Differential susceptibility of Onchocerca ochengi adult male worms to flubendazole in gerbils and hamsters.

Onchocerciasis is a devastating skin and eye disease that afflicts about 21 million people, most of whom live in sub-Saharan Africa. Its control with the microfilaricidal drug ivermectin is limited, thus necessitating the development of preclinical animal models to aid in the discovery of a macrofilaricide. Previously, we found that Onchocerca ochengi (the closest relative of the human O. volvulus) worm masses survive better in hamsters than in gerbils. The aim of this study was to compare the survival of O. ochengi adult male worms and their susceptibility to flubendazole (FBZ, a macrofilaricide) in gerbils and hamsters. The animals were intraperitoneally implanted with O. ochengi male worms, treated with FBZ, and sacrificed 35 days post-implantation. Unlike gerbils which had some worms moving freely in the peritoneum and some in newly formed nodules (neo-nodules), all the worms in the hamsters were found in neo-nodules. FBZ significantly decreased worm burden, motility, and viability in gerbils whereas it had no significant effect in hamsters. These results highlight a major difference in how O. ochengi adult male worms are sustained and affected by FBZ in gerbils compared to hamsters. Understanding the difference between these two models is important in the development of effective macrofilaricides for onchocerciasis.

Exposure of Male Farmers and Nonfarmers to Neonicotinoid Pesticides in the South-West and Littoral Regions of Cameroon: A Comparative Study.

Pesticides, especially the newly developed neonicotinoids, are increasingly used in many countries around the world, including Cameroon, to control pests involved in crop destruction or disease transmission. Unfortunately, the pesticides also pose tremendous environmental problems because a predominant amount of their residues enter environmental matrices to affect other nontargeted species including humans. This therefore calls for continuous biomonitoring of these insecticides in human populations. The present study sought to assess the neonicotinoid insecticide exposures in two agrarian regions of Cameroon, the South-West region and Littoral region. The study involved 188 men, including 125 farmers and 63 nonfarmers. Spot urine samples were obtained from these subjects and subjected to liquid chromatographic-tandem mass spectrometric analysis for concentrations of neonicotinoid compounds, including acetamiprid, clothianidin, dinotefuran, imidacloprid, thiacloprid, nitenpyram, thiamethoxam, and N-dm-acetamiprid. Neonicotinoid compounds were detected in all study participants, and residues of all the screened pesticides were detected among participants. N-dm-Acetamiprid and imidacloprid were the most prevalent among the subjects (100.0% and 93.1%, respectively), whereas nitenpyram was less common (3.2%). The median values of imidacloprid and total urinary neonicotinoid concentrations were elevated among farmers (0.258 vs. 0.126 µg/L and 0.829 vs. 0.312 µg/L, respectively). Altogether the findings showed that both the farmer and nonfarmer study populations of Cameroon were exposed to multiple residues of neonicotinoids, with relatively higher levels of pesticides generally recorded among farmers. Although exposure levels of the neonicotinoids were generally lower than their respective reference doses, these results warrant further research on the health risk evaluation of multiple residues of the pesticides and reinforcement of control measures to minimize the exposure risks, especially among farmers. Environ Toxicol Chem 2024;43:952-964. © 2024 SETAC.

Survival of worm masses of Onchocerca ochengi in gerbils and hamsters: implications for the development of an in vivo macrofilaricide screening model.

Onchocerciasis, the second leading infectious cause of blindness, afflicts approximately 21 million people globally. Its control is limited to the use of the microfilaricidal drugs, ivermectin and moxidectin. Both drugs are unable to kill the adult worms which can survive for up to 15 years in patients, justifying the urgent need for potent and novel macrofilaricides that kill adult worms. The development of such drugs has been hindered by the lack of an appropriate small laboratory animal model to evaluate potential drug candidates in vivo. This study assessed the survival of O. ochengi female worms and their embryos over time in two laboratory rodents: gerbils and hamsters and tested using "proof-of-concept" studies, whether known macrofilaricidal drugs can kill these worms. Animals were surgically implanted with mechanical or collagenase-liberated O. ochengi worm masses, and necropsied at various time points to test for survival. Recovered worm masses were assessed for viability by biochemical analysis (MTT/formazan assay) or fecundity (embryogram). Flubendazole (FBZ) administered at 20 mg/kg body weight was used to validate both rodent models. By day 26 post-implantation of 15 worm masses, a median of 7.00 (4.00-10.00) was recovered from hamsters, and 2.50 (2.00-4.00) from gerbils. Worm masses recovered from gerbils were mostly disintegrated or fragmented, with significantly higher fragmentation observed with collagenase-liberated worm masses. FBZ had no significant effect on the number of worm masses recovered, but enhanced embryo degradation in gerbils and reduced worm mass viability in hamsters. This exploratory study has revealed the gerbil and hamster as permissible rodents to adult female worms of O. ochengi. The hamsters appeared to maintain the worms longer, compared to gerbils.

