Multimodal biomarker discovery for active Onchocerca volvulus infection.

The neglected tropical disease onchocerciasis, or river blindness, is caused by infection with the filarial nematode Onchocerca volvulus. Current estimates indicate that 17 million people are infected worldwide, the majority of them living in Africa. Today there are no non-invasive tests available that can detect ongoing infection, and that can be used for effective monitoring of elimination programs. In addition, to enable pharmacodynamic studies with novel macrofilaricide drug candidates, surrogate endpoints and efficacy biomarkers are needed but are non-existent. We describe the use of a multimodal untargeted mass spectrometry-based approach (metabolomics and lipidomics) to identify onchocerciasis-associated metabolites in urine and plasma, and of specific lipid features in plasma of infected individuals (O. volvulus infected cases: 68 individuals with palpable nodules; lymphatic filariasis cases: 8 individuals; non-endemic controls: 20 individuals). This work resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine (CCG) as biomarker for O. volvulus. During the targeted validation study, metabolite-specific cutoffs were determined (inosine: 34.2 ng/ml; hypoxanthine: 1380 ng/ml; CCG: 29.7 ng/ml) and sensitivity and specificity profiles were established. Subsequent evaluation of these biomarkers in a non-endemic population from a different geographical region invalidated the urine metabolite CCG as biomarker for O. volvulus. The plasma metabolites inosine and hypoxanthine were confirmed as biomarkers for filarial infection. With the availability of targeted LC-MS procedures, the full potential of these 2 biomarkers in macrofilaricide clinical trials, MDA efficacy surveys, and epidemiological transmission studies can be investigated.

Urine metabolites for the identification of Onchocerca volvulus infections in patients from Cameroon.

BACKGROUND: The tropical disease onchocerciasis (river blindness), caused by Onchocerca volvulus filarial nematodes, is targeted for elimination by mass treatment with nematocidal and antimicrobial drugs. Diagnosis of O. volvulus infections is based on counts of skin-borne microfilariae, but additional diagnostic tools, e.g. worm- or host-derived small RNAs, proteins or metabolites, are required for high-throughput screening. N-acetyltyramine-O,ß-glucuronide (NATOG) was suggested as a biomarker for onchocerciasis but its viability as diagnostic tool has been challenged. METHODS: We performed a screening program of urine samples from individuals from Cameroon infected with O. volvulus, Loa loa, Mansonella perstans or a combination thereof. Urine metabolites were measured by liquid chromatography-mass spectrometry (LC-MS). Principle component analysis (PCA) revealed that onchocerciasis causes complex changes of the urine metabolome. RESULTS: The mean NATOG content was elevated in urine of O. volvulus-infected compared with non-infected individuals, but NATOG levels showed considerable variation. However, 13.8% of all O. volvulus-infected individuals had high NATOG levels never reached by individuals without filarial infections or only infected with L. loa or M. perstans. Therefore, the identification of individuals with high NATOG levels might be used to screen for the elimination of onchocerciasis after mass drug application. Additional metabolites, including a compound identified as cinnamoylglycine, had high PC1/PC2 loadings in the data set. Mean levels of cinnamoylglycine were increased in O. volvulus-infected individuals, and 17.2% of all O. volvulus individuals had elevated cinnamoylglycine levels not reached by the controls. CONCLUSIONS: On an individual level, NATOG alone had poor discriminative power distinguishing infected from non-infected individuals. However, 13.8% of all O. volvulus-infected individuals had NATOG levels never reached by individuals without filarial infections or infected with only L. loa or M. perstans. Discrimination of O. volvulus infections from controls or individuals suffering from multiple infections was improved by the measurement of additional metabolites, e.g. cinnamoylglycine. Thus, measuring a combination of urine metabolites may provide a way to assess onchocerciasis on the population level. This provides the possibility to design a strategy for large-scale onchocerciasis epidemiological screening programs based on urine rather than invasive techniques.

Development of Onchocerca volvulus in humanized NSG mice and detection of parasite biomarkers in urine and serum.

BACKGROUND: The study of Onchocerca volvulus has been limited by its host range, with only humans and non-human primates shown to be susceptible to the full life cycle infection. Small animal models that support the development of adult parasites have not been identified. METHODOLOGY/PRINCIPAL FINDINGS: We hypothesized that highly immunodeficient NSG mice would support the survival and maturation of O. volvulus and alteration of the host microenvironment through the addition of various human cells and tissues would further enhance the level of parasite maturation. NSG mice were humanized with: (1) umbilical cord derived CD34+ stem cells, (2) fetal derived liver, thymus and CD34+ stem cells or (3) primary human skeletal muscle cells. NSG and humanized NSG mice were infected with 100 O. volvulus infective larvae (L3) for 4 to 12 weeks. When necropsies of infected animals were performed, it was observed that parasites survived and developed throughout the infection time course. In each of the different humanized mouse models, worms matured from L3 to advanced fourth stage larvae, with both male and female organ development. In addition, worms increased in length by up to 4-fold. Serum and urine, collected from humanized mice for identification of potential biomarkers of infection, allowed for the identification of 10 O. volvulus-derived proteins found specifically in either the urine or the serum of the humanized O. volvulus-infected NSG mice. CONCLUSIONS/SIGNIFICANCE: The newly identified mouse models for onchocerciasis will enable the development of O. volvulus specific biomarkers, screening for new therapeutic approaches and potentially studying the human immune response to infection with O. volvulus.

Noninvasive Urine Biomarker Lateral Flow Immunoassay for Monitoring Active Onchocerciasis.

The parasitic disease onchocerciasis is the second leading cause of preventable blindness, afflicting more than 18 million people worldwide. Despite an available treatment, ivermectin, and control efforts by the World Health Organization, onchocerciasis remains a burden in many regions. With an estimated 120 million people living in areas at risk of infection, efforts are now shifting from prevention to surveillance and elimination. The lack of a robust, point-of-care diagnostic for an active Onchocerca infection has been a limiting factor in these efforts. Previously, we reported the discovery of the biomarker N-acetyl-tyramine- O-glucuronide (NATOG) in human urine samples and its ability to track treatment progression between medicated patients relative to placebo; we also established its capability to monitor disease burden in a jird model. NATOG is a human-produced metabolite of tyramine, which itself is produced as a nematode neurotransmitter. The ability of NATOG to distinguish between active and past infection overcomes the limitations of antibody biomarkers and PCR methodologies. Lateral flow immunoassay (LFIA) diagnostics offer the versatility and simplicity to be employed in the field and are inexpensive enough to be utilized in large-scale screening efforts. Herein, we report the development and assessment of a NATOG-based urine LFIA for onchocerciasis, which accurately identified 85% of analyzed patient samples ( N = 27).

Validation of onchocerciasis biomarker N-acetyltyramine-O-glucuronide (NATOG).

The Neglected Tropical Disease onchocerciasis is a parasitic disease. Despite many control programmes by the World Health Organization (WHO), large communities in West and Central Africa are still affected. Besides logistic challenges during biannual mass drug administration, the lack of a robust, point-of-care diagnostic is limiting successful eradication of onchocerciasis. Towards the implementation of a non-invasive and point-of-care diagnostic, we have recently reported the discovery of the biomarker N-acetyltyramine-O-glucuronide (NATOG) in human urine samples using a metabolomics-mining approach. NATOG's biomarker value was enhanced during an investigation in a rodent model. Herein, we further detail the specificity of NATOG in active onchocerciasis infections as well as the co-infecting parasites Loa loa and Mansonella perstans. Our results measured by liquid chromatography coupled with mass spectrometry (LC-MS) reveal elevated NATOG values in mono- and co-infection samples only in the presence of the nematode Onchocerca volvulus. Metabolic pathway investigation of l-tyrosine/tyramine in all investigated nematodes uncovered an important link between the endosymbiotic bacterium Wolbachia and O. volvulus for the biosynthesis of NATOG. Based on these extended studies, we suggest NATOG as a biomarker for tracking active onchocerciasis infections and provide a threshold concentration value of NATOG for future diagnostic tool development.

Human infection with Onchocerca volvulus in Asir District (Saudi Arabia).

During one academic year, three patients were referred to Parasitology Laboratory from Dermatology Outpatients Clinics in King Abdulaziz University Hospitals. They were diagnosed as Sowda (chronic hyperactive form of onchocerciasis volvulus). The patients came from Asir Region in the Southern of the Kingdom. The lesion was characterized by a sever papule dermatitis localized to the lower limbs, with marked skin darkening. There was extensive follicular hyperplasia of the regional lymph nodes in two cases only. The skin snips taken from the three patients were positive microfilariae. On the other hand, the urine sample of one patient was positive. Six months after the onset of treatment by the clinicians in the Specialized Hospital, skin snips and urine samples were negative.

Eosinophils, eosinophil cationic protein and eosinophil-derived neurotoxin in serum and urine of patients with onchocerciasis coinfected with intestinal nematodes and in urinary schistosomiasis.

Eosinophils, eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN/EPX), myeloperoxidase (MPO) and IgE were measured in blood, serum and/or urine in Schistosoma haematobium- and Onchocerca volvulus-infected Guineans and O. volvulus- and S. haematobium-negative Guineans coinfected or infected with intestinal nematodes. The number of eosinophils and levels of eosinophil granule proteins but not of MPO were found to be strongly elevated in all Africans as compared to European controls. The highest serum ECP and serum and urinary EDN/EPX levels were observed in the hyperreactive form of onchocerciasis (sowda). Onchocerciasis patients and O. volvulus-negative Africans coinfected or infected with intestinal nematodes (hookworm and/or Ascaris lumbricoides) revealed higher serum granule protein concentrations and/or absolute eosinophil counts and urinary ECP than those without nematode infections. Statistical differences between both sections were found for the absolute eosinophil counts and for serum EDN/EPX and IgE in generalized onchocerciasis, and for urinary ECP in sowda, indicating stimulation of the eosinophil potential of O. volvulus-positive patients by coexistent hookworm infection. This worm species, in contrast to A. lumbricoides, causes especially high eosinophil counts and EDN/EPX and IgE levels. From these results it is concluded that in nematode diseases, ECP and EDN/EPX levels reflect the degree of antigenic stimulation, eosinophil activation and eosinophil turnover rates. Serum ECP and serum and urinary EDN/EPX may, therefore, serve as parameters to monitor helminth infection. Urinary ECP may be a marker of eosinophiluria secondary to urogenital manifestation of S. haematobium. It is elevated in hyperreactive onchocerciasis activated by intestinal nematodes.

Microfilaruria in an area of Nigeria with hyperendemic onchocerciasis.

Urine samples collected from sixty-four Nigerians living in a community which is hyperendemic for onchocerciasis were examined for microfilaria. Each urine donor was examined for the physical symptoms of long-standing onchocerciasis. The microfilaruria rate was 23.4%. Males had a higher rate of microfilaruria (27.5%) than females (16.7%). The prevalence rates of leopard skin and nodules were 18.8% and 46.9%, respectively. Thirteen persons had microfilaruria and had nodules. Seventeen showed no microfilaruria but had nodules. Only two of those without nodules were without microfilaruria. A very strong association was found between nodule-possession, nodule-load and microfilaruria (coefficient of association 0.85). Nodule load seemed to be the major factor in having microfilaria in urine. Microfilaruria is not common in Nigeria and the present finding is the first report of microfilaruria in the Taraba river valley which is a continuous onchocerciasis zone with the Western Cameroun-Southern Chad focus of transmission where microfilaruria has been reported. This paper discusses the implications of the results of this investigation with respect to monitoring the impact of ivermectin distribution in the Taraba river valley of Nigeria.

Albendazole in the treatment of onchocerciasis: double-blind clinical trial in Venezuela.

A double-blind clinical trial was conducted in Monagas State, Venezuela to assess the tolerance and efficacy of albendazole in the therapy of Onchocerca volvulus infection. Forty-nine patients (26 treated and 23 controls) received a 10-day course of albendazole (400 mg/day) or a placebo. Consistent with the excellent tolerance observed, albendazole did not kill microfilariae. However, analysis of changes in microfilarial densities (mf/mg of skin) over one year showed that albendazole was active against O. volvulus, presumably by interfering with embryogenesis. The nature, degree, and duration of this effect remain to be determined.

Pharmacokinetics of CGP 6140 (amocarzine) after oral administration of single 100-1600 mg doses to patients with onchocerciasis.

The concentrations of CGP 6140 [4-nitro-4'-(N-methyl-piperazinylthiocarbonylamido)-diphenylamine] and of its N-oxide metabolite, CGP 13,231, were measured in plasma and urine after single oral dose of 100-1600 mg of CGP 6140 to 41 fasted Ghanaian patients with Onchocerca volvulus infections. The absorption of CGP 6140 was rapid and its terminal elimination half-life was about 3 h. The plasma concentrations of CGP 6140 were essentially proportional to the dose. A greater variability in plasma concentrations was apparent after the 800 and 1600 mg doses indicating a poor bioavailability of the drug administered in fasting conditions to several patients. In plasma, the concentrations of CGP 13,231 were similar to those of CGP 6140. The amount of CGP 13,231 excreted in urine was 25-40% of the dose of CGP 6140 whereas only 1.5% was excreted as unchanged drug. If a single dose of drug is used for the treatment, the plasma concentration would be maintained for 3-4 h at a high level. At 8 h, the concentration falls to about 10% of the Cmax. If sustained plasma concentrations of the drug are needed for efficacy, twice daily administration would maintain the minimum concentration at about 10% of the Cmax.

