Onchocerciasis Drug Discovery: In Vitro Evaluation of FDA-Approved Drugs against Onchocerca gutturosa in Gambia.

Onchocerciasis treatment and control relies mainly on the use of ivermectin which has high activity against the microfilarial stage of Onchocerca volvulus but limited activity against the long-lived, tissue dwelling adult nematodes. As this neglected tropical disease has now been targeted for elimination, there is an urgent need for new drugs to combat these parasites, ideally with macrofilaricidal activity. In this study, we have examined the anti-Onchocerca activity of a range of existing FDA-approved drugs with a view to repurposing, which can lead to rapid and relatively inexpensive development. From the Pharmakon-1600 library, 106 drugs were selected and tested against O. gutturosa adult male parasites using a concentration of 1.25 × 10-5 M in an in vitro 5-day standard assay to assess motility and viability (using MTT/formazan colorimetry). The findings revealed that 44 drugs produced marginal/moderate activity (50-99% motility and/or MTT reductions) including cefuroxime sodium, methenamine, primaquine phosphate and rivastigmine tartrate, while 23 drugs produced good activity (100% motility reductions and significant MTT reductions), including atovaquone, isradipine, losartan, rifaximin, cefaclor and pyrantel pamoate. Although this study represents only a first step, some of the identified hits indicate there are potential anti-Onchocerca drug candidates worthy of further investigation.

Hepatocellular carcinoma presenting as right shoulder pain.

We report an uncommon presentation of liver cancer in a 33-year-old woman who presented with persistent right shoulder pain with a normal physical examination of her shoulder and normal X-rays. An abdominal ultrasound scan and a computed tomography scan subsequently revealed a large liver cancer in this patient who was subsequently found to be hepatitis B positive. Extrinsic causes of shoulder pain should be considered when shoulder movement is normal and does not alter the character of the pain. Subdiaphragmatic liver lesions should be considered in the differential diagnosis of right shoulder pain. In any geographical area with a high incidence of hepatitis B infection, hepatocellular carcinoma should be included in the differential diagnosis of shoulder pain if a clear local cause is not identified.

Résumé Nous rapportons une présentation peu fréquente du cancer du foie chez une femme de 33 ans qui présentait une douleur persistante à l'épaule droite avec une douleur normale examen physique de son épaule et radiographies normales. Une échographie abdominale et une tomodensitométrie ont par la suite révélé un gros cancer du foie chez ce patient qui s'est avéré par la suite positif à l'hépatite B. Les causes extrinsèques de la douleur à l'épaule doivent être prises en compte lorsque le mouvement de l'épaule est normal et ne modifie pas le caractère de la douleur. Des lésions hépatiques sous-diaphragmatiques doivent être envisagées dans le diagnostic différentiel de la douleur à l'épaule droite. Dans toute zone géographique où l'incidence de l'hépatite B est élevée, carcinome hépatocellulaire devrait être inclus dans le diagnostic différentiel de la douleur à l'épaule si une cause locale claire n'est pas identifiée. Mots-clés: Hépatite B, cancer du foie, douleur à l'épaule.

Severe Electrolyte Disturbances Due to Proton Pump Inhibitor Therapy: An Underrecognized Problem with Potentially Severe Sequelae.

