rounds of mass drug administration (MDA). In Guinea, there is evidence of ongoing LF transmission, but little is known about the most densely populated parts of the country, including the capital Conakry. In order to guide the LF control and elimination efforts, serological and entomological surveys were carried out to determine whether or not LF transmission occurs in Conakry.; The prevalence of circulating filarial antigen (CFA) of Wuchereria bancrofti was assessed by an immuno-chromatography test (ICT) in people recruited from all five districts of Conakry. Mosquitoes were collected over a 1-year period, in 195 households in 15 communities. A proportion of mosquitoes were analysed for W. bancrofti, using dissection, loop-mediated isothermal amplification (LAMP) assay and conventional polymerase chain reaction (PCR).; CFA test revealed no infection in the 611 individuals examined. A total of 14,334 mosquitoes were collected; 14,135 Culex (98.6 %), 161 Anopheles (1.1 %) and a few other species. Out of 1,312 Culex spp. (9.3 %) and 51 An. gambiae (31.7 %) dissected, none was infected with any stage of the W. bancrofti parasite. However, the LAMP assay revealed that 1.8 % of An. gambiae and 0.31 % of Culex spp. were positive, while PCR determined respective prevalences of 0 % and 0.19 %.; This study revealed the presence of W. bancrofti DNA in mosquitoes, despite the apparent absence of infection in the human population. Although MDA interventions are not recommended where the prevalence of ICT is below 1 %, the entomological results are suggestive of the circulation of the parasite in the population of Conakry. Therefore, rigorous surveillance is still warranted so that LF transmission in Conakry would be identified rapidly and adequate responses being implemented

Ivermectin treatment in humans for reducing malaria transmission.

BACKGROUND: Malaria is transmitted through the bite of Plasmodium-infected adult female Anopheles mosquitoes. Ivermectin, an anti-parasitic drug, acts by killing mosquitoes that are exposed to the drug while feeding on the blood of people (known as blood feeds) who have ingested the drug. This effect on mosquitoes has been demonstrated by individual randomized trials. This effect has generated interest in using ivermectin as a tool for malaria control. OBJECTIVES: To assess the effect of community administration of ivermectin on malaria transmission. SEARCH METHODS: We searched the Cochrane Infectious Diseases Group (CIDG) Specialized Register, CENTRAL, MEDLINE, Embase, LILACS, Science Citation index - expanded, the World Health Organization (WHO) International Clinical Trials Registry Platform, ClinicalTrials.gov, and the National Institutes of Health (NIH) RePORTER database to 14 January 2021. We checked the reference lists of included studies for other potentially relevant studies, and contacted researchers working in the field for unpublished and ongoing trials. SELECTION CRITERIA: We included cluster-randomized controlled trials (cRCTs) that compared ivermectin, as single or multiple doses, with a control treatment or placebo given to populations living in malaria-endemic areas, in the context of mass drug administration. Primary outcomes were prevalence of malaria parasite infection and incidence of clinical malaria in the community. DATA COLLECTION AND ANALYSIS: Two review authors independently extracted data on the number of events and the number of participants in each trial arm at the time of assessment. For rate data, we noted the total time at risk in each trial arm. To assess risk of bias, we used Cochrane's RoB 2 tool for cRCTs. We documented the method of data analysis, any adjustments for clustering or other covariates, and recorded the estimate of the intra-cluster correlation (ICC) coefficient. We re-analysed the trial data provided by the trial authors to adjust for cluster effects. We used a Poisson mixed-effect model with small sample size correction, and a cluster-level analysis using the linear weighted model to adequately adjust for clustering.  MAIN RESULTS: We included one cRCT and identified six ongoing trials.  The included cRCT examined the incidence of malaria in eight villages in Burkina Faso, randomized to two arms. Both trial arms received a single dose of ivermectin 150 µg/kg to 200 µg/kg, together with a dose of albendazole. The villages in the intervention arm received an additional five doses of ivermectin, once every three weeks. Children were enrolled into an active cohort, in which they were repeatedly screened for malaria infection.  The primary outcome was the cumulative incidence of uncomplicated malaria in a cohort of children aged five years and younger, over the 18-week study. We judged the study to be at high risk of bias, as the analysis did not account for clustering or correlation between participants in the same village. The study did not demonstrate an effect of Ivermectin on the cumulative incidence of uncomplicated malaria in the cohort of children over the 18-week study (risk ratio 0.86, 95% confidence interval (CI) 0.62 to 1.17; P = 0.2607; very low-certainty evidence). AUTHORS' CONCLUSIONS: We are uncertain whether community administration of ivermectin has an effect on malaria transmission, based on one trial published to date