Development and validation of small animal models for onchocerciasis and loiasis microfilaricide discovery.

BACKGROUND: Onchocerciasis (river blindness) caused by the filarial worm Onchocerca volvulus is a neglected tropical disease that affects the skin and eyes of humans. Mass drug administration with ivermectin (IVM) to control the disease often suffers from severe adverse events in individuals co-injected with high loads of Loa loa microfilariae (mf). Thus loiasis animal models for counter-screening of compounds effective against onchocerciasis are needed, as are the corresponding onchocerciasis screening models. The repertoire of such models is highly limiting. Therefore, this study was aimed at developing and validating mf immunocompetent small animal models to increase tools for onchocerciasis drug discovery. METHODOLOGY/PRINCIPAL FINDINGS: O. ochengi mf from cattle skin and L. loa mf from human blood were used to infect BALB/c mice and Mongolian gerbils, and IVM was used for model validation. O. ochengi mf were given subcutaneously to both rodents while L. loa mf were administered intravenously to mice and intraperitoneally to gerbils. IVM was given orally. In an 8-day model of O. ochengi mf in BALB/c mice, treatment with IVM depleted all mf in the mice, unlike the controls. Also, in a 2.5-day model of L. loa mf in BALB/c, IVM significantly reduced mf in treated mice compared to the untreated. Furthermore, the gerbils were very susceptible to O. ochengi mf and IVM eradicated all mf in the treated animals. In the peritoneal L. loa mf gerbil model, IVM reduced mf motility in treated animals compared to the controls. In a 30-day gerbil co-injection model, IVM treatment cleared all O. ochengi mf and reduced motility of L. loa mf. Both mf survived for up to 50 days in a gerbil co-injection model. CONCLUSIONS/SIGNIFICANCE: We have developed two immunocompetent small animal models for onchocerciasis and loiasis that can be used for microfilaricide discovery and to counter-screen onchocerciasis macrofilarides.

Correction: Antischistosomal, antionchocercal and antitrypanosomal potentials of some Ghanaian traditional medicines and their constituents.

[This corrects the article DOI: 10.1371/journal.pntd.0008919.].

Anti-Onchocercal Properties of Extracts of Scoparia dulcis and Cylicodiscus gabunensis.

Introduction: The elimination of onchocerciasis is hampered by the absence of suitable drugs that are effective against adult filariae. This study is aimed at assessing the anti-onchocercal effects of extracts of Scoparia dulcis and Cylicodiscus gabunensis that could serve as drug leads against onchocerciasis. Methods: Different parts of the plants (Scoparia dulcis and Cylicodiscus gabunensis) were extracted with hexane, methylene chloride, and methanol. The extracts were tested in vitro against the bovine model parasite, Onchocerca ochengi. Adult female worm viability was determined biochemically by MTT/formazan colorimetry, while the adult male and microfilariae viability were determined by microscopy based on % inhibition of worm motility score. Cytotoxicity and acute toxicity of active extracts were tested on monkey kidney epithelial cells (LLC-MK2) and Balb/C mice, respectively. Results: The hexane extract of Scoparia dulcis recorded the highest activity, with IC50s of 50.78 µg/ml on both adult male and female worms and 3.91 µg/ml on microfilariae. For Cylicodiscus gabunensis extract, the highest activity was seen with the methylene chloride extract, with IC50s of 50.78 µg/ml, 62.50 µg/ml, and 16.28 µg/ml on, respectively, adult male, female, and microfilariae. The 50% cytotoxic concentration on the LLC-MK2 cells was 31.25 µg/ml for the most active extracts. No acute toxicity was recorded for the extracts. Phytochemical analysis of the extracts revealed the presence of alkaloids, flavonoids, sterols, saponins, phenols, and glycosides. Conclusion: This study validates the traditional use of these plants in treating onchocerciasis and suggests S. dulcis and C. gabunensis as new potential sources for the isolation of anti-onchocerca lead compounds.

Diversity and behavioral activity of Anopheles mosquitoes on the slopes of Mount Cameroon.

