Doxycycline Leads to Sterility and Enhanced Killing of Female Onchocerca volvulus Worms in an Area With Persistent Microfilaridermia After Repeated Ivermectin Treatment: A Randomized, Placebo-Controlled, Double-Blind Trial.

BACKGROUND: Ivermectin (IVM) has been the drug of choice for the treatment of onchocerciasis. However, there have been reports of persistent microfilaridermia in individuals from an endemic area in Ghana after many rounds of IVM, raising concerns of suboptimal response or even the emergence of drug resistance. Because it is considered risky to continue relying only on IVM to combat this phenomenon, we assessed the effect of targeting the Onchocerca volvulus Wolbachia endosymbionts with doxycycline for these individuals with suboptimal response. METHODS: One hundred sixty-seven patients, most of them with multiple rounds of IVM, were recruited in areas with IVM suboptimal response and treated with 100 mg/day doxycycline for 6 weeks. Three and 12 months after doxycycline treatment, patients took part in standard IVM treatment. RESULTS: At 20 months after treatment, 80% of living female worms from the placebo group were Wolbachia positive, whereas only 5.1% in the doxycycline-treated group contained bacteria. Consistent with interruption of embryogenesis, none of the nodules removed from doxycycline-treated patients contained microfilariae, and 97% of those patients were without microfilaridermia, in contrast to placebo patients who remained at pretreatment levels (P < .001). Moreover, a significantly enhanced number of dead worms were observed after doxycycline. CONCLUSIONS: Targeting the Wolbachia in O. volvulus is effective in clearing microfilariae in the skin of onchocerciasis patients with persistent microfilaridermia and in enhanced killing of adult worms after repeated standard IVM treatment. Strategies can now be developed that include doxycycline to control onchocerciasis in areas where infections persist despite the frequent use of IVM. CLINICAL TRIALS REGISTRATION: ISRCTN 66649839.

Palp ratio as a field identification tool for two members of the Anopheles gambiae complex in Ghana (A. melas and A. gambiae).

BACKGROUND: The Anopheles gambiae Giles complex is the most widely studied and the most important insect vector group. We explored the use of the palp ratio method as a field tool to identify A. melas and A. gambiae in Ghana. METHODS: Human landing catches were conducted to collect mosquitoes in the coastal area of Western Region of Ghana. Palps were removed and segments 3 and 4 + 5 measured using a compound microscope. DNA extraction and downstream PCR for species identification was carried out using the legs and wings. Known A. gambiae collected from the Ashanti Region of Ghana were used for comparison. RESULTS: A total of 2120 A. gambiae were collected. Lengths of segments 3 and 4 + 5 were significantly correlated in samples from both regions. Using a palp ratio of 0.81 as the cut-off value, 14.9% outliers (≥0.81) from our study area were confirmed by PCR as A. melas. PCR also confirmed outliers from the Ashanti Region with palp ratio < 0.81 (10.2%) as A. gambiae. CONCLUSION: The palp ratio method proved to be a useful tool to identify populations of salt and freshwater A. melas and A. gambiae.

Effectiveness of routine BCG vaccination on buruli ulcer disease: a case-control study in the Democratic Republic of Congo, Ghana and Togo.

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

Geographically weighted regression of land cover determinants of Plasmodium falciparum transmission in the Ashanti Region of Ghana.