The impact of residual infections on Anopheles-transmitted Wuchereria bancrofti after multiple rounds of mass drug administration

Background Many countries have made significant progress in the implementation of World Health Organization recommended preventive chemotherapy strategy, to eliminate lymphatic filariasis (LF). However, pertinent challenges such as the existence of areas of residual infections in disease endemic districts pose potential threats to the achievements made. Thus, this study was undertaken to assess the importance of these areas in implementation units (districts) where microfilaria (MF) positive individuals could not be found during the mid-term assessment after three rounds of mass drug administration. Methods This study was undertaken in Bo and Pujehun, two LF endemic districts of Sierra Leone, with baseline MF prevalence of 2 % and 0 % respectively in sentinel sites for monitoring impact of the national programme. Study communities in the districts were purposefully selected and an assessment of LF infection prevalence was conducted together with entomological investigations undertaken to determine the existence of areas with residual MF that could enable transmission by local vectors. The transmission Assessment Survey (TAS) protocol described by WHO was applied in the two districts to determine infection of LF in 6–7 year old children who were born before MDA against LF started. Results The results indicated the presence of MF infected children in Pujehun district. An. gambiae collected in the district were also positive for W. bancrofti, even though the prevalence of infection was below the threshold associated with active transmission. Conclusions Residual infection was detected after three rounds of MDA in Pujehun – a district of 0 % Mf prevalence at the sentinel site. Nevertheless, our results showed that the transmission was contained in a small area. With the scale up of vector control in Anopheles transmission zones, some areas of residual infection may not pose a serious threat for the resurgence of LF if the prevalence of infections observed during TAS are below the threshold required for active transmission of the parasite. However, robust surveillance strategies capable of detecting residual infections must be implemented, together with entomological assessments to determine if ongoing vector control activities, biting rates and infection rates of the vectors can support the transmission of the disease. Furthermore, in areas where mid-term assessments reveal MF prevalence below 1 % or 2 % antigen level, in Anopheles transmission areas with active and effective malaria vector control efforts, the minimum 5 rounds of MDA may not be required before implementing TAS. Thus, we propose a modification of the WHO recommendation for the timing of sentinel and spot-check site assessments in national programs

Lymphedema in a 7-year-old boy infected with Wuchereria bancrofti in Sierra Leone: A case report

We present a case of congenital lymphedema in a 7-year-old boy in Sierra Leone with active filarial infection and penile edema. The genital edema with onset at 6 months of age may have been due to a congenital abnormality in lymphatic drainage. Other possible causes of childhood lymphedema, including Milroy's disease, are discussed

Strengthening Visceral Leishmaniasis Diagnosis Capacity to Improve Access to Care in Kenya: The Example of Marsabit County

Background: Visceral leishmaniasis (VL), also known as kala-azar, is a neglected tropical disease (NTD) that is fatal if not treated early. The WHO targets the elimination of VL as a public health problem in its 2030 NTD road map. However, improving access to VL diagnosis and treatment remains a major challenge in many VL-endemic countries. Kenya is endemic for VL and is among the top 6 high-disease burden countries in the world. Methods: FIND, through its activities in improving the diagnosis of VL and supporting the elimination of the disease in Kenya, has worked with various county ministries of health (MOH) and central MOH over the last couple of years. FIND’s activities in Marsabit county started in 2018. In this work, we present the implementation of activities and the impacts in Marsabit county. We reviewed the data for 2017 and 2019 outbreaks (before and after the implementation of FIND’s activities) and assessed the importance of improving access and community sensitization to VL diagnosis. We assessed the contribution of each facility to the total distance traveled from a perspective of location optimization. Results: There was a sharp increase in the number of people tested in the 2017 outbreak compared to the 2019 outbreak. In 2017, 437 people were tested compared to 2,338 in 2019. The county reported 234 and 688 VL cases in 2017 and 2019, respectively. The data revealed a shift in the demographic structures of cases toward the younger population (mean age in 2017 was 17.6 years and 15.3 years in 2019), with more female cases reported in 2019 compared to 2017. In 2017, 44.4% were 10 years of age or under. In 2019, the proportion 10 years or below was 52.2%. The addition of two new diagnosis facilities in 2018 resulted in a decrease in the distance traveled by confirmed VL cases from 28.1 km in 2017 to 10.8 km in 2019. Assessing the impact of facility placement indicated the most optimal facilities to provide VL diagnostic services and minimize the distance traveled by patients. Adding new facilities reduces the travel distance until a point where the addition of a new facility provides no additional impact. Conclusion: The results from this study indicate the need to carefully consider the placement of health facilities in improving access to VL diagnosis and treatment and could serve as an investment case in deciding when to stop adding new facilities in a particular setting. Extending the activities in Kenya to other VL-endemic countries in East Africa will contribute significantly toward the elimination of the disease, addressing the needs of marginalized populations and leaving no one behind.Funding for the VL activities in Kenya presented in this paper was provided by the Swiss Agency for Development and Cooperation (SDC) and the Fundación Probitas.S

Is mass drug administration against lymphatic filariasis required in urban settings? The experience in Kano, Nigeria.

