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.

Diversity of Culicoides in the middle belt of Ghana with Implications on the transmission of Mansonella perstans; a molecular approach.

BACKGROUND: Culicoides, also known as biting midges, carry pathogens which include Mansonella perstans. Mansonella perstans is a nematode parasite implicated in a number of disease outcomes. Even though a high prevalence of about 75% M. perstans infection has been recorded in some communities in the middle belt of Ghana, and a wide diversity of Culicoides species has been identified, the exact Culicoides species transmitting M. perstans in Ghana has not yet been deciphered. This study therefore aimed at assessing the species diversity of Culicoides and their role in the transmission of M. perstans in the middle belt of Ghana. METHODS: Culicoides species were sampled from 11 communities in the Asante-Akim North and Sene West districts in the middle belt of Ghana. Centre for Disease Control (CDC) UV light traps, as well as human bait (i.e. human landing catch and engorged catch) methods were used to assess the species abundance and diversity of Culicoides in the study communities in the wet and dry season. A colorimetric Loop-Mediated Isothermal Amplification (LAMP) assay was performed to assess the vector competence of the various Culicoides species. RESULTS: A total of 4810 Culicoides from 6 species were sampled. These included Culicoides inornatipennis, C. milnei, C. schultzei, C. grahamii, C. neavei, and C. imicola. Culicoides imicola was the most abundant species (56%) followed by C. grahamii (16%). Light traps sampled the most diverse species (6 species). Human landing catch and engorged catch methods identified three anthropophilic species, C. grahamii, C. milnei, and C. inornatipennis, with C. grahamii being the most anthropophilic with a peak biting time between the hours of 5 p.m. to 6 p.m. Generally, there was relatively higher species abundance in the wet than dry season. LAMP assay identified C. grahamii as the potential vector for M. perstans transmission in the middle belt of Ghana. CONCLUSIONS: For the first time, we have demonstrated that C. grahamii is the potential competent vector for M. perstans transmission in the middle belt of Ghana. It is more abundant in the rainy season and has a peak biting time between the hours of 5 and 6 p.m.

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.

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.

The Efficacy of Doxycycline Treatment on Mansonella perstans Infection: An Open-Label, Randomized Trial in Ghana.

Treating Mansonella perstans is challenged by the low efficacy of registered antihelminthics. Wolbachia endobacteria provide an alternative treatment target because depletion results in amicrofilaremia in filarial infections with Wuchereria bancrofti and Onchocerca volvulus infections. This open-label, randomized study sought to confirm that i) Wolbachia are present in M. perstans in Ghana and ii) doxycycline treatment will deplete Wolbachia and cause a slow, sustained decline in microfilariae (MF). Two hundred and two Ghanaians with M. perstans infection were randomized into early (immediate) and delayed (6 months deferred) treatment groups, given doxycycline 200 mg/day for 6 weeks, and monitored for MF and Wolbachia levels at baseline, 4, 12, and 24 months after the study onset (= time of randomization and start of treatment for the early group). Per protocol analysis revealed that the median MF/mL in the early group declined from 138 at baseline to 64 at month 4 and further to 0 at month 12. In the delayed group, MF load did not change from a baseline median of 97 to 102 at month 4 but declined to 42 at month 12, that is, 6 months after receiving treatment, trailing the early group as expected. By month 24, both treatment groups had reached a median MF level of 0. After treatment, Wolbachia were depleted from MF by ≥ 1-log drop compared with baseline levels. We conclude that M. perstans in Ghana harbor Wolbachia that are effectively depleted by doxycycline with subsequent reduction in MF loads, most likely because of interruption of fertility of adult worms.

Update on the biology and ecology of Culicoides species in the South-West region of Cameroon with implications on the transmission of Mansonella perstans.

BACKGROUND: Culicoides (Diptera; Ceratoponidae) are tiny, stout, blood-sucking flies with a near worldwide distribution. When present, they are often considered a biting nuisance but in addition, they are involved in the transmission of pathogens to humans, domestic and wild animals. Data on Culicoides species in the South-West region of Cameroon dates back to the 1950s. Over the decades, ecological transformation due to agriculture and deforestation may have affected the population dynamics of Culicoides and therefore our study provides an update of their bio-ecology in the region. Furthermore, the role of various Culicoides species in the transmission of parasitic filariae of the genus Mansonella remains inconclusive in this region. This study was designed to address these unknown issues and expand on current scientific knowledge. RESULTS: Eight species of Culicoides (C. bedfordi, C. inornatipennis, C. fulvithorax, C. grahamii, C. imicola, C. milnei, C. neavei and C. kumbaensis) were collected using light traps and human baits. Culicoides grahamii was the most abundant species, followed closely by C. milnei. Three species (C. milnei, C. grahamii and C. inornatipennis) were common in all observed larval development sites. Only four species (C. inornatipennis, C. fulvithorax, C. grahamii and C. milnei) were collected on humans. Anthropophilic species were more abundant (P < 0.001) in the evening (4-7 pm) when compared to the morning collections (6-9 am). After overnight fly collections using a drop trap with a human microfilaremic donor, C. milnei emerged as the potential host for transmitting Mansonella perstans. Substantial heterogeneity was observed between the trap visiting cycles of the various species (P < 0.001). The biting cycle of the main vector, C. milnei, showed two peaks (10-11 pm and 4-5 am), the highest being 10-11 pm. CONCLUSIONS: The Culicoides fauna of the South-West region of Cameroon has not changed significantly since the 1950s. Culicoides milnei was demonstrated to be the major vector of M. perstans in this part of Cameroon. It is essentially a nocturnal species which peaks in abundance between 10 and 11 pm.

Epidemiology of Mansonella perstans in the middle belt of Ghana.

BACKGROUND: Mansonellosis was first reported in Ghana by Awadzi in the 1990s. Co-infections of Mansonella perstans have also been reported in a small cohort of patients with Buruli ulcer and their contacts. However, no study has assessed the exact prevalence of the disease in a larger study population. This study therefore aimed to find out the prevalence of M. perstans infection in some districts in Ghana and to determine the diversity of Culicoides that could be potential vectors for transmission. METHODS: From each participant screened in the Asante Akim North (Ashanti Region), Sene West and Atebubu Amantin (Brong Ahafo Region) districts, a total of 70 µl of finger prick blood was collected for assessment of M. perstans microfilariae. Centre for Disease Control (CDC) light traps as well as the Human Landing Catch (HLC) method were used to assess the species diversity of Culicoides present in the study communities. RESULTS: From 2,247 participants, an overall prevalence of 32% was recorded although up to 75% prevalence was demonstrated in some of the communities. Culicoides inornatipennis was the only species of Culicoides caught with the HLC method. By contrast, C. imicola (47%), C. neavei (25%) and C. schultzei (15%) were caught by the CDC light trap method. A wide diversity of other Culicoides spp. was also identified but correlation was only found between the prevalence of C. inornatipennis and M. perstans during the dry season. CONCLUSIONS: Here we demonstrate for the first time that M. perstans is highly prevalent in three districts in Ghana. We found a wide spectrum of Culicoides spp. Culicoides inornatipennis was the most anthropophilic and is therefore likely to be the species responsible for transmission of infection but formal proof has yet to be obtained. TRIAL REGISTRATION: NCT02281643 . Registered October 26, 2014. 'Retrospectively registered'. TRIAL REGISTRY: ClinicalTrials.gov.

Increased pfmdr1 gene copy number and the decline in pfcrt and pfmdr1 resistance alleles in Ghanaian Plasmodium falciparum isolates after the change of anti-malarial drug treatment policy.

BACKGROUND: With the introduction of artemisinin-based combination therapy (ACT) in 2005, monitoring of anti-malarial drug efficacy, which includes the use of molecular tools to detect known genetic markers of parasite resistance, is important for first-hand information on the changes in parasite susceptibility to drugs in Ghana. This study investigated the Plasmodium falciparum multidrug resistance gene (pfmdr1) copy number, mutations and the chloroquine resistance transporter gene (pfcrt) mutations in Ghanaian isolates collected in seven years to detect the trends in prevalence of mutations. METHODS: Archived filter paper blood blots collected from children aged below five years with uncomplicated malaria in 2003-2010 at sentinel sites were used. Using quantitative real-time polymerase chain reaction (qRT-PCR), 756 samples were assessed for pfmdr1 gene copy number. PCR and restriction fragment length polymorphism (RFLP) were used to detect alleles of pfmdr1 86 in 1,102 samples, pfmdr1 184, 1034, 1042 and 1246 in 832 samples and pfcrt 76 in 1,063 samples. Merozoite surface protein 2 (msp2) genotyping was done to select monoclonal infections for copy number analysis. RESULTS: The percentage of isolates with increased pfmdr1 copy number were 4, 27, 9, and 18% for 2003-04, 2005-06, 2007-08 and 2010, respectively. Significant increasing trends for prevalence of pfmdr1 N86 (×(2) = 96.31, p <0.001) and pfcrt K76 (×(2) = 64.50, p <0.001) and decreasing trends in pfmdr1 Y86 (x(2) = 38.52, p <0.001) and pfcrt T76 (x(2) = 43.49, p <0.001) were observed from 2003-2010. The pfmdr1 F184 and Y184 prevalence showed an increasing and decreasing trends respectively but were not significant (×(2) = 7.39,p=0.060; ×(2) = 7.49, p = 0.057 respectively). The pfmdr1 N86-F184-D1246 haplotype, which is alleged to be selected by artemether-lumefantrine showed a significant increasing trend (×(2) = 20.75, p < 0.001). CONCLUSION: Increased pfmdr1 gene copy number was observed in the isolates analysed and this finding has implications for the use of ACT in the country although no resistance has been reported. The decreasing trend in the prevalence of chloroquine resistance markers after change of treatment policy presents the possibility for future introduction of chloroquine as prophylaxis for malaria risk groups such as children and pregnant women in Ghana.