BACKGROUND Proton pump inhibitors are increasingly being recognized as a cause of multiple electrolyte disturbances, including hypomagnesemia, hypocalcemia, hypophosphatasemia, hypokalemia and hyponatremia, particularly in persons on long-term therapy. The mechanisms, consequences, and management of these electrolyte disturbances are discussed below. CASE REPORT A 55-year-old woman was seen by various clinicians, with a variety of clinical presentations, over the space of a couple of years. During each visit, she had electrolyte disturbances and was on proton pump inhibitor therapy, which were either continued or changed to a different proton pump inhibitor. She had presented variously with diarrhea and weight loss due to microscopic colitis, confusion, and grand mal seizures on separate occasions. Changing the proton pump inhibitor did not alleviate her profound electrolyte disturbances, which completely resolved shortly after stopping drug therapy. CONCLUSIONS It is important for clinicians to be aware of the electrolyte disturbances that can be caused by these medications, and to actively monitor patients on long-term therapy for these disturbances, thus avoiding potentially severe consequences. Electrolyte disturbances are more likely to arise in patients who are prescribed concomitant diuretic treatment or who overuse alcohol. The incidental finding of hypocalcemia in persons on proton pump inhibitors may be secondary to hypomagnesemia, and hypomagnesemia may be a consequence of an underlying otherwise symptomless genetic disorders. Clinicians should be encouraged to deprescribe these drugs after 4 weeks of treatment in patients with mild symptoms or mild disease.

Evaluation of the in vitro susceptibility of various filarial nematodes to emodepside.

Filariae are vector-borne nematodes responsible for an enormous burden of disease. Human lymphatic filariasis, caused by Wuchereria bancrofti, Brugia malayi, and Brugia timori, and onchocerciasis (caused by Onchocerca volvulus) are neglected parasitic diseases of major public health significance in tropical regions. To date, therapeutic efforts to eliminate human filariasis have been hampered by the lack of a drug with sufficient macrofilaricidal and/or long-term sterilizing effects that is suitable for use in mass drug administration (MDA) programs, particularly in areas co-endemic with Loa loa, the causative agent of loiasis. Emodepside, a semi-synthetic cyclooctadepsipeptide, has been shown to have broad-spectrum efficacy against gastrointestinal nematodes in a variety of mammalian hosts, and has been approved as an active ingredient in dewormers for cats and dogs. This paper evaluates, compares (where appropriate) and summarizes the in vitro effects of emodepside against a range of filarial nematodes at various developmental stages. Emodepside inhibited the motility of all tested stages of filariae frequently used as surrogate species for preclinical investigations (Acanthocheilonema viteae, Brugia pahangi, Litomosoides sigmodontis, Onchocerca gutturosa, and Onchocerca lienalis), human-pathogenic filariae (B. malayi) and filariae of veterinary importance (Dirofilaria immitis) in a concentration-dependent manner. While motility of all filariae was inhibited, both stage- and species-specific differences were observed. However, whether these differences were detected because of stage- and/or species-specific factors or as a consequence of variations in protocol parameters among the participating laboratories (such as purification of the parasites, read-out units, composition of media, incubation conditions, duration of incubation etc.) remains unclear. This study, however, clearly shows that emodepside demonstrates broad-spectrum in vitro activity against filarial nematode species across different genera and can therefore be validated as a promising candidate for the treatment of human filariases, including onchocerciasis and lymphatic filariasis.

Gametocyte Development and Carriage in Ghanaian Individuals with Uncomplicated Plasmodium falciparum Malaria.

Plasmodium falciparum gametocytes develop over 9-12 days while sequestered in deep tissues. On emergence into the bloodstream, they circulate for varied amounts of time during which certain host factors might influence their further development. We aimed to evaluate the potential association of patient clinical parameters with gametocyte development and carriage via in vivo methods. Seventy-two patients were enrolled from three hospitals in the Volta region of Ghana in 2016. Clinical parameters were documented for all patients, and gametocyte prevalence by microscopy was estimated at 12.5%. By measuring RNA transcripts representing two distinct gametocyte developmental stages using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), we obtained a more precise estimate of gametocyte carriage while also inferring gametocyte maturation. Fifty-three percent of the study participants harbored parasites expressing transcripts of the immature gametocyte-specific gene (PF3D7_1477700), whereas 36% harbored PF3D7_1438800 RNA-positive parasites, which is enriched in mid and mature gametocytes, suggesting the presence of more immature stages. Linear logistic regression showed that patients older than 5 years but less than 16 years were more likely to carry gametocytes expressing both PF3D7_1477700 and PF3D7_1438800 compared with younger participants, and gametocytemia was more likely in mildly anemic individuals compared with those with severe/moderate anemia. These data provide further evidence that a greater number of malaria patients harbor gametocytes than typically estimated by microscopy and suggest a possible association between age, fever, anemia, and gametocytemia.

Comparison of malaria diagnostic methods in four hospitals in the Volta region of Ghana.

Background: Rapid diagnostic tests (RDTs) and microscopy are routinely used for the diagnosis of malaria in Ghana. DNA-based polymerase chain reaction (PCR) is not yet used routinely. We compared diagnostic methods and tested the sensitivities of different malaria diagnostic methods against PCR. Materials and methods: Study participants from four hospitals with a suspicion of malaria donated finger -prick blood for RDT and blood film examination. In addition, a blood spot was collected for PCR analysis, prior to treatment. Retrospective species-specific PCR was performed on all samples collected. Results: Using PCR we found an overall malaria prevalence of 39% among the 211 evaluable blood spots (83/211) and this ranged between 6-61% across the four hospitals. Of the 164 participants with RDT data, malaria prevalence was 57% (94/164), ranging from 3-100% from the four hospitals. Microscopy was the least sensitive with a parasite prevalence of 21% (25/119) of the evaluable 119 participants, varying from 9 to 35% across three health facilities. By comparison, we found the sensitivities and specificities of RDT results when compared to PCR to be slightly higher than microscopy compared to PCR. These were 56.4% versus 41.7% and 90% versus 81.9%, respectively, but generally lower than expected. Ninety-five percent of the PCR-detected infections were P. falciparum, while 4% were mixed species infections of P. falciparum and P. malariae, with the remaining being a mono-infection of P. malariae. Conclusions: While using PCR as a gold standard, we found RDT to be more reliable in diagnosing malaria than microscopy. In addition, a majority of malaria-treated cases were not supported by PCR diagnosis, leading to possible overtreatment. Pragmatic strategies are needed to ensure suspected malaria cases are accurately diagnosed before treatment.

Progressive dyspnoea following the treatment of Mycobacterium abscessus infection in an individual with relapsing granulamatosis with polyangitis (Wegener's), complicated by hearing loss requiring cochlear implantation.

BACKGROUND: Granulomatosis with polyangitis (Wegener's) is a vasculitic disease predominantly affecting the lungs, skin, kidneys, ears, nose and throat. Mycobacterium abscessus is an uncommon rapidly growing mycobacterium causing sporadic lung disease. This is the first report of both GPA and Mycobacterium abscessus pulmonary disease reported in literature. CASE PRESENTATION: We present a case report of a 33 year old Caucasian man with relapsing disease complicated by pulmonary infection with Mycobacterium abscessus. He subsequently required bilateral cochlear implantation for progressive sensori-neural hearing loss. His M. abscessus was treated successfully with a prolonged course of antimicrobial therapy. His Granulomatosis with polyangitis (Wegener's) relapsed towards the end of antimicrobial therapy and required treatment. Shortly after completing his antimicrobial therapy and relapse, he developed progressive dyspnea due to pulmonary fibrosis. CONCLUSION: The potential causes of his progressive dyspnoea are discussed including the potential role of his underlying disease and treatment.

Over-representation of diabetic patients with renal anaemia in the primary care setting.

UNLABELLED: AIMS. Anaemia is a complication of chronic kidney disease (CKD); the National Institute for Clinical Excellence (NICE) have defined renal anaemia as estimated glomerular filtration rate (eGFR) <60 ml/min/1.73 m2 and haemoglobin (HB) <11.0 g/dl. The purpose of this study was to see if diabetic patients have a higher prevalence in primary care of this new anaemia definition. Furthermore, we wished to determine whether diabetic patients were over-represented above HB 11.0 g/dl, which may lead to developing renal anaemia. METHODS: We used an observational prevalence study in primary care from one Staffordshire practice in 2005-2006. Egton Medical Information Systems Ltd computer database was searched for patients with two Modification of Diet in Renal Disease eGFRs separated by 3 months, HB levels and medications. RESULTS: From a list size of 1830 patients, 362 had two eGFRs <60; of those, 308 had a HB available. In all, 29 (9.4%) patients had NICE renal anaemia, with over-representation of diabetic patients, 13 (16%) against 16 (7%) without diabetes (P < 0.02). We found that diabetic patients were also over-represented at HB 11.0 to <12.5 g/dl, 26 (32%) with diabetes and 39 (17.6%) without (P < 0.001). Mean HB was significantly lower for the diabetic group (n = 81, 26%), 12.8 g/dl (95% Confidence Intervals (CI) 12.4-13.1) against non-diabetic group (n = 227, 74%), 13.4 g/dl (95% CI 13.2-13.6), P < 0.01. Predictors of HB on multivariate regression analysis were female gender, eGFR and diabetes (all P < 0.001). CONCLUSIONS: Diabetic patients were more likely to have NICE defined renal anaemia in this primary care population with CKD stages 3-5. This is similar to observations in secondary care settings. We observed over-representation of diabetic patients above NICE definition at HB 11.0 to <12.5 g/dl.

The efficacy of a single dose of intravenous ferric carboxymaltose (Ferinject) on anaemia in a pre-dialysis population of chronic kidney disease patients.

Anaemia due to chronic kidney disease (CKD) is common and may be managed with erythropoiesis stimulating agents and/or iron preparations. Iron preparations may be administered orally, intravenously or by intramuscular injection. Oral preparations pose a significant tablet burden on patients who are often taking multiple medications and may have undesirable gastrointestinal side effects. The existing intravenous preparation Venofer requires multiple doses of drug (typically 100-200 mg) at multiple clinic visits. The preparation Cosmofer may be given as a single dose, but this requires four to six hours to administer. For these reasons, their use in pre-dialysis patients remains limited in practice. The new intravenous iron preparation Ferric Carboxymaltose (Ferinject) may be given as a single dose of up to 1000 mg (but not exceeding 15 mg/kg/week) as an infusion over 6-15 minutes. This offers a significant advance in the management of these patients. We describe our initial experience with using this drug in a non-dialysis patient population with chronic kidney disease.

Steatorrhoea complicating post-infectious diarrhoea in a renal transplant patient on mycophenolate mofetil therapy.

Mycophenolate mofetil (MMF) is licensed as a prophylaxis in combination therapy to prevent renal transplant rejection. Gastrointestinal side effects are fairly common and include diarrhoea, abdominal discomfort, nausea, vomiting, gastritis and constipation. This drug has recently been described as causing villous atrophy, nutrient malabsorption and colonic mucosal changes. We present a case of reversible steatorrhoea occurring in a patient treated with MMF following an episode of infections diarrhoea.

Intravenous iron in chronic kidney disease: haemoglobin change shortly after treatment of patients neither on dialysis nor on erythropoietin.

Anaemia is a common in chronic kidney disease. Although erythropoietin and iron supplementation are established treatments, knowledge on the use of IV iron alone in patients not on dialysis or erythropoietin is incomplete. The responses of 82 patients referred to the renal anaemia service with haemoglobin of 11.5 g/dl or less were assessed 1 week after completing four once weekly doses of 200 mg of venofer. No patients were on dialysis or erythropoietin. The haemoglobin rise 1 week after treatment was 0.53 g/dl. Ferritin levels improved from 110.8 to 410.2 ng/l and transferrin saturation from 17.7 to 27.3%. Ferritin levels remained below our target range (200-500 ng/l) in 7.7% while 25.6% had levels above this. Ferritin levels remained less than 800 ng/l in nearly all patients. Intravenous iron is cost effective and should be considered for use in patients with renal anaemia. Patients with CKD stage 5 appeared to respond less well.

Fibrillary glomerulonephritis with small fibrils in a patient with the antiphospholipid antibody syndrome successfully treated with immunosuppressive therapy.