BACKGROUND: Malaria is a mosquito-borne parasitic disease that causes severe mortality and morbidity, particularly in Sub-Saharan Africa. As the vectors predominantly bite between dusk and dawn, risk of infection is determined by the abundance of P. falciparum infected mosquitoes in the surroundings of the households. Remote sensing is commonly employed to detect associations between land use/land cover (LULC) and mosquito-borne diseases. Due to challenges in LULC identification and the fact that LULC merely functions as a proxy for mosquito abundance, assuming spatially homogenous relationships may lead to overgeneralized conclusions. METHODS: Data on incidence of P. falciparum parasitaemia were recorded by active and passive follow-up over two years. Nine LULC types were identified through remote sensing and ground-truthing. Spatial associations of LULC and P. falciparum parasitaemia rate were described in a semi-parametric geographically weighted Poisson regression model. RESULTS: Complete data were available for 878 individuals, with an annual P. falciparum rate of 3.2 infections per person-year at risk. The influences of built-up areas (median incidence rate ratio (IRR): 0.94, IQR: 0.46), forest (median IRR: 0.9, IQR: 0.51), swampy areas (median IRR: 1.15, IQR: 0.88), as well as banana (median IRR: 1.02, IQR: 0.25), cacao (median IRR: 1.33, IQR: 0.97) and orange plantations (median IRR: 1.11, IQR: 0.68) on P. falciparum rate show strong spatial variations within the study area. Incorporating spatial variability of LULC variables increased model performance compared to the spatially homogenous model. CONCLUSIONS: The observed spatial variability of LULC influence in parasitaemia would have been masked by traditional Poisson regression analysis assuming a spatially constant influence of all variables. We conclude that the spatially varying effects of LULC on P. falciparum parasitaemia may in fact be associated with co-factors not captured by remote sensing, and suggest that future studies assess small-scale spatial variation of vegetation to circumvent generalised assumptions on ecological associations that may in fact be artificial.

Infection with Mansonella perstans Nematodes in Buruli Ulcer Patients, Ghana.

During August 2010-December 2012, we conducted a study of patients in Ghana who had Buruli ulcer, caused by Mycobacterium ulcerans, and found that 23% were co-infected with Mansonella perstans nematodes; 13% of controls also had M. perstans infection. M. perstans co-infection should be considered in the diagnosis and treatment of Buruli ulcer.

Immunoepidemiological profiling of onchocerciasis patients reveals associations with microfilaria loads and ivermectin intake on both individual and community levels.

Mass drug administration (MDA) programmes against Onchocerca volvulus use ivermectin (IVM) which targets microfilariae (MF), the worm's offspring. Most infected individuals are hyporesponsive and present regulated immune responses despite high parasite burden. Recently, with MDA programmes, the existence of amicrofilaridermic (a-MF) individuals has become apparent but little is known about their immune responses. Within this immunoepidemiological study, we compared parasitology, pathology and immune profiles in infection-free volunteers and infected individuals that were MF(+) or a-MF. The latter stemmed from villages in either Central or Ashanti regions of Ghana which, at the time of the study, had received up to eight or only one round of MDA respectively. Interestingly, a-MF patients had fewer nodules and decreased IL-10 responses to all tested stimuli. On the other hand, this patient group displayed contrary IL-5 profiles following in vitro stimulation or in plasma and the dampened response in the latter correlated to reduced eosinophils and associated factors but elevated neutrophils. Furthermore, multivariable regression analysis with covariates MF, IVM or the region (Central vs. Ashanti) revealed that immune responses were associated with different covariates: whereas O. volvulus-specific IL-5 responses were primarily associated with MF, IL-10 secretion had a negative correlation with times of individual IVM therapy (IIT). All plasma parameters (eosinophil cationic protein, IL-5, eosinophils and neutrophils) were highly associated with MF. With regards to IL-17 secretion, although no differences were observed between the groups to filarial-specific or bystander stimuli, these responses were highly associated with the region. These data indicate that immune responses are affected by both, IIT and the rounds of IVM MDA within the community. Consequently, it appears that a lowered infection pressure due to IVM MDA may affect the immune profile of community members even if they have not regularly participated in the programmes.

Comparison of two assays for molecular determination of rifampin resistance in clinical samples from patients with Buruli ulcer disease.

This study evaluates a novel assay for detecting rifampin resistance in clinical Mycobacterium ulcerans isolates. Although highly susceptible for PCR inhibitors in 50% of the samples tested, the assay was 100% M. ulcerans specific and yielded >98% analyzable sequences with a lower limit of detection of 100 to 200 copies of the target sequence.

Insertion sequence element single nucleotide polymorphism typing provides insights into the population structure and evolution of Mycobacterium ulcerans across Africa.

Buruli ulcer is an indolent, slowly progressing necrotizing disease of the skin caused by infection with Mycobacterium ulcerans. In the present study, we applied a redesigned technique to a vast panel of M. ulcerans disease isolates and clinical samples originating from multiple African disease foci in order to (i) gain fundamental insights into the population structure and evolutionary history of the pathogen and (ii) disentangle the phylogeographic relationships within the genetically conserved cluster of African M. ulcerans. Our analyses identified 23 different African insertion sequence element single nucleotide polymorphism (ISE-SNP) types that dominate in different areas where Buruli ulcer is endemic. These ISE-SNP types appear to be the initial stages of clonal diversification from a common, possibly ancestral ISE-SNP type. ISE-SNP types were found unevenly distributed over the greater West African hydrological drainage basins. Our findings suggest that geographical barriers bordering the basins to some extent prevented bacterial gene flow between basins and that this resulted in independent focal transmission clusters associated with the hydrological drainage areas. Different phylogenetic methods yielded two well-supported sister clades within the African ISE-SNP types. The ISE-SNP types from the "pan-African clade" were found to be widespread throughout Africa, while the ISE-SNP types of the "Gabonese/Cameroonian clade" were much rarer and found in a more restricted area, which suggested that the latter clade evolved more recently. Additionally, the Gabonese/Cameroonian clade was found to form a strongly supported monophyletic group with Papua New Guinean ISE-SNP type 8, which is unrelated to other Southeast Asian ISE-SNP types.

The causal effect of malaria on stunting: a Mendelian randomization and matching approach.

BACKGROUND: Previous studies on the association of malaria and stunted growth delivered inconsistent results. These conflicting results may be due to different levels of confounding and to considerable difficulties in elucidating a causal relationship. Randomized experiments are impractical and previous observational studies have not fully controlled for potential confounding including nutritional deficiencies, breastfeeding habits, other infectious diseases and socioeconomic status. METHODS: This study aims to estimate the causal effect between malaria episodes and stunted growth by applying a combination of Mendelian randomization, using the sickle cell trait, and matching. We demonstrate the method on a cohort of children in the Ashanti Region, Ghana. RESULTS: We found that the risk of stunting increases by 0.32 (P-value: 0.004, 95% CI: 0.09, 1.0) for every malaria episode. The risk estimate based on Mendelian randomization substantially differs from the multiple regression estimate of 0.02 (P-value: 0.02, 95% CI: 0.003, 0.03). In addition, based on the sensitivity analysis, our results were reasonably insensitive to unmeasured confounders. CONCLUSIONS: The method applied in this study indicates a causal relationship between malaria and stunting in young children in an area of high endemicity and demonstrates the usefulness of the sickle cell trait as an instrument for the analysis of conditions that might be causally related to malaria.

Doxycycline improves filarial lymphedema independent of active filarial infection: a randomized controlled trial.

BACKGROUND: The aim of this study was to determine whether improvement of filarial lymphedema (LE) by doxycycline is restricted to patients with ongoing infection (positive for circulating filarial antigen [CFA]), or whether the majority of CFA-negative patients with LE would also show a reduction in LE severity. METHODS: One hundred sixty-two Ghanaian participants with LE stage 1-5 (Dreyer) were randomized blockwise into 2 groups (CFA positive or negative) and allocated to 3 treatment arms of 6 weeks: (1) amoxicillin (1000 mg/d), (2) doxycycline (200 mg/d), or (3) placebo matching doxycycline. All groups received standard hygiene morbidity management. The primary outcome was reduction of LE stages. Secondary outcomes included frequency of acute attacks and ultrasonographic assessment of skin thickness at the ankles. Parameters were assessed before treatment and after 3, 12, and 24 months. RESULTS: Doxycycline-treated patients with LE stage 2-3 showed significant reductions in LE severity after 12 and 24 months, regardless of CFA status. Improvement was observed in 43.9% of doxycycline-treated patients, compared with only 3.2% and 5.6% in the amoxicillin and placebo arms, respectively. Skin thickness was correlated with LE stage improvement. Both doxycycline and amoxicillin were able to reduce acute dermatolymphangioadenitis attacks. CONCLUSIONS: Doxycycline treatment improves mild to moderate LE independent of ongoing infection. This finding expands the benefits of doxycycline to the entire population of patients suffering from LE. Patients with LE stage 1-3 should benefit from a 6-week course of doxycycline every other year or yearly, which should be considered as an improved tool to manage morbidity in filarial LE. Clinical Trials Registration. ISRCTN 90861344.

Elevated adaptive immune responses are associated with latent infections of Wuchereria bancrofti.

In order to guarantee the fulfillment of their complex lifecycle, adult filarial nematodes release millions of microfilariae (MF), which are taken up by mosquito vectors. The current strategy to eliminate lymphatic filariasis as a public health problem focuses upon interrupting this transmission through annual mass drug administration (MDA). It remains unclear however, how many rounds of MDA are required to achieve low enough levels of MF to cease transmission. Interestingly, with the development of further diagnostic tools a relatively neglected cohort of asymptomatic (non-lymphedema) amicrofilaremic (latent) individuals has become apparent. Indeed, epidemiological studies have suggested that there are equal numbers of patent (MF(+)) and latent individuals. Since the latter represent a roadblock for transmission, we studied differences in immune responses of infected asymptomatic male individuals (n = 159) presenting either patent (n = 92 MF(+)) or latent (n = 67 MF(-)) manifestations of Wuchereria bancrofti. These individuals were selected on the basis of MF, circulating filarial antigen in plasma and detectable worm nests. Immunological profiles of either Th1/Th17, Th2, regulatory or innate responses were determined after stimulation of freshly isolated PBMCs with either filarial-specific extract or bystander stimuli. In addition, levels of total and filarial-specific antibodies, both IgG subclasses and IgE, were ascertained from plasma. Results from these individuals were compared with those from 22 healthy volunteers from the same endemic area. Interestingly, we observed that in contrast to MF(+) patients, latent infected individuals had lower numbers of worm nests and increased adaptive immune responses including antigen-specific IL-5. These data highlight the immunosuppressive status of MF(+) individuals, regardless of age or clinical hydrocele and reveal immunological profiles associated with latency and immune-mediated suppression of parasite transmission.

Paradoxical responses after start of antimicrobial treatment in Mycobacterium ulcerans infection.

BACKGROUND: Antimicrobial killing in mycobacterial infections may be accompanied by (transient) clinical deterioration, known as paradoxical reaction. To search for patterns reflecting such reactions in the treatment of Buruli ulcer (Mycobacterium ulcerans infection), the evolution of lesions of patients treated with antimicrobials was prospectively assessed. METHODS: The lesion size of participants of the BURULICO antimicrobial trial (with lesions ≤10 cm cross-sectional diameter) was assessed by careful palpation and recorded by serial acetate sheet tracings. Patients were treated with antimicrobials for 8 weeks. For the size analysis, participants whose treatment had failed, had skin grafting, or were coinfected with human immunodeficiency virus were excluded. For every time point, surface area was compared with the previous assessment. A generalized additive mixed model was used to study lesion evolution. Nonulcerative lesions were studied using digital images recording possible subsequent ulceration. RESULTS: Of 151 participants, 134 were included in the lesion size analysis. Peak paradoxical response occurred at week 8; >30% of participants showed an increase in lesion size as compared with the previous (week 6) assessment. Seventy-five of 90 (83%) of nonulcerative lesions ulcerated after start of treatment. Nine participants developed new lesions during or after treatment. All lesions subsequently healed. CONCLUSIONS: After start of antimicrobial treatment for Buruli ulcer, new or progressive ulceration is common before healing sets in. This paradoxical response, most prominent at the end of the 8-week antimicrobial treatment, should not be misinterpreted as failure to respond to treatment. Clinical Trials Registration. ClinicalTrials.gov, NCT00321178.

Transforming growth factor-ß1 variant Leu10Pro is associated with both lack of microfilariae and differential microfilarial loads in the blood of persons infected with lymphatic filariasis.

Antigen testing and ultrasound detection have shown that many persons are infected with Wuchereria bancrofti even though they do not have microfilariae (Mf) in the blood. To ascertain the role of human host immunogenetics on the lack of circulating Mf in the blood, 152 lymphatic filariasis (LF)-infected patients comprising 118 patients with microfilaremic (Mf+, patent) infection and 34 patients with latent (Mf-, antigen-positive) infection were recruited and genotyped for association of single nucleotide polymorphisms of TGF-ß1 and differential Mf load and/or lack of Mf in the blood from infected persons in Ghana. An association was found between the TGF-ß1 Leu10Pro variant and lack of Mf in the blood. Patients with latent infection had a higher frequency of the Leu/Leu genotype than patients with patent infection (p = 0.03). Secondary analysis revealed an association among the three possible Leu10Pro genotypes and different Mf loads in the blood. In conclusion, the differential Mf loads and the lack of Mf in the blood of patients is likely to have a genetic basis. Because the adult worms are responsible for pathology, these results underscore the need for a review of using only Mf detection in blood smears for diagnosis of LF infection in endemic areas. This information is also important for the mapping and surveillance activities of national and global programs for elimination of LF.

Macrofilaricidal Activity in Wuchereria bancrofti after 2 Weeks Treatment with a Combination of Rifampicin plus Doxycycline.

Infection with the filarial nematode Wuchereria bancrofti can lead to lymphedema, hydrocele, and elephantiasis. Since adult worms cause pathology in lymphatic filariasis (LF), it is imperative to discover macrofilaricidal drugs for the treatment of the infection. Endosymbiotic Wolbachia in filariae have emerged as a new target for antibiotics which can lead to macrofilaricidal effects. In Ghana, a pilot study was carried out with 39 LF-infected men; 12 were treated with 200 mg doxycycline/day for 4 weeks, 16 were treated with a combination of 200 mg doxycycline/day + 10 mg/kg/day rifampicin for 2 weeks, and 11 patients received placebo. Patients were monitored for Wolbachia and microfilaria loads, antigenaemia, and filarial dance sign (FDS). Both 4-week doxycycline and the 2-week combination treatment reduced Wolbachia load significantly. At 18 months posttreatment, four-week doxycycline resulted in 100% adult worm loss, and the 2-week combination treatment resulted in a 50% adult worm loss. In conclusion, this pilot study with a combination of 2-week doxycycline and rifampicin demonstrates moderate macrofilaricidal activity against W. bancrofti.

Follow-up survey of children who received sulfadoxine-pyrimethamine for intermittent preventive antimalarial treatment in infants.

Recently, the World Health Organization emphasized the potential benefit of intermittent preventive treatment in infants (IPTi) to control malaria and officially recommended implementation of IPTi with sulfadoxine-pyrimethamine (SP) in areas with moderate and high transmission, where SP resistance is not high. As reported rebound effects make further observation mandatory, we performed a survey of participants of a former IPTi trial. Malariometric parameters were similar in the SP and the placebo group. In contrast, anti-Plasmodium falciparum lysate immunoglobulin G antibody levels, a proxy measure for preceding malaria episodes, remained lower in the SP arm. The most likely explanation is a lower overall exposure to parasitic antigens after IPTi.

IL3 variant on chromosomal region 5q31-33 and protection from recurrent malaria attacks.