BACKGROUND The Global Programme to Eliminate Lymphatic Filariasis (GPELF), launched in 2000, has the target of eliminating the disease as a public health problem by the year 2020. The strategy adopted is mass drug administration (MDA) to all eligible individuals in endemic communities and the implementation of measures to reduce the morbidity of those suffering from chronic disease. Success has been recorded in many rural endemic communities in which elimination efforts have centered. However, implementation has been challenging in several urban African cities. The large cities of West Africa, exemplified in Nigeria in Kano are challenging for LF elimination program because reaching 65% therapeutic coverage during MDA is difficult. There is therefore a need to define a strategy which could complement MDA. Thus, in Kano State, Nigeria, while LF MDA had reached 33 of the 44 Local Government Areas (LGAs) there remained eleven 'urban' LGAs which had not been covered by MDA. Given the challenges of achieving at least 65% coverage during MDA implementation over several years in order to achieve elimination, it may be challenging to eliminate LF in such settings. In order to plan the LF control activities, this study was undertaken to confirm the LF infection prevalence in the human and mosquito populations in three urban LGAs. METHODS The prevalence of circulating filarial antigen (CFA) of Wuchereria bancrofti was assessed by an immuno-chromatography test (ICT) in 981 people in three urban LGAs of Kano state, Nigeria. Mosquitoes were collected over a period of 4 months from May to August 2015 using exit traps, gravid traps and pyrethrum knock-down spray sheet collections (PSC) in different households. A proportion of mosquitoes were analyzed for W. bancrofti, using dissection, loop-mediated isothermal amplification (LAMP) assay and conventional polymerase chain reaction (PCR). RESULTS The results showed that none of the 981 subjects (constituted of <21% of children 5-10 years old) tested had detectable levels of CFA in their blood. Entomological results showed that An. gambiae s.l. had W. bancrofti DNA detectable in pools in Kano; W. bancrofti DNA was detected in between 0.96% and 6.78% and to a lesser extent in Culex mosquitoes where DNA was detected at rates of between 0.19% and 0.64%. DNA analysis showed that An. coluzzii constituted 9.9% of the collected mosquitoes and the remaining 90.1% of the mosquitoes were Culex mosquitoes. CONCLUSION Despite detection of W. bancrofti DNA within mosquito specimens collected in three Kano urban LGAs, we were not able to find a subject with detectable level of CFA. Together with other evidence suggesting that LF transmission in urban areas in West Africa may not be of significant importance, the Federal Ministry of Health advised that two rounds of MDA be undertaken in the urban areas of Kano. It is recommended that the prevalence of W. bancrofti infection in the human and mosquito populations be re-assessed after a couple of years

Assessing Anopheles vector species diversity and transmission of malaria in four health districts along the borders of Côte d’Ivoire