BACKGROUND: Fibrillary glomerulonephritis is a rare cause of progressive renal dysfunction, often leading to the need for dialysis within a few years. The role of immunosuppressive treatment is still uncertain although this has been tried with variable success. CASE PRESENTATION: A 56 year old woman with the antiphospholipid antibody syndrome (IgM anticardiolipin antibodies) was seen in the nephrology clinic with haematuria, proteinuria, and worsening renal function. A renal biopsy demonstrated a mesangial proliferative glomerulonephritis on light microscopy and smaller fibrils (10.6-13.8 nm in diameter) than is usual for fibrillary glomerulonephritis (typically 18-22 nm) on electron microscopy. Amyloidosis was excluded following detailed evaluation. On account of rapidly worsening renal failure she was started on cyclophosphamide and prednisolone which led to the partial recovery and stabilization of her renal function. CONCLUSION: This case highlights the need for routine electron microscopy in native renal biopsies, where the differential diagnosis is wide and varied and the light and immunofluorescence microscopic findings may be non specific.

Onchocerca parasites and Wolbachia endosymbionts: evaluation of a spectrum of antibiotic types for activity against Onchocerca gutturosa in vitro.

BACKGROUND: The filarial parasites of major importance in humans contain the symbiotic bacterium Wolbachia and recent studies have shown that targeting of these bacteria with antibiotics results in a reduction in worm viability, development, embryogenesis, and survival. Doxycycline has been effective in human trials, but there is a need to develop drugs that can be given for shorter periods and to pregnant women and children. The World Health Organisation-approved assay to screen for anti-filarial activity in vitro uses male Onchocerca gutturosa, with effects being determined by worm motility and viability as measured by reduction of MTT to MTT formazan. Here we have used this system to screen antibiotics for anti-filarial activity. In addition we have determined the contribution of Wolbachia depletion to the MTT reduction assay. METHODS: Adult male O. gutturosa were cultured on a monkey kidney cell (LLCMK 2) feeder layer in 24-well plates with antibiotics and antibiotic combinations (6 to 10 worms per group). The macrofilaricide CGP 6140 (Amocarzine) was used as a positive control. Worm viability was assessed by two methods, (i) motility levels and (ii) MTT/formazan colorimetry. Worm motility was scored on a scale of 0 (immotile) to 10 (maximum) every 5 days up to 40 days. On day 40 worm viability was evaluated by MTT/formazan colorimetry, and results were expressed as a mean percentage reduction compared with untreated control values at day 40. To determine the contribution of Wolbachia to the MTT assay, the MTT formazan formation of an insect cell-line (C6/36) with or without insect Wolbachia infection and treated or untreated with tetracycline was compared. RESULTS: Antibiotics with known anti-Wolbachia activity were efficacious in this system. Rifampicin (5 x 10(-5) M) was the most effective anti-mycobacterial agent; clofazimine (1.25 x 10(-5) M and 3.13 x 10(-6) M) produced a gradual reduction in motility and by 40 days had reduced worm viability. The other anti-mycobacterial drugs tested had limited or no activity. Doxycycline (5 x 10(-5) M) was filaricidal, but minocycline was more effective and at a lower concentration (5 x 10(-5) M and 1.25 x 10(-5) M). Inactive compounds included erythromycin, oxytetracycline, trimethoprim and sulphamethoxazole. The MTT assay on the insect cell-line showed that Wolbachia made a significant contribution to the metabolic activity within the cells, which could be reduced when they were exposed to tetracycline. CONCLUSION: The O. gutturosa adult male screen for anti-filarial drug activity is also valid for the screening of antibiotics for anti-Wolbachia activity. In agreement with previous findings, rifampicin and doxycycline were effective; however, the most active antibiotic was minocycline. Wolbachia contributed to the formation of MTT formazan in the MTT assay of viability and is therefore not exclusively a measure of worm viability and indicates that Wolbachia contributes directly to the metabolic activity of the nematode.