Using segregation analyses, control of malaria parasites has previously been linked to a major gene within the chromosomal region 5q31-33, but also to complex genetic factors in which effects are under substantial age-dependent influence. However, the responsible gene variants have not yet been identified for this chromosomal region. In order to perform association analyses of 5q31-33 locus candidate single nucleotide polymorphisms (SNPs), 1015 children were recruited at the age of 3 months and followed monthly until the age of 2 years in an area holoendemic for Plasmodium falciparum malaria in Ghana. Quantitative (incidence rates of malaria episodes) and qualitative phenotypes (i.e. 'more than one malaria episode' or 'not more than one malaria episode') were used in population- and family-based analyses. The strongest signal was observed for the interleukin 3 gene (IL3) SNP rs40401 (P = 3.4 × 10(-7), P(c)= 1.4 × 10(-4)). The IL3 genotypes rs40401(CT) and rs40401(TT) were found to exert a protective effect of 25% [incidence rate ratio (IRR) 0.75, P = 4.1 × 10(-5)] and 33% (IRR 0.67, P = 3.2 × 10(-8)), respectively, against malaria attacks. The association was confirmed in transmission disequilibrium tests (TDT, qTDT). The results could argue for a role of IL3 in the pathophysiology of falciparum malaria.

The role of ultrasonography in the differentiation of the various types of filaricele due to bancroftian filariasis.

OBJECTIVE: (i) To determine the frequencies of urogenital pathologies in men infected with bancroftian filariasis, and (ii) to evaluate the role of ultrasonography (USG) as a diagnostic tool to differentiate between diverse pathologies with different clinical implications. To date, all types of scrotal enlargement resulting from lymphatic filariasis (LF) have been summarized under one term: "filaricele". PATIENTS AND METHODS: Data were compiled from recruitment phases for field trials in an endemic area for LF in Ghana. 1453 men aged 18 years and above underwent ultrasound examinations of the scrotum. Observation parameters were: Filaria Dance Sign (FDS), dilation of supratesticular lymphatic vessels, thickness of scrotal skin, occurrence and amount of fluid accumulation, echogenicity of the fluid between the layers of the tunica vaginalis, as well as position and homogenicity of testis, epididymis and spermatic cord. In 1132 men, blood samples were taken for parasitological analysis. RESULTS: In 56% of examined patients, fluid accumulation around the testis was detected (38% subclinical-, 18% clinical stages). Differentiation of the echogenicity of the fluid revealed echo-free hydrocele (EFH) in 47% and echo-dense hydrocele (EDH) in 9%. Patients without hydrocele and subclinical stages had a thinner scrotal skin than those in clinical stages or with lymphscrotum (P < 0.001). In the EDH group the scrotal skin was thicker than in the EFH group (P < 0.001). 1.4% had a lymphscrotum. FDS was detected in 24% of all 1453 volunteers who underwent USG. The number of worm nests correlated with microfilarial load and levels of circulating filarial antigen (P < 0.001; 20% microfilaremic, 48% antigen positive). CONCLUSION: In an unexpected high number of men (56%) fluid accumulation around the testis was detected by USG of which more than one third (38%) presented with subclinical stages. The study showed that echo-dense and echo-free fluid could be differentiated and that a considerable number of cases had EDH (9%) posing a risk to develop necrotic testis and infertility and therefore requiring immediate surgical intervention. USG thus turned out to be a useful diagnostic technique to differentiate between those cases requiring immediate surgical intervention from those that can be treated with (anti-wolbachial and hyperpermeability reducing) drugs that ameliorate or halt progression of the disease.

A genotypic approach for detection, identification, and characterization of drug resistance in Mycobacterium ulcerans in clinical samples and isolates from Ghana.

Standardized antimycobacterial therapy is considered the treatment of choice for Buruli ulcer disease. To assess the prevalence of drug resistance among clinical Mycobacterium ulcerans isolates in Ghana, we conducted a sequence-based approach to detect mutations associated with drug resistance. We subjected clinical samples to direct DNA sequencing of rpoB and rpsL genes and compared culture and whole-genome extracts regarding the efficiency of sequence analysis; 99.1% (rpoB) and 100% (rpsL) of the patients harbored M. ulcerans wild type. In one isolate (0.9%), a point mutation of the rpoB gene at codon Ser522 leading to an amino acid change was detected. Culture extracts yielded a significantly higher sequencing efficiency than whole-genome extracts. Our data suggest a low level of drug resistance in Ghana. However, mutations associated with drug resistance do occur and require monitoring. Improved techniques are necessary to enhance the efficiency of sequence analysis of whole-genome extracts.