Characterising co-infections with Plasmodium spp., Mansonella perstans or Loa loa in asymptomatic children,adults and elderly people living on Bioko Island using nucleic acids extracted from malaria rapid diagnostic tests

BackgroundRegular and comprehensive epidemiological surveys of the filarial nematodes Mansonella perstans and Loa loa in children, adolescents and adults living across Bioko Island, Equatorial Guinea are lacking. We aimed to demonstrate that blood retained on malaria rapid diagnostic tests, commonly deployed for malaria surveys, could be used as a source of nucleic acids for molecular based detection of M. perstans and L. loa. We wanted to determine the positivity rate and distribution of filarial nematodes across different age groups and geographical areas as well as to understand level of co-infections with malaria in an asymptomatic population.MethodologyM. perstans, L. loa and Plasmodium spp. parasites were monitored by qPCR in a cross-sectional study using DNA extracted from a subset malaria rapid diagnostic tests (mRDTs) collected during the annual malaria indicator survey conducted on Bioko Island in 2018.Principal findingsWe identified DNA specific for the two filarial nematodes investigated among 8.2% (263) of the 3214 RDTs screened. Positivity rates of M. perstans and L. loa were 6.6% and 1.5%, respectively. M. perstans infection were more prominent in male (10.5%) compared to female (3.9%) survey participants. M. perstans parasite density and positivity rate was higher among older people and the population living in rural areas. The socio-economic status of participants strongly influenced the infection rate with people belonging to the lowest socio-economic quintile more than 3 and 5 times more likely to be L. loa and M. perstans infected, respectively. No increased risk of being co-infected with Plasmodium spp. parasites was observed among the different age groups.Conclusions/SignificanceWe found otherwise asymptomatic individuals were infected with M. perstans and L. loa. Our study demonstrates that employing mRDTs probed with blood for malaria testing represents a promising, future tool to preserve and ship NAs at room temperature to laboratories for molecular, high-throughput diagnosis and genotyping of blood-dwelling nematode filarial infections. Using this approach, asymptomatic populations can be reached and surveyed for infectious diseases beyond malaria.     

Diagnostic performance of capillary and venous blood samples in the detection of Loa loa and Mansonella perstans microfilaraemia using light microscopy

BackgroundLoa loa and Mansonella perstans–the causative agents of loiasis and mansonellosis—are vector-borne filarial parasites co-endemic in sub-Saharan Africa. Diagnosis of both infections is usually established by microscopic analysis of blood samples. It was recently established that the odds for detecting Plasmodium spp. is higher in capillary (CAP) blood than in venous (VEN) blood. In analogy to this finding this analysis evaluates potential differences in microfilaraemia of L. loa and M. perstans in samples of CAP and VEN blood.MethodsRecruitment took place between 2015 and 2019 at the CERMEL in Lambaréné, Gabon and its surrounding villages. Persons of all ages presenting to diagnostic services of the research center around noon were invited to participate in the study. A thick smear of each 10 microliters of CAP and VEN blood was prepared and analysed by a minimum of two independent microscopists. Differences of log2-transformed CAP and VEN microfilaraemia were computed and expressed as percentages. Furthermore, odds ratios for paired data were computed to quantify the odds to detect microfilariae in CAP blood versus in VEN blood.ResultsA total of 713 participants were recruited among whom 52% were below 30 years of age, 27% between 30–59 years of age and 21% above 60 years of age. Male-female ratio was 0.84. Among 152 participants with microscopically-confirmed L. loa infection median (IQR) microfilaraemia was 3,650 (275–11,100) per milliliter blood in CAP blood and 2,775 (200–8,875) in VEN blood (p<0.0001), while among 102 participants with M. perstans this was 100 (0–200) and 100 (0–200), respectively (p = 0.44). Differences in linear models amount up to an average of +34.5% (95% CI: +11.0 to +63.0) higher L. loa microfilaria quantity in CAP blood versus VEN blood and for M. perstans it was on average higher by +24.8% (95% CI: +0.0 to +60.5). Concordantly, the odds for detection of microfilaraemia in CAP samples versus VEN samples was 1.24 (95% CI: 0.65–2.34) and 1.65 (95% CI: 1.0–2.68) for infections with L. loa and M. perstans, respectively.ConclusionThis analysis indicates that average levels of microfilaraemia of L. loa are higher in CAP blood samples than in VEN blood samples. This might have implications for treatment algorithms of onchocerciasis and loiasis, in which exact quantification of L. loa microfilaraemia is of importance. Furthermore, the odds for detection of M. perstans microfilariae was higher in CAP than in VEN blood which may pre-dispose CAP blood for detection of M. perstans infection in large epidemiological studies when sampling of large blood quantities is not feasible. No solid evidence for a higher odds of L. loa microfilariae detection in CAP blood was revealed, which might be explained by generally high levels of L. loa microfilaraemia in CAP and VEN blood above the limit of detection of 100 microfilariae/ml. Yet, it cannot be excluded that the study was underpowered to detect a moderate difference.     

Mansonella,including a Potential New Species,as Common Parasites in Children in Gabon

Background

Like other tropical African countries, Gabon is afflicted by many parasitic diseases, including filariases such as loiasis and mansonellosis. This study aimed to assess the prevalence of these two filarial diseases in febrile and afebrile children using quantitative real-time PCR and standard PCR assays coupled with sequencing.

Methodology/Principal Findings

DNA from blood specimens of 1,418 Gabonese children (1,258 febrile and 160 afebrile) were analyzed. Overall, filarial DNA was detected in 95 (6.7%) children, including 67 positive for M. perstans (4.7%), which was the most common. M. perstans was detected in 61/1,258 febrile children (4.8%) and 6/160 afebrile children (3.8%, P = 0.6). Its prevalence increased statistically with age: 3.5%, 7.7% and 10.6% in children aged ≤5, 6–10 and 11–15 years, respectively. M. perstans prevalence was significantly higher in Koulamoutou and Lastourville (12% and 10.5%, respectively) than in Franceville and Fougamou (2.6% and 2.4%, respectively). Loa loa was detected in seven febrile children including one co-infection with M. perstans. Finally, 21 filarial DNA positive were negative for M. perstans and Loa loa, but ITS sequencing could be performed for 12 and allowed the identification of a potential new species of Mansonella provisionally called “DEUX”. Mansonella sp. “DEUX” was detected only in febrile children.

Conclusions/Significance

Further study should be performed to characterize Mansonella sp. “DEUX” and evaluate the clinical significance of mansonellosis in humans.     

Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification

Loa loa infections have emerged as a serious public health problem in patients co-infected with Onchocerca volvulus or Wuchereria bancrofti because of severe adverse neurological reactions after treatment with ivermectin. Accurate diagnostic tests are needed for careful mapping in regions where mass drug administration is underway. Loop-mediated isothermal amplification (LAMP) has become a widely adopted screening method because of its operational simplicity, rapidity and versatility of visual detection readout options. Here, we present a multi-step bioinformatic pipeline to generate diagnostic candidates suitable for LAMP and experimentally validate this approach using one of the identified candidates to develop a species-specific LAMP assay for L. loa. The pipeline identified ~140 new L. loa specific DNA repeat families as putative biomarkers of infection. The consensus sequence of one family, repeat family 4 (RF4), was compiled from ~ 350 sequences dispersed throughout the L. loa genome and maps to a L. loa-specific region of the long terminal repeats found at the boundaries of Bel/Pao retrotransposons. PCR and LAMP primer sets targeting RF4 specifically amplified L. loa but not W. bancrofti, O. volvulus, Brugia malayi, human or mosquito DNA. RF4 LAMP detects the DNA equivalent of one microfilaria (100 pg) in 25–30 minutes and as little as 0.060 pg of L. loa DNA (~1/1600^th of a microfilaria) purified from spiked blood samples in approximately 50 minutes. In summary, we have successfully employed a bioinformatic approach to mine the L. loa genome for species-specific repeat families that can serve as new DNA biomarkers for LAMP. The RF4 LAMP assay shows promise as a field tool for the implementation and management of mass drug administration programs and warrants further testing on clinical samples as the next stage in development towards this goal.     

Mapping of Bancroftian Filariasis in Cameroon: Prospects for Elimination

BackgroundLymphatic filariasis (LF) is one of the most debilitating neglected tropical diseases (NTDs). It still presents as an important public health problem in many countries in the tropics. In Cameroon, where many NTDs are endemic, only scant data describing the situation regarding LF epidemiology was available. The aim of this study was to describe the current situation regarding LF infection in Cameroon, and to map this infection and accurately delineate areas where mass drug administration (MDA) was required.MethodologyThe endemicity status and distribution of LF was assessed in eight of the ten Regions of Cameroon by a rapid-format card test for detection of W. bancrofti antigen (immunochromatographic test, ICT). The baseline data required to monitor the effectiveness of MDA was collected by assessing microfilariaemia in nocturnal calibrated thick blood smears in sentinel sites selected in the health districts where ICT positivity rate was ≥ 1%.Conclusion/significanceICT card test results showed that LF was endemic in all the Regions and in about 90% of the health districts surveyed. All of these health districts qualified for MDA (i.e. ICT positivity rate ≥ 1%). Microfilariaemia data collected as part of this study provided the national program with baseline data (sentinel sites) necessary to measure the impact of MDA on the endemicity level and transmission of LF important for the 2020 deadline for global elimination.     

Epidemiology of concomitant infection due to Loa loa and Mansonella perstans in Gabon

Background

The filarial parasites Loa loa and Mansonnella perstans are endemic in the central and western African forest block. Loa loa is pathogenic and represents a major obstacle to the control of co-endemic filariae because its treatment can cause fatal complications such as encephalitis.

Methodology/Principal Findings

4392 individuals aged over 15 years were studied both by direct examination and a concentration technique. The overall prevalence rates were 22.4% for Loa loa microfilaremia, 10.2% for M. perstans microfilaremia, and 3.2% for mixed infection. The prevalence of both filariae was higher in the forest ecosystem than in savannah and lakeland (p<0.0001). The intensity of microfilariae (mf) was also higher in the forest ecosystem for both parasites. The prevalence and intensity of microfilaria were both influenced by age and gender. Correlations were found between the prevalence and intensity of Loa loa microfilariae (r = 0.215 p = 0.036), and between the prevalence of Loa loa and the prevalence of individuals with microfilaria >8000 mf/ml (r = 0.624; p<0.0001) and microfilariae >30 000 mf/ml (r = 0.319, p = 0.002). In contrast, the prevalence of pruritis and Calabar swellings correlated negatively with the prevalence of Loa loa microfilaria (r = −0.219, p = 0.032; r = −0.220; p = 0.031, respectively). Pruritis, Calabar swellings and eye worm were not associated with L. loa mf intensity (r = −0.144, p = 0.162; r–0.061, p = 0.558; and r = 0.051, p = 0.624, respectively), or with the prevalence or intensity of M. perstans microfilariae.

Conclusions/Significance

This map of the distribution of filariae in Gabon should prove helpful for control programs. Our findings confirm the spatial uniformity of the relationship between parasitological indices. Clinical manifestations point to a relationship between filariae and allergy.     

Ecological Drivers of Mansonella perstans Infection in Uganda and Patterns of Co-endemicity with Lymphatic Filariasis and Malaria

Background

Mansonella perstans is a widespread, but relatively unknown human filarial parasite transmitted by Culicoides biting midges. Although it is found in many parts of sub-Saharan Africa, only few studies have been carried out to deepen the understanding of its ecology, epidemiology, and health consequences. Hence, knowledge about ecological drivers of the vector and parasite distribution, integral to develop spatially explicit models for disease prevention, control, and elimination strategies, is limited.

Methodology

We analyzed data from a comprehensive nationwide survey of M. perstans infection conducted in 76 schools across Uganda in 2000–2003, to identify environmental drivers. A suite of Bayesian geostatistical regression models was fitted, and the best fitting model based on the deviance information criterion was utilized to predict M. perstans infection risk for all of Uganda. Additionally, we investigated co-infection rates and co-distribution with Wuchereria bancrofti and Plasmodium spp. infections observed at the same survey by mapping geographically overlapping areas.

Principal Findings

Several bioclimatic factors were significantly associated with M. perstans infection levels. A spatial Bayesian regression model showed the best fit, with diurnal temperature range, normalized difference vegetation index, and cattle densities identified as significant covariates. This model was employed to predict M. perstans infection risk at non-sampled locations. The level of co-infection with W. bancrofti was low (0.3%), due to limited geographic overlap. However, where the two infections did overlap geographically, a positive association was found.

Conclusions/Significance

This study presents the first geostatistical risk map for M. perstans in Uganda. We confirmed a widespread distribution of M. perstans, and identified important potential drivers of risk. The results provide new insight about the ecologic preferences of this otherwise poorly known filarial parasite and its Culicoides vector species in Uganda, which might be relevant for other settings in sub-Saharan Africa.     

Detection and discrimination of Loa loa, Mansonella perstans and Wuchereria bancrofti by PCR–RFLP and nested-PCR of ribosomal DNA ITS1 region

The ribosomal deoxyribonucleic acid (DNA) internal transcribed spacer region (ITS1) of two filarial nematodes, Loa loa and Mansonella perstans, was amplified and further sequenced to develop an species-specific polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) protocol for the differentiation of both species from Wuchereria bancrofti, three filarial nematodes with blood circulating microfilariae. The ITS1–PCR product digested with the restriction endonuclease Ase I generated an specific diagnostic pattern for each of the three species. Moreover, three new specific nested-PCRs, targeting the ITS1 region, for differential detection of L. loa, M. perstans and W. bancrofti were developed and used when the ITS1–PCR products were insufficient for the Ase I enzymatic digestion. These filarial species-specific molecular protocols were evaluated in forty blood samples from African adult immigrants attending in the Hospital Insular of Gran Canaria, Canarias, Spain.     

Impact of Anti-Retroviral Treatment and Cotrimoxazole Prophylaxis on Helminth Infections in HIV-Infected Patients in Lambaréné, Gabon

Background

Foci of the HIV epidemic and helminthic infections largely overlap geographically. Treatment options for helminth infections are limited, and there is a paucity of drug-development research in this area. Limited evidence suggests that antiretroviral therapy (ART) reduces prevalence of helminth infections in HIV-infected individuals. We investigated whether ART exposure and cotrimoxazole preventive therapy (CTX-P) is associated with a reduced prevalence of helminth infections.

Methodology and Principal Findings

This cross-sectional study was conducted at a primary HIV-clinic in Lambaréné, Gabon. HIV-infected adults who were ART-naïve or exposed to ART for at least 3 months submitted one blood sample and stool and urine samples on 3 consecutive days. Outcome was helminth infection with intestinal helminths, Schistosoma haematobium, Loa loa or Mansonella perstans. Multivariable logistic regression was used to assess associations between ART or CTX-P and helminth infection. In total, 408 patients were enrolled. Helminth infection was common (77/252 [30.5%]). Filarial infections were most prevalent (55/310 [17.7%]), followed by infection with intestinal helminths (35/296 [11.8%]) and S. haematobium (19/323 [5.9%]). Patients on CTX-P had a reduced risk of Loa loa microfilaremia (adjusted odds ratio (aOR) 0.47, 95% CI 0.23-0.97, P = 0.04), also in the subgroup of patients on ART (aOR 0.36, 95% CI 0.13-0.96, P = 0.04). There was no effect of ART exposure on helminth infection prevalence.

Conclusions/Significance

CTX-P use was associated with a decreased risk of Loa loa infection, suggesting an anthelminthic effect of antifolate drugs. No relation between ART use and helminth infections was established.     

Treatment of W. bancrofti (Wb) in HIV/Wb Coinfections in South India

BackgroundThe disease course of human immunodeficiency virus (HIV) is often altered by existing or newly acquired coincident infections.Conclusions/SignificanceWe were unable to find a significant effect of W. bancrofti infection or its treatment on HIV clinical course or surrogate markers of HIV disease progression though we recognized that our study was limited by the smaller than predicted sample size and by the use of ART in half of the patients. Treatment of W. bancrofti coinfection in HIV positive subjects (as is usual in mass drug administration campaigns) did not represent an increased risk to the subjects, and should therefore be considered for PLWHA living in W. bancrofti endemic areas.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00344279","term_id":"NCT00344279"}}NCT00344279     

Contributions of different mosquito species to the transmission of lymphatic filariasis in central Nigeria: implications for monitoring infection by PCR in mosquito pools

Background

Monitoring and evaluation are essential to the successful implementation of mass drug administration programmes for LF elimination. Monitoring transmission when it is low requires both large numbers of mosquito vectors and sensitive methods for detecting Wuchereria bancrofti infections in them. PCR-based methods are preferred over classical dissections but the best protocol so far achieved detection of one L₃ Wuchereria bancrofti larva in a pool of 35–50 Anopheles mosquitoes. It also lacks consistency and remains still a costly tool. Hence we decided to improve upon this to achieve detection in a pool of 100 or more by enhancing the quality of the template DNA. Prior to this we also evaluated three vector sampling methods in the context of numbers for monitoring.

Methods

Human landing, pyrethrium spray and light traps catches were conducted concurrently at sites in an LF endemic district in Ghana and the numbers obtained compared. Two DNA extraction methods; Bender buffer and phenol/chloroform purification, and DNAeasy Tissue kit (Quaigen Inc) were used on pools of 25, 50, 75 100 and 150 mosquitoes each seeded with one L₃ or its quivalent amount of DNA. Then another set of extracted DNA by the two methods was subjected to Dynal bead purification method (using capture oligonucleotide primers). These were used as template DNA in PCR to amplify W. bancrofti sequences. The best PCR result was then evaluated in the field at five sites by comparing its results (infections per 1000 mosquitoes) with that of dissection of roughly equal samples sizes.

Results

The largest numbers of mosquitoes were obtained with the human landing catches at all the sites sampled. Although PCR detection of one L₃ in pools of 25, 50 and 75 mosquitoes was consistent irrespective of the extraction method, that of one L₃ in 100 was only achieved with the kit-extracted DNA/Dynal bead purification method. Infections were found at only two sites by both dissection and pool-screening being 14.3 and 19 versus 13.4 and 20.1 per 1000 Anopheles mosquitoes respectively, which were not statistically significant

Discussion and conclusion

HLC still remains the best option for sampling for the large numbers of mosquitoes required for monitoring transmission during MDA programmes, when vector population densities are high and classical indices of transmission are required. One – in – 100 detection is an improvement on previous PCR pool-screening methods, which in our opinion was a result of the introduction of the extra step of parasite DNA capture using Dynal/beads. As pool sizes increase the insects DNA will swamp parasite DNA making the latter less available for an efficient PCR, therefore we propose either additional steps of parasite DNA capture or real-time PCR to improve further the pool screening method. The study also attests also to the applicability of Katholi et al's algorithm developed for determining onchocerciasis prevalence in LF studies.     

An open label,randomized clinical trial to compare the tolerability and efficacy of ivermectin plus diethylcarbamazine and albendazole vs. diethylcarbamazine plus albendazole for treatment of brugian filariasis in Indonesia

Improved treatments for lymphatic filariasis (LF) could accelerate the global elimination program for this disease. A triple drug combination of the anti-filarial drugs ivermectin, diethylcarbamazine (DEC) and albendazole (IDA) has been shown to be safe and effective for achieving sustained clearance of microfilariae (Mf) of the filarial parasite Wuchereria bancrofti from human blood. However, the triple drug combination has not been previously been evaluated for treatment of brugian filariasis, which accounts for about 10% of the global LF burden. This hospital-based clinical trial compared the safety and efficacy of IDA with that of the standard treatment (DEC plus albendazole, DA) in persons with Brugia timori infections on Sumba island, Indonesia. Fifty-five asymptomatic persons with B. timori Mf were treated with either a single oral dose of IDA (28 subjects) or with DEC plus albendazole (DA, 27 subjects). Participants were actively monitored for adverse events (AE) for two days after treatment by nurses and physicians who were masked regarding treatment assignments. Passive monitoring was performed by clinical teams that visited participant’s home villages for an additional five days. Microfilaremia was assessed by membrane filtration of 1 ml night blood at baseline, at 24h and one year after treatment. IDA was more effective than DA for completely clearing Mf at 24 hours (25/28, 89% vs. 8/27, 30%, P < 0.001). By 12 months after treatment, only one of 27 IDA recipients had Mf in their blood (4%) vs. 10 of 25 (40%) in persons treated with DA (P = 0.002). Approximately 90% of participants had antibodies to recombinant filarial antigen BmR1 at baseline. Antibody prevalence decreased to approximately 30% in both treatment groups at 12 months. About 45% of persons in both treatment groups experienced AE such as fever, muscle aches, lower back, joint and abdominal pain. These were mostly mild and most common during the first two days after treatment. No participant experienced a severe or serious AE. This study showed that IDA was well-tolerated and significantly more effective for clearing B. timori Mf from the blood than DA. Larger studies should be performed to further assess the safety and efficacy of IDA as a mass drug administration regimen to eliminate brugian filariasis.Trial Registration: {"type":"clinical-trial","attrs":{"text":"NCT02899936","term_id":"NCT02899936"}}NCT02899936.     

No Evidence for Lymphatic Filariasis Transmission in Big Cities Affected by Conflict Related Rural-Urban Migration in Sierra Leone and Liberia

Background

In West Africa, the principal vectors of lymphatic filariasis (LF) are Anopheles species with Culex species playing only a minor role in transmission, if any. Being a predominantly rural disease, the question remains whether conflict-related migration of rural populations into urban areas would be sufficient for active transmission of the parasite.

Methodology/Principal Findings

We examined LF transmission in urban areas in post-conflict Sierra Leone and Liberia that experienced significant rural-urban migration. Mosquitoes from Freetown and Monrovia, were analyzed for infection with Wuchereria bancrofti. We also undertook a transmission assessment survey (TAS) in Bo and Pujehun districts in Sierra Leone. The majority of the mosquitoes collected were Culex species, while Anopheles species were present in low numbers. The mosquitoes were analyzed in pools, with a maximum of 20 mosquitoes per pool. In both countries, a total of 1731 An. gambiae and 14342 Culex were analyzed for W. bancrofti, using the PCR. Two pools of Culex mosquitoes and 1 pool of An. gambiae were found infected from one community in Freetown. Pool screening analysis indicated a maximum likelihood of infection of 0.004 (95% CI of 0.00012–0.021) and 0.015 (95% CI of 0.0018–0.052) for the An. gambiae and Culex respectively. The results indicate that An. gambiae is present in low numbers, with a microfilaria prevalence breaking threshold value not sufficient to maintain transmission. The results of the TAS in Bo and Pujehun also indicated an antigen prevalence of 0.19% and 0.67% in children, respectively. This is well below the recommended 2% level for stopping MDA in Anopheles transmission areas, according to WHO guidelines.

Conclusions

We found no evidence for active transmission of LF in cities, where internally displaced persons from rural areas lived for many years during the more than 10 years conflict in Sierra Leone and Liberia.     

Step towards elimination of Wuchereria bancrofti in Southwest Tanzania 10 years after mass drug administration with Albendazole and Ivermectin

BackgroundLymphatic filariasis is a mosquito transmitted parasitic infection in tropical regions. Annual mass treatment with ivermectin and albendazole is used for transmission control of Wuchereria bancrofti, the infective agent of lymphatic filariasis in many African countries, including Tanzania.MethodologyIn a general population study in Southwest Tanzania, individuals were tested for circulating filarial antigen, an indicator of W. bancrofti adult worm burden in 2009 before mass drug administration commenced in that area. Seven annual rounds with ivermectin and albendazole were given between 2009 and 2015 with a population coverage of over 70%. Participants of the previous study took part in a follow-up activity in 2019 to measure the effect of this governmental activity.FindingsOne thousand two hundred and ninety nine inhabitants of Kyela district in Southwest Tanzania aged 14 to 65 years who had participated in the study activities in 2009 were revisited in 2010/11 and 2019. Among this group, the prevalence of lymphatic filariasis of the 14–65 years olds in 2009 was 35.1%. A follow-up evaluation in 2010/11 had shown a reduction to 27.7%. In 2019, after 7 years of annual treatment and an additional three years of surveillance, the prevalence had dropped to 1.7%, demonstrating successful treatment by the national control programme. Risk factors for W. bancrofti-infection were the occupation as farmer, male sex, and older age. Most infected individuals in the 2019 follow-up study already had a positive test for filarial antigen in 2009 and/or 2010/11.ConclusionsThis data supports the findings of the Tanzanian Neglected Tropical Disease Control Programme (NTDCP), who conducted Transmission Assessment Surveys and found an impressive reduction in the prevalence of LF in children. Our results complement this data by showing a similar decrease in prevalence of LF in the adult population in the same area. The elimination of LF seems achievable in the near future.     

Report of a Scientific Working Group on Serious Adverse Events following Mectizan<Superscript>®</Superscript> treatment of onchocerciasis in <Emphasis Type="Italic">Loa loa</Emphasis> endemic areas

The occurrence of Serious Adverse Experiences (SAEs) following Mectizan^® treatment of onchocerciasis in Loa loa endemic areas has been increasingly reported over the past decade. These SAEs include a severely disabling, and potentially fatal, encephalopathy, which appears to correlate with a high load of L. loa microfilariae (> 30,000 mf/ml).Previous consultations organized by the Mectizan^® Donation Program (MDP) in 1995 and 1999 have developed useful "case" definitions of encephalopathic SAEs following Mectizan^® treatment and have summarized available evidence on its pathogenesis and optimal clinical management. At both meetings, the need for better understanding of the pathogenesis of the encephalopathy was emphasized, including the need for biological and autopsy specimens from the affected cases.Following a recommendation at the Joint Action Forum of the African Programme for Onchocerciasis Control in December 2001, the MDP, on behalf of the Mectizan^® Expert Committee, organized a Scientific Working Group on L. loa associated SAEs following Mectizan^® treatment in May 2002. The present report includes the background, new evidence, conclusions and recommendations from that Scientific Working Group. The following points represent a summary of the present status:1. Although there are more and better quality clinical and epidemiological data on L. loa, the pathogenesis of the Mectizan^®-related L. loa encephalopathy remains obscure.2. Very limited progress has been made in research on the pathogenesis of encephalopathy, because of the lack of specimens from cases, and the lack of animal models.3. There has been no particular breakthrough in terms of the medical management of patients with L. loa encephalopathy; however, a favorable outcome usually results from prompt general nursing and nutritional care which remain the major interventions.The main recommendations for future actions are as follows:1. Validate and update the mapping of L. loa with a combination of remote sensing and RAPLOA techniques.2. Conduct an expert analysis of the apparent clustering of encephalopathic SAEs reported so far.3. Investigate a possible "pre-treatment" scheme with high-dose albendazole in L. loa endemic communities at high risk of encephalopathic SAEs if treated with Mectizan^®; this study will be conducted in collaboration with WHO/TDR.4. Establish a post of Loiasis Technical Advisor for research and operational support in Cameroon, to conduct population surveys and to facilitate better data collection from SAE cases, including postmortem studies as appropriate.5. Investigate the possibility of developing an animal model of L. loa encephalopathy; this activity would be linked to the above-mentioned research agenda in Cameroon.6. Investigate the best care model for encephalopathic SAEs, including identification of early warning signs and therapeutic interventions.7. Develop further models for health education messages needed for community compliance with Mectizan^® treatment, and family support for SAE cases.8. Conduct research studies on the safety of combination therapy of Mectizan^® and albendazole in areas co-endemic for L. loa and lymphatic filariasis (LF) with coordination from the relevant technical bodies that oversee these issues.The above recommendations will be implemented through a continuing collaboration between the interested parties represented at the Scientific Working Group, involved in onchocerciasis control and/or the Global Programme to Eliminate Lymphatic Filariasis.     

Unique Insights in the Cervicovaginal Lactobacillus iners and L. crispatus Proteomes and Their Associations with Microbiota Dysbiosis

Background

A Lactobacillus-dominated cervicovaginal microbiota (VMB) protects women from adverse reproductive health outcomes, but the role of L. iners in the VMB is poorly understood. Our aim was to explore the association between the cervicovaginal L. iners and L. crispatus proteomes and VMB composition.

Methods

The vaginal proteomes of 50 Rwandan women at high HIV risk, grouped into four VMB groups (based on 16S rDNA microarray results), were investigated by mass spectrometry using cervicovaginal lavage (CVL) samples. Only samples with positive 16S results for L. iners and/or L. crispatus within each group were included in subsequent comparative protein analyses: Lactobacillus crispatus-dominated VMB cluster (with 16S-proven L. iners (n_i) = 0, and with 16S-proven L. crispatus (n_c) = 5), L. iners-dominated VMB cluster (n_i = 11, n_c = 4), moderate dysbiosis (n_i = 12, n_c = 2); and severe dysbiosis (n_i = 8, n_c = 2). The relative abundances of proteins that were considered specific for L. iners and L. crispatus were compared among VMB groups.

Results

Forty Lactobacillus proteins were identified of which 7 were specific for L. iners and 11 for L. crispatus. The relative abundances of L. iners DNA starvation/stationary phase protection protein (DPS), and the glycolysis enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glucose-6-phosphate isomerase (GPI), were significantly decreased in women with L. iners-containing dysbiosis compared to women with a L. iners-dominated VMB, independent of vaginal pH and L. iners abundance. Furthermore, L. iners DPS, GAPDH, GPI, and fructose-bisphosphate aldolase (ALDO) were significantly negatively associated with vaginal pH. Glycolysis enzymes of L. crispatus showed a similar negative, but nonsignificant, trend related to dysbiosis.

Conclusions

Most identified Lactobacillus proteins had conserved intracellular functions, but their high abundance in CVL supernatant might imply an additional extracellular (moonlighting) role. Our findings suggest that these proteins can be important in maintaining a Lactobacillus-dominated VMB. Functional studies are needed to investigate their roles in vaginal bacterial communities and whether they can be used to prevent vaginal dysbiosis.     

Brugia malayi Microfilariae Induce a Regulatory Monocyte/Macrophage Phenotype That Suppresses Innate and Adaptive Immune Responses

Background

Monocytes and macrophages contribute to the dysfunction of immune responses in human filariasis. During patent infection monocytes encounter microfilariae in the blood, an event that occurs in asymptomatically infected filariasis patients that are immunologically hyporeactive.

Aim

To determine whether blood microfilariae directly act on blood monocytes and in vitro generated macrophages to induce a regulatory phenotype that interferes with innate and adaptive responses.

Methodology and principal findings

Monocytes and in vitro generated macrophages from filaria non-endemic normal donors were stimulated in vitro with Brugia malayi microfilarial (Mf) lysate. We could show that monocytes stimulated with Mf lysate develop a defined regulatory phenotype, characterised by expression of the immunoregulatory markers IL-10 and PD-L1. Significantly, this regulatory phenotype was recapitulated in monocytes from Wuchereria bancrofti asymptomatically infected patients but not patients with pathology or endemic normals. Monocytes from non-endemic donors stimulated with Mf lysate directly inhibited CD4⁺ T cell proliferation and cytokine production (IFN-γ, IL-13 and IL-10). IFN-γ responses were restored by neutralising IL-10 or PD-1. Furthermore, macrophages stimulated with Mf lysate expressed high levels of IL-10 and had suppressed phagocytic abilities. Finally Mf lysate applied during the differentiation of macrophages in vitro interfered with macrophage abilities to respond to subsequent LPS stimulation in a selective manner.

Conclusions and significance

Conclusively, our study demonstrates that Mf lysate stimulation of monocytes from healthy donors in vitro induces a regulatory phenotype, characterized by expression of PD-L1 and IL-10. This phenotype is directly reflected in monocytes from filarial patients with asymptomatic infection but not patients with pathology or endemic normals. We suggest that suppression of T cell functions typically seen in lymphatic filariasis is caused by microfilaria-modulated monocytes in an IL-10-dependent manner. Together with suppression of macrophage innate responses, this may contribute to the overall down-regulation of immune responses observed in asymptomatically infected patients.     

Safety and efficacy of mass drug administration with a single-dose triple-drug regimen of albendazole + diethylcarbamazine + ivermectin for lymphatic filariasis in Papua New Guinea: An open-label,cluster-randomised trial

BackgroundPapua New Guinea (PNG) has a high burden of lymphatic filariasis (LF) caused by Wuchereria bancrofti, with an estimated 4.2 million people at risk of infection. A single co-administered dose of ivermectin, diethylcarbamazine and albendazole (IDA) has been shown to have superior efficacy in sustained clearance of microfilariae compared to diethylcarbamazine and albendazole (DA) in small clinical trials. A community-based cluster-randomised trial of DA versus IDA was conducted to compare the safety and efficacy of IDA and DA for LF in a moderately endemic, treatment-naive area in PNG.MethodologyAll consenting, eligible residents of 24 villages in Bogia district, Madang Province, PNG were enrolled, screened for W. bancrofti antigenemia and microfilaria (Mf) and randomised to receive IDA (N = 2382) or DA (N = 2181) according to their village of residence. Adverse events (AE) were assessed by active follow-up for 2 days and passive follow-up for an additional 5 days. Antigen-positive participants were re-tested one year after MDA to assess treatment efficacy.Principal findingsOf the 4,563 participants enrolled, 96% were assessed for AEs within 2 days after treatment. The overall frequency of AEs were similar after either DA (18%) or IDA (20%) treatment. For those individuals with AEs, 87% were mild (Grade 1), 13% were moderate (Grade 2) and there were no Grade 3, Grade 4, or serious AEs (SAEs). The frequency of AEs was greater in Mf-positive than Mf-negative individuals receiving IDA (39% vs 20% p<0.001) and in Mf-positive participants treated with IDA (39%), compared to those treated with DA (24%, p = 0.023). One year after treatment, 64% (645/1013) of participants who were antigen-positive at baseline were re-screened and 74% of these participants (475/645) remained antigen positive. Clearance of Mf was achieved in 96% (52/54) of infected individuals in the IDA arm versus 84% (56/67) of infected individuals in the DA arm (relative risk (RR) 1.15; 95% CI, 1.02 to 1.30; p = 0.019). Participants receiving DA treatment had a 4-fold higher likelihood of failing to clear Mf (RR 4.67 (95% CI: 1.05 to 20.67; p = 0.043). In the DA arm, a significant predictor of failure to clear was baseline Mf density (RR 1.54; 95% CI, 1.09 to 2.88; p = 0.007).ConclusionIDA was well tolerated and more effective than DA for clearing Mf. Widespread use of this regimen could accelerate LF elimination in PNG.Trial registrationRegistration number {"type":"clinical-trial","attrs":{"text":"NCT02899936","term_id":"NCT02899936"}}NCT02899936; https://clinicaltrials.gov/ct2/show/{"type":"clinical-trial","attrs":{"text":"NCT02899936","term_id":"NCT02899936"}}NCT02899936.     

Rapid molecular assays for specific detection and quantitation of Loa loa microfilaremia

Background

Accurate diagnosis of Loa loa infection is essential to the success of mass drug administration efforts to eliminate onchocerciasis and lymphatic filariasis, due to the risk of fatal encephalopathic reactions to ivermectin occurring among highly microfilaremic Loa-infected individuals living in areas co-endemic for multiple filarial species.

Methodology/Principal Findings

From a pool of over 1,800 L. loa microfilaria (mf) expressed sequence tags, 18 candidate L. loa mf-specific PCR targets were identified. Real-time PCR (qPCR) assays were developed for two targets (LLMF72 and LLMF269). The qPCR assays were highly specific for L. loa compared with related filariae and also highly sensitive, with detection limits of 0.1 pg genomic DNA, or 1% of DNA extracted from normal blood spiked with a single L. loa microfilaria. Using various DNA extraction methods with dried blood spots obtained from Cameroonian subjects with parasitologically proven loiasis, the LLMF72 qPCR assay successfully estimated mf burden in 65 of 68 samples (50–96,000 mf/mL by microscopy), including all 12 samples subjected to a simple 10-minute boiling extraction. Additionally, the assay detected low-level microfilaremia among 5 of 16 samples from patients thought to be amicrofilaremic by microscopy.

Conclusions/Significance

This novel, rapid, highly sensitive and specific qPCR assay is an important step forward in the laboratory diagnosis of L. loa infection.