The effect of moderate urine alkalinisation on low dose diethylcarbamazine therapy in patients with onchocerciasis.

Twenty-one patients with moderate to heavy infections with O. volvulus were treated with 25 mg of diethylcarbamazine (DEC) citrate twice daily for 10 days. In 11 patients the urine was made alkaline with sodium bicarbonate, 2 g, administered 6 hourly for three doses daily beginning 1 day before DEC was started and continued throughout the DEC therapy. Ten patients served as controls. The mean pre-dose plasma DEC concentration during treatment and the mean plasma DEC half-life were significantly higher in bicarbonate treated patients as compared to controls. Total urinary excretion of DEC was significantly less in the bicarbonate treated group than in controls. Mean overall total reaction was higher in bicarbonate-treated patients but the difference was not significant. The bicarbonate-treated group achieved a significantly greater reduction in skin microfilarial counts than the control group as assessed 1 week after completion of therapy, but there was little difference at 1 month. Microfilarial killing was associated with microfilarial mobilisation, alteration in peripheral leucocytes and elevation in serum aminotransferases in both groups. There was no effect of DEC on the number of adult worms recovered in nodules removed at the end of the therapy. This study indicates that moderate urinary alkalinisation alters the kinetics of DEC and the therapeutic response. However the severity of clinical reaction coupled with the inadequate level of microfilarial killing achieved make it unlikely that manipulation of urinary pH will be of practical value in onchocerciasis chemotherapy.

[Microfilaruria and ocular onchocerciasis (author's transl)]. / Microfilarurie et lesions ocularies onchocerquiennes.

A study of the relationship between microfilaruria and onchocercal eye lesions has been carried out among 619 persons in 5 villages of North Benin. There is evidence of a significant relationship between microfilaruria and irreversible eye lesions in men aged 15-34 years. The association of microfilaruria with high numbers of microfilariae in the anterior segment of the eye occurs more in older men.

Factors influencing the passage of Onchocerca volvulus microfilariae into the urine.

The effect of various substances on the output of Onchocerca volvulus microfilariae in the urine was investigated in volunteers infected with the Cameroon forest and Sudan-savanna strains of the parasite. Output of microfilariae in the urine tended to be higher during periods of normal activity than during sleep. During waking hours, the rate of output remained generally steady, but in some patients occasional showers of microfilariae appeared in the urine, possibly associated with the intake of food and drink. Drinking 1.2-2.5 litres water produced a shower of microfilariae in the urine of some subjects. This began within an hour of drinking and its onset preceeded that of the diuresis. Thiazide diuretics, acting on the convoluted tubules, produced no increase in microfilaruria. In savanna subjects intravenous injection of DT TAB vaccine caused pyrexia, and simultaneously large numbers of microfilariae appeared in the urine. There was no associated diuresis, and no increase in the concentration of microfilariae in the venous blood. In forest subjects DT TAB caused no increase in microfilaruria. In all subjects 25-50 mg diethylcarbamazine (DEC) caused large numbers of microfilariae to appear in the urine on day 0, within a few hours of the first dose; and there was an increased output of urine over the first 24 hours. Microfilaruria declined sharply on day 1 of treatment, but in subjects developing a high microfilaraemia, it rose again on day 2, and declined more slowly thereafter. Betamethazone, given in conjunction with DEC, appeared to slow the rate of destruction of microfilariae in the skin and lymph glands, and to prolong the duration of microfilaraemia and microfilaruria. The findings suggest that there is a reservoir of microfilariae in the glomerular capillaries, which fills slowly by accumulating microfilariae from the circulating blood. The microfilariae probably enter the urine by penetrating the glomerular capillary.

Onchocerciasis in Guatemala. I. Epidermiological studies of microfilaruria.

Microfilariae of Onchocerca volvulus were detected in the urine of 65 residents of three coffee plantations near Yepocapa, Guatemala. In this area the prevalence of microfilaruria is estimated to be between 17% and 30% of the population 10 years of age and older. Almost all of the people examined had clinical manifestations of onchocerciasis and 80% of them had microfilariae in skin snips. The frequency of microfilaruria is associated with the number of microfilariae in the skin. Within each age group those who had lived longer on the coffee plantations were more likely to have microfilariae in a skin snip and more likely to have microfilariae in their urine. The presence of subcutaneous nodules or history of prior nodulectomy did not reduce the incidence of microfilaruria nor did the presence of subcutaneous nodules increase the incidence of microfilariae in the urine.