Although malaria and Anopheles mosquito vectors are highly prevalent in Côte d’Ivoire, limited data are available to help understand the malaria vector density and transmission dynamics in areas bordering the country. To address this gap, the Anopheles mosquito species diversity, the members of the Anopheles gambiae complex and the transmission of malaria were assessed in four health districts along the borders of Côte d’Ivoire. Methods From July 2016 through December 2016 and July 2017 through December 2017, adult Anopheles mosquitoes were collected in four health districts of Côte d’Ivoire (Aboisso, Bloléquin, Odienné and Ouangolodougou) using standardized window exit trap (WET) and pyrethrum knockdown spray collection (PSC) methods. The collected mosquitoes were identified morphologically at species level and the members of the An. gambiae complex were separated using short interspersed nuclear element-based polymerase chain reaction (SINE-PCR). Anopheles gambiae sensu lato (s.l.), Anopheles funestus s.l. and Anopheles nili specimens were analysed for malaria Plasmodium parasite detection using the cytochrome oxidase I gene (COX-I), and malaria prevalence among human population through local Ministry of Health (MoH) statistical yearbooks. Results A total of 281 female Anopheles were collected in Aboisso, 754 in Bloléquin, 1319 in Odienné and 2443 in Ouangolodougou. Seven Anopheles species were recorded including An. gambiae s.l. (94.8–99.1%) as the main vector, followed by An. funestus s.l. (0.4–4.3%) and An. nili (0–0.7%). Among An. gambiae s.l., Anopheles coluzzii represented the predominant species in Aboisso (89.2%) and Bloléquin (92.2%), while An. gambiae sensu stricto (s.s.) was the major species in Odienné (96.0%) and Ouangolodougou (94.2%). The Plasmodium sporozoite infection rate in An. gambiae s.l. was highest in Odienné (11.0%; n = 100) followed by Bloléquin (7.8%, n = 115), Aboisso (3.1%; n = 65) and Ouangologoudou (2.5%; n = 120). In An. funestus s.l., Plasmodium falciparum sporozoite infection rate was estimated at 6.2% (n = 32) in Bloléquin, 8.7% (n = 23) in Odienné. No An. funestus s.l. specimens were found infected with P. falciparum sporozoite infection in Ouangolodougou and Aboisso. No P. falciparum sporozoite was detected in An. nili specimens in the four health districts. Among the local human populations, malaria incidence was higher in Odienné (39.7%; n = 45,376) and Bloléquin (37.6%; n = 150,205) compared to that in Ouangolodougou (18.3%; n = 131,629) and Aboisso (19.7%; n = 364,585). Conclusion Anopheles vector species diversity, abundance and Plasmodium sporozoite infection were high within the health districts along the borders of the country of Côte d’Ivoire, resulting in high malaria transmission among the local populations. Anopheles gambiae s.l. and An. funestus s.l. were found to be highly infected with Plasmodium in the health districts of Bloléquin and Odienné where higher malaria incidence was observed than the other districts. This study provides important information that can be used to guide Côte d’Ivoire National Malaria Control Programme for vector control decision-making, mainly in districts that are at the country borders

Elimination of trachoma as a public health problem in Ghana: Providing evidence through a pre-validation survey.

BACKGROUND: In order to achieve elimination of trachoma, a country needs to demonstrate that the elimination prevalence thresholds have been achieved and then sustained for at least a two-year period. Ghana achieved the thresholds in 2008, and since 2011 has been implementing its trachoma surveillance strategy, which includes community and school screening for signs of follicular trachoma and trichiasis, in trachoma-endemic districts. In 2015-2016, the country conducted a district level population-based survey to validate elimination of trachoma as a public health problem. METHODS: As per WHO recommendations, a cross-sectional survey, employing a two-stage cluster random sampling methodology, was used across 18 previously trachoma endemic districts (evaluation units (EUs) in the Upper West and Northern Regions of Ghana. In each EU 24 villages were selected based on probability proportional to estimated size. A minimum of 40 households were targeted per village and all eligible residents were examined for clinical signs of trachoma, using the WHO simplified grading system. The number of trichiasis cases unknown to the health system was determined. Household environmental risk factors for trachoma were also assessed. RESULTS: Data from 45,660 individuals were examined from 11,099 households across 18 EUs, with 27,398 (60.0%) children aged 1-9 years and 16,610 (36.4%) individuals 15 years and above All EUs had shown to have maintained the WHO elimination threshold for Trachomatous inflammation-Follicular (TF) (<5.0% prevalence) in children aged 1-9 years old. The EU TF prevalence in children aged 1-9 years old ranged from between 0.09% to 1.20%. Only one EU (Yendi 0.36%; 95% CI: 0.0-1.01) failed to meet the WHO TT elimination threshold (< 0.2% prevalence in adults aged 15 and above). The EU prevalence of trichiasis (TT) unknown to the health system in adults aged ≥15 years, ranged from 0.00% to 0.36%. In this EU, the estimated TT backlog is 417 All TT patients identified in the study, as well as through on-going surveillance efforts will require further management. A total of 75.9% (95% CI 72.1-79.3, EU range 29.1-92.6) of households defecated in the open but many households had access to an improved water source 75.9% (95%CI: 71.5-79.8, EU range 47.4-90.1%), with 45.5% (95% CI 41.5-49.7%, EU range 28.4-61.8%) making a round trip of water collection < 30 minutes. CONCLUSION: The findings from this survey indicate elimination thresholds have been maintained in Ghana in 17 of the 18 surveyed EUs. Only one EU, Yendi, did not achieve the TT elimination threshold. A scheduled house-by-house TT case search in this EU coupled with surgery to clear the backlog of cases is necessary in order for Ghana to request validation of elimination of trachoma as a public health problem