BACKGROUND: Malaria remains endemic in Cameroon, with heterogeneous transmission related to eco-climatic variations, vector diversity and spatial distribution. The intensification of malaria prevention and control through the free distribution of insecticide-treated nets in recent years may have altered the composition, geographic distribution and natural infection rate of Anopheles species, with implications for malaria transmission dynamics. The present study seeks to assess the vectorial diversity, dynamics and infectivity across different seasons and altitudes in relationship to parasite prevalence around the slopes of Mount Cameroon, southwestern region. METHOD: Mosquitoes were sampled (indoors and outdoors) in 11 eco-epidemiological settings at low (18-197 m), intermediate (371-584 m) and high (740-1067 m) altitude by nightly human landing catches. The mosquitoes were identified morphologically and Anopheles gambiae sibling species identified by PCR. Parity status was ascertained by examining the ovaries and the entomological inoculation rates (EIR) determined by Plasmodium falciparum circumsporozoite antigen ELISA of the head-thorax. The prevalence of Plasmodium infection across target communities was assessed using rapid diagnostic tests. RESULTS: A total of 7327 (18.0 mosquitoes/trap/night) mosquitoes were trapped, mainly during the rainy season (5678, 77.5%) and at low altitude (3669, 50.1%). Anopheles spp. (5079, 69.3%) was the most abundant genera and An. gambiae complex (2691, 36.7%) the major vector, varying with altitude (χ2 = 183.87, df = 8, P < 0.001) and season (χ2 = 28.14, df = 4, P < 0.001). Only An. gambiae (s.s.) was identified following molecular analysis of An. gambiae complex siblings. The overall biting peak for An. gambiae complex was 2-3 a.m. Anopheles cinctus was the most abundant secondary vector in the area. The average EIR in the area was 2.08 infective bites per person per night (ib/p/n), higher at low (2.45 ib/p/n) than at intermediate altitude (1.39 ib/p/n) and during the rainy (1.76 ib/p/n) compared to the dry season (0.34 ib/p/n). Anopheles funestus was most infectious overall (28.1%, 16/57) while An. gambiae had the highest inoculation rates averaging 1.33 ib/p/n. Most Anopheles species across all altitudes and seasons were parous, highest in communities with the highest proportion of malaria parasite infections. CONCLUSION: Anopheles gambiae (s.s.) remains the major malaria vector in the area and An. cinctus possibly a secondary vector of the disease in the slopes of Mt. Cameroon. The seasonal and altitudinal effects on the distribution of these mosquitoes may have implications for the transmission of malaria and its control strategies in the area. Regular monitoring of the bionomics of local Anopheles vector species and targeted control interventions in the 'hotspots' is necessary to curb the prevalence of the infection and incidence of disease.

Pyrvinium Pamoate and Structural Analogs Are Early Macrofilaricide Leads.

Onchocerciasis and lymphatic filariasis are neglected tropical diseases caused by infection with filarial worms. Annual or biannual mass drug administration with microfilaricidal drugs that kill the microfilarial stages of the parasites has helped reduce infection rates and thus prevent transmission of both infections. However, success depends on high population coverage that is maintained for the duration of the adult worm's lifespan. Given that these filarial worms can live up to 14 years in their human hosts, a macrofilaricidal drug would vastly accelerate elimination efforts. Here, we have evaluated the repurposed drug pyrvinium pamoate as well as newly synthesized analogs of pyrvinium for their efficacy against filarial worms in vitro and in vivo. We found that pyrvinium pamoate, tetrahydropyrvinium and one of the analogs were highly potent in inhibiting worms in in vitro whole-worm screening assays, and that all three compounds reduced female worm fecundity and inhibited embryogenesis in the Brugia pahangi-gerbil in vivo model of infection.

Filaricidal activity of Daniellia oliveri and Psorospermum febrifugum extracts.

BACKGROUND: Drugs currently used for controlling onchocerciasis and lymphatic filariasis (LF) are mainly microfilaricidal, with minimal or no effect on the adult worms. For efficient management of these diseases, it is necessary to search for new drugs with macrofilaricidal activities that can be used singly or in combination with existing ones. Daniellia oliveri and Psorospermum febrifugum are two plants commonly used in the local management of these infections in Bambui, a township in the North West Region of Cameroon, but there is currently no documented scientific evidence to support their claimed anthelmintic efficacy and safety. The aim of this study was to provide evidence in support of the search for means to eliminate these diseases by screening extracts and chromatographic fractions isolated from these plants for efficacy against the parasitic roundworms Onchocerca ochengi and Brugia pahangi. METHODS: The viability of O. ochengi adult worms was assessed using the MTT/formazan assay. Fully confluent monkey kidney epithelial cells (LLC-MK2) served as the feeder layer for the O. ochengi microfilariae (mfs) assays. Viability of the mfs was assessed by microscopic examination for mean motility scoring (relative to the negative control) