Association of haemato-biochemical indices and blood composite ratios with microfilaridermia in Onchocerciasis patients.

BACKGROUND: Onchocerciasis causes chronic systemic inflammation. Several studies have used markers such as haemato-biochemical indices to predict the occurrence of systemic inflammation. This study assessed the variability and predictability of haemato-biochemical indices and blood composite ratios (BCRs) in microfilariae positive (MF+) and microfilariae negative (MF-) subgroups of onchocercomata participants. METHODS: One hundred and five (105) MF + and 34 MF- participants were retrospectively recruited into the study. Screening for the presence of O. volvulus microfilariae was done from skin snips taken from the left and right iliac crests of participants using established and approved protocols. Haematological and biochemical indices were measured using standard laboratory automated analyzers. Blood composite ratios (BCRs) were calculated as ratios of the absolute parameters involved. RESULTS: A significantly increased total WBC, absolute eosinophil, eosinophil percent and absolute basophil were observed in the MF + participants compared to MF- participants. Reduced gamma-glutamyl transferase (GGT) with increased estimated glomerular filtration rate (eGFR) was significantly associated with MF + participants compared to MF- participants. BCRs were significantly higher for eosinophil-to-neutrophil ratio (ENR), eosinophil-to-monocyte ratio (EMR), eosinophil-to-basophil ratio (EBR) and eosinophil-to-lymphocyte ratio (ELR) in MF + participants compared to MF- participants. After multivariate adjustment, onchocercomata participants with increased eosinophil counts (aOR = 13.86, 95% CI [2.07-92.90], p = 0.007), ENR x10 (aOR = 1.42, 95% CI [1.05-1.93], p = 0.025), EMR (aOR = 2.64, 95% CI [1.25-5.60], p = 0.011), EBR (aOR = 1.07, 95% CI [1.01-1.10], p = 0.020) and ELR x10 (aOR = 1.69, 95% CI [1.14-2.51], p = 0.009) were more likely to have microfilaridermia. CONCLUSIONS: Elevated eosinophil counts with higher ENR, EMR, EBR and ELR levels are significantly associated with microfilaridermia in onchocercomata participants. Combining BCRs with eosinophil count significantly led to an improvement in the conventional model for predicting microfilaridermia.

Stage-Dependent Increase of Systemic Immune Activation and CCR5+CD4+ T Cells in Filarial Driven Lymphedema in Ghana and Tanzania.

Chronic lymphedema caused by infection of Wuchereria bancrofti is a disfiguring disease that leads to physical disability, stigmatization, and reduced quality of life. The edematous changes occur mainly on the lower extremities and can progress over time due to secondary bacterial infections. In this study, we characterized participants with filarial lymphedema from Ghana and Tanzania as having low (stage 1-2), intermediate (stage 3-4), or advanced (stage 5-7) lymphedema to determine CD4+ T cell activation patterns and markers associated with immune cell exhaustion. A flow cytometry-based analysis of peripheral whole blood revealed different T cell phenotypes within participants with different stages of filarial lymphedema. In detail, increased frequencies of CD4+HLA-DR+CD38+ T cells were associated with higher stages of filarial lymphedema in patients from Ghana and Tanzania. In addition, significantly increased frequencies of CCR5+CD4+ T cells were seen in Ghanaian participants with advanced LE stages, which was not observed in the Tanzanian cohort. The frequencies of CD8+PD-1+ T cells were augmented in individuals with higher stage lymphedema in both countries. These findings show distinct activation and exhaustion patterns in lymphedema patients but reveal that immunological findings differ between West and East African countries.

Reduced Type 2 Innate Lymphocyte Cell Frequencies in Patent Wuchereria bancrofti-Infected Individuals.

Approximately 51 million individuals suffer from lymphatic filariasis (LF) caused mainly by the filarial worm Wuchereria bancrofti. Mass drug administration (MDA) programs led to a significant reduction in the number of infected individuals, but the consequences of the treatment and clearance of infection in regard to host immunity remain uncertain. Thus, this study investigates the composition of myeloid-derived suppressor cells (MDSCs), macrophage subsets and innate lymphoid cells (ILCs), in patent (circulating filarial antigen (CFA)+ microfilariae (MF)+) and latent (CFA+MF-) W. bancrofti-infected individuals, previously W. bancrofti-infected (PI) individuals cured of the infection due to MDA, uninfected controls (endemic normal (EN)) and individuals who suffer from lymphoedema (LE) from the Western Region of Ghana. Frequencies of ILC2 were significantly reduced in W. bancrofti-infected individuals, while the frequencies of MDSCs, M2 macrophages, ILC1 and ILC3 were comparable between the cohorts. Importantly, clearance of infection due to MDA restored the ILC2 frequencies, suggesting that ILC2 subsets might migrate to the site of infection within the lymphatic tissue. In general, the immune cell composition in individuals who cured the infection were comparable to the uninfected individuals, showing that filarial-driven changes of the immune responses require an active infection and are not maintained upon the clearance of the infection.

First genome-wide association study for lymphatic filariasis in a West African population points to a human leukocyte antigen-mediated disease pathophysiology.

OBJECTIVES: Lymphatic filariasis (LF) represents a parasitic disease caused by filarial nematodes. Although some infected individuals present an asymptomatic course, others suffer severe chronic lymphatic pathology, including lymphedema, hydrocele, and elephantiasis. Several studies have shown that host genetic factors influence LF susceptibility and chronic pathology. The current study aimed to conduct the first genome-wide association study to systematically determine LF susceptibility. METHODS: We analyzed genome-wide single-nucleotide polymorphism data from 1459 LF cases and 1492 asymptomatic controls of West African (Ghanaian) descent. RESULTS: We identified two independent genome-wide significant associated genetic variants near the genes HLA-DQB2 (rs7742085) and HLA-DQA1 (rs4959107) contributing to LF and/or lymphedema susceptibility (P <5.0 × 10-8, odds ratios [ORs] >1.30). We also observed suggestive evidence of LF associations (P <1.0 × 10-6) at two non-HLA loci, near the genes ZFHX4-AS1 (rs79562145) and CHP2 (rs12933387). In contrast, we could not replicate any previously reported LF associations drawn from candidate gene association studies. On the polygenic level, we show that our genome-wide association study data explain 24-42% of LF heritability, depending on an assumed population prevalence of 0.5-5.0%. CONCLUSION: Our findings point to an involvement of HLA-mediated immune mechanisms in LF pathophysiology.

Occurrence of Lymphatic Filariasis infection after 15 years of mass drug administration in two hotspot districts in the Upper East Region of Ghana.

BACKGROUND: Lymphatic filariasis (LF) causes chronic morbidity, which usually manifests as lymphedema or hydrocele. Mass drug administration (MDA) began in Kassena Nankana East Municipal (KNEM) and Nabdam, two hotspot districts in the Upper East Region in Ghana, in 2000 and 2005, respectively. This cross-sectional study evaluated the impact of 15 years of MDA on the control of LF as determined by circulating filarial antigen (CFA) and microfilariae assessment in the KNEM and the Nabdam districts. METHODOLOGY/PRINCIPAL FINDINGS: A total of 7,453 participants from eight sub-districts in the two hotspot districts (KNEM: N = 4604; Nabdam: N = 2849) were recruited into the study. The overall CFA prevalence as determined by the FTS was 19.6% and 12.8% in the KNEM and Nabdam districts, respectively. Manyoro, a sub-district on the border with Burkina Faso, recorded the highest CFA prevalence of 26% in the KNEM. Assessment of microfilariae and Og4C3 antigen was done from 1009 (KNEM: N = 799 (79.2%); Nabdam: N = 210 (20.8%)) randomly selected FTS-positive (N = 885) and FTS-negative (N = 124) individuals. The Og4C3 antigen was found in 22.6%/23.0% of the selected individuals (KNEM/Nabdam), whereas the night blood revealed microfilariae in only 0.7%/0.5%. CONCLUSIONS/SIGNIFICANCE: Using the WHO endorsed FTS, CFA prevalence exceeded the long-standing <2% threshold-which may need revision and validation. Surprisingly, the Og4C3 ELISA showed positive results in only about one-fifth of the FTS positive samples. However, even this result would not have met the <2% CFA criteria for LF elimination. In contrast, projections from the microfilariae results revealed a halt in LF transmission. The global elimination target was due in 2020 but has been extended to 2030 since this could not be met. Focused MDA intervention intensification on seasonal migrants and non-compliers, and implementation of alternative treatment strategies may suffice for the elimination of the disease.

Distinct Immune Profiles of Exhausted Effector and Memory CD8+ T Cells in Individuals With Filarial Lymphedema.

CD8+ T cells are crucial for the clearance of viral infections, and current research begins to highlight their importance in parasitic diseases too. In-depth research about characteristics of CD8+ T-cell subsets and exhaustion remains uncertain, especially during filariasis, a chronic helminth infection. Lymphatic filariasis, elicited by Wuchereria bancrofti, remains a serious health problem in endemic areas in Ghana, especially in those suffering from morbidity due to lymphedema (LE). In this observational study, the characteristics and profiles of CD8+ T cells were compared between asymptomatic Wuchereria bancrofti-infected individuals, uninfected endemic normals, and those with LE (grades 2-6). Focusing on exhausted memory (CD8+exmem: CD8+ T-betdimEomeshi) and effector (CD8+exeff: CD8+T-bethiEomesdim) CD8+ T-cell subsets, advanced flow cytometry revealed that LE individuals presented reduced frequencies of IFN-γ+CD8+exmem T cells expressing Tim-3 or LAG-3 which negatively correlated to the presence of LE. Moreover, the LE cohort further showed significantly higher frequencies of IL-10+CD8+exeff T cells expressing either Tim-3, LAG-3, CD39, KLRG-1, or PD-1, all associated markers of exhaustion, and that these frequencies positively correlated with the presence of LE. In summary, this study shows that distinct exhausted CD8+ T-cell subsets are prominent in individuals suffering from LE, suggesting that enhanced inflammation and constant immune activation might drive exhaustion of CD8+ T cells. Since T-cell exhaustion is known to be associated with insufficient control of persisting antigen, the data presented here reveals that these CD8+ T-cell exhaustion patterns in filarial LE should be taken into consideration for prevention and control management of LE.

Flow cytometric analysis of cell lineage and immune activation markers using minimal amounts of human whole blood-Field method for remote settings.

Remote laboratory settings - such as those where studies on neglected tropical diseases are performed - often lack specialized equipment required for flow cytometric analysis of immune cell subsets, which complicates evaluations on a single cell level using peripheral blood. Our aim was to establish a method to use whole blood for phenotypic characterization of T-cells for specific markers including CD3, CD4, HLA-DR, CD38, CCR5, CD27, CD45RA, CD25, and FoxP3. This method uses 100 µL whole blood which is stained for extracellular markers, lysed, and cryopreserved at -20 °C at a field laboratory before transferring to liquid nitrogen for long-term storage and transportation. Cells can then be transported to a central laboratory for flow cytometry analysis. The method was initially established using samples from healthy donors; expression levels after cryopreservation were comparable to fresh whole blood samples from the same individuals. Moreover, data sets were also comparable to those which were stored in liquid nitrogen for up to one year. The method was then transferred to field studies in a remote area of Ghana which was used to observe its practicality and robustness in limited resource settings. Collectively, the low amount of whole blood (such as that taken from a finger prick), lack of any specialized equipment, and ease of use make this method suitable for utilization in remote field locations.

Filarial Lymphedema Patients Are Characterized by Exhausted CD4+ T Cells.

Worldwide, more than 200 million people are infected with filariae which can cause severe symptoms leading to reduced quality of life and contribute to disability-adjusted life years (DALYs). In particular, lymphatic filariasis (LF) caused by Wuchereria bancrofti can lead to lymphedema (LE) and consequently presents a serious health problem. To understand why only a fraction of the infected individuals develop pathology, it is essential to understand how filariae regulate host immunity. The central role of T cells for immunity against filariae has been shown in several studies. However, there is little knowledge about T cell exhaustion, which causes T cell dysfunction and impaired immune responses, in this group of individuals. Recently, we showed that LE patients from Ghana harbor distinct patterns of exhausted effector and memory CD8+ T cell subsets. Based on these findings, we now characterized CD4+ T cell subsets from the same Ghanaian patient cohort by analyzing distinct markers within a 13-colour flow cytometry panel. We revealed that LE patients had increased frequencies of CD4+ T cells expressing exhaustion-associated receptors such as KLRG-1, TIM-3 and PD-1 compared to healthy endemic normal and W. bancrofti-infected individuals. Moreover, CD4+ T cells in LE patients were characterized by distinct co-expression patterns of inhibitory receptors. Collectively with the previous findings on CD8+ T cell exhaustion patterns, the data shown here demonstrates that filarial LE patients harbor distinct subsets of exhausted T cells. Thus, T cell exhaustion patterns in LE patients need attention especially in regards to susceptibility of concomitant infections and should be taken into consideration for LE management measures.

Morbidity management and surveillance of lymphatic filariasis disease and acute dermatolymphangioadenitis attacks using a mobile phone-based tool by community health volunteers in Ghana.

Morbidity burden of lymphatic filariasis (LF) relies on the information from the Mass Drug Administration (MDA) programme where Community Health Volunteers (CHVs) passively report cases identified. Consequently, the exact prevalence of morbidity cases is not always accurate. The use of mobile phone technology to report morbidity cases was piloted in Ghana using a text-based short messaging service (SMS) tool by CHVs. Though successful, illiterate CHVs could not effectively use the SMS tool. The aim of this study was to evaluate the use of a mobile phone-based Interactive Voice Response System (mIVRS) by CHVs in reporting LF morbidity cases and acute dermatolymphangioadenitis (ADLA) attacks in Ghana. The mIVRS was designed as a surveillance tool to capture LF data in Kassena Nankana Districts of Ghana. One hundred CHVs were trained to identify and report lymphedema and hydrocele cases as well as ADLA attacks by calling a hotline linked to the mIVRS. The system asked a series of questions about the disease condition. The ability of the CHV to report accurately was assessed and the data from the mIVRS were compared with the paper records from the CHVs and existing MDA programme records from the same communities and period. Higher numbers of lymphedema and hydrocele cases were recorded by the CHVs using the mIVRS (n = 590 and n = 103) compared to the paper-based reporting (n = 417 and n = 76) and the MDA records (n = 154 and n = 84). Female CHVs, CHVs above 40 years, and CHVs with higher educational levels were better at paper-based reporting (P = 0.007, P = 0.001, P = 0.049 respectively). The system, when fully developed and linked to national databases, may help to overcome underreporting of morbidity cases and ADLA attacks in endemic communities. The system has the potential to be further expanded to other diseases.

Comparison of Repeated Doses of Ivermectin Versus Ivermectin Plus Albendazole for the Treatment of Onchocerciasis: A Randomized, Open-label, Clinical Trial.

BACKGROUND: Improved treatment for onchocerciasis is needed to accelerate onchocerciasis elimination in Africa. Aiming to better exploit registered drugs, this study was undertaken to determine whether annual or semiannual treatment with ivermectin (IVM; 200 µg/kg) plus albendazole (ALB; 800 mg single dose) is superior to IVM alone. METHODS: This trial was performed in Ghana and included 272 participants with microfilariae (MF), who were randomly assigned to 4 treatment arms: (1) IVM annually at 0, 12, and 24 months; (2) IVM semiannually at 0, 6, 12, 18, and 24 months; (3) IVM+ALB annually; or (4) IVM+ALB semiannually. Microfiladermia was determined pretreatment and at 6, 18, and 36 months. The primary outcome was the proportion of fertile and viable female worms in onchocercomata excised at 36 months. RESULTS: Posttreatment nodule histology showed that 15/135 (11.1%), 22/155 (14.2%), 35/154 (22.7%), and 20/125 (16.0%) living female worms had normal embryogenesis in the IVM annual, IVM semiannual, IVM+ALB annual, and IVM+ALB semiannual groups, respectively (P = .1229). Proportions of dead worms also did not differ between the 4 groups (P = .9198). Proportions of patients without MF at 36 months (1 year after the last treatment) were 35/56 (63%) after annual IVM, 42/59 (71%) after semiannual IVM, 39/64 (61%) after annual IVM+ALB, and 43/53 (81%) after semiannual IVM+ALB. CONCLUSIONS: The combination treatment of IVM plus ALB was no better than IVM alone for sterilizing, killing adult worms, or achieving sustained MF clearance. However, semiannual treatment was superior to annual treatment for achieving sustained clearance of Onchocerca volvulus MF from the skin (P = .024). CLINICAL TRIALS REGISTRATION: ISRCTN50035143.

Wuchereria bancrofti-infected individuals harbor distinct IL-10-producing regulatory B and T cell subsets which are affected by anti-filarial treatment.

Despite worldwide mass drug administration, it is estimated that 68 million individuals are still infected with lymphatic filariasis with 19 million hydrocele and 17 million lymphedema reported cases. Despite the staggering number of pathology cases, the majority of LF-infected individuals do not develop clinical symptoms and present a tightly regulated immune system characterized by higher frequencies of regulatory T cells (Treg), suppressed proliferation and Th2 cytokine responses accompanied with increased secretion of IL-10, TGF-ß and infection-specific IgG4. Nevertheless, the filarial-induced modulation of the host`s immune system and especially the role of regulatory immune cells like regulatory B (Breg) and Treg during an ongoing LF infection remains unknown. Thus, we analysed Breg and Treg frequencies in peripheral blood from Ghanaian uninfected endemic normals (EN), lymphedema (LE), asymptomatic patent (CFA+MF+) and latent (CFA+MF-) W. bancrofti-infected individuals as well as individuals who were previously infected with W. bancrofti (PI) but had cleared the infection due to the administration of ivermectin (IVM) and albendazole (ALB). In summary, we observed that IL-10-producing CD19+CD24highCD38dhigh Breg were specifically increased in patently infected (CFA+MF+) individuals. In addition, CD19+CD24highCD5+CD1dhigh and CD19+CD5+CD1dhighIL-10+ Breg as well as CD4+CD127-FOXP3+ Treg frequencies were significantly increased in both W. bancrofti-infected cohorts (CFA+MF+ and CFA+MF-). Interestingly, the PI cohort presented frequency levels of all studied regulatory immune cell populations comparable with the EN group. In conclusion, the results from this study show that an ongoing W. bancrofti infection induces distinct Breg and Treg populations in peripheral blood from Ghanaian volunteers. Those regulatory immune cell populations might contribute to the regulated state of the host immune system and are probably important for the survival and fertility (microfilaria release) of the helminth.

Single nucleotide polymorphisms in the angiogenic and lymphangiogenic pathways are associated with lymphedema caused by Wuchereria bancrofti.

BACKGROUND: Lymphedema (LE) is a chronic clinical manifestation of filarial nematode infections characterized by lymphatic dysfunction and subsequent accumulation of protein-rich fluid in the interstitial space-lymphatic filariasis. A number of studies have identified single nucleotide polymorphisms (SNPs) associated with primary and secondary LE. To assess SNPs associated with LE caused by lymphatic filariasis, a cross-sectional study of unrelated Ghanaian volunteers was designed to genotype SNPs in 285 LE patients as cases and 682 infected patients without pathology as controls. One hundred thirty-one SNPs in 64 genes were genotyped. The genes were selected based on their roles in inflammatory processes, angiogenesis/lymphangiogenesis, and cell differentiation during tumorigenesis. RESULTS: Genetic associations with nominal significance were identified for five SNPs in three genes: vascular endothelial growth factor receptor-3 (VEGFR-3) rs75614493, two SNPs in matrix metalloprotease-2 (MMP-2) rs1030868 and rs2241145, and two SNPs in carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM-1) rs8110904 and rs8111171. Pathway analysis revealed an interplay of genes in the angiogenic/lymphangiogenic pathways. Plasma levels of both MMP-2 and CEACAM-1 were significantly higher in LE cases compared to controls. Functional characterization of the associated SNPs identified genotype GG of CEACAM-1 as the variant influencing the expression of plasma concentration, a novel finding observed in this study. CONCLUSION: The SNP associations found in the MMP-2, CEACAM-1, and VEGFR-3 genes indicate that angiogenic/lymphangiogenic pathways are important in LE clinical development.

Comparison of Doxycycline, Minocycline, Doxycycline plus Albendazole and Albendazole Alone in Their Efficacy against Onchocerciasis in a Randomized, Open-Label, Pilot Trial.

The search for new macrofilaricidal drugs against onchocerciasis that can be administered in shorter regimens than required for doxycycline (DOX, 200mg/d given for 4-6 weeks), identified minocycline (MIN) with superior efficacy to DOX. Further reduction in the treatment regimen may be achieved with co-administration with standard anti-filarial drugs. Therefore a randomized, open-label, pilot trial was carried out in an area in Ghana endemic for onchocerciasis, comprising 5 different regimens: the standard regimen DOX 200mg/d for 4 weeks (DOX 4w, N = 33), the experimental regimens MIN 200mg/d for 3 weeks (MIN 3w; N = 30), DOX 200mg/d for 3 weeks plus albendazole (ALB) 800mg/d for 3 days (DOX 3w + ALB 3d, N = 32), DOX 200mg/d for 3 weeks (DOX 3w, N = 31) and ALB 800mg for 3 days (ALB 3d, N = 30). Out of 158 randomized participants, 116 (74.4%) were present for the follow-up at 6 months of whom 99 participants (63.5%) followed the treatment per protocol and underwent surgery. Histological analysis of the adult worms in the extirpated nodules revealed absence of Wolbachia in 98.8% (DOX 4w), 81.4% (DOX 3w + ALB 3d), 72.7% (MIN 3w), 64.1% (DOX 3w) and 35.2% (ALB 3d) of the female worms. All 4 treatment regimens showed superiority to ALB 3d (p < 0.001, p < 0.001, p = 0.002, p = 0.008, respectively), which was confirmed by real-time PCR. Additionally, DOX 4w showed superiority to all other treatment arms. Furthermore DOX 4w and DOX 3w + ALB 3d showed a higher amount of female worms with degenerated embryogenesis compared to ALB 3d (p = 0.028, p = 0.042, respectively). These results confirm earlier studies that DOX 4w is sufficient for Wolbachia depletion and the desired parasitological effects. The data further suggest that there is an additive effect of ALB (3 days) on top of that of DOX alone, and that MIN shows a trend for stronger potency than DOX. These latter two results are preliminary and need confirmation in a fully randomized controlled phase 2 trial. TRIAL REGISTRATION: ClinicalTrials.gov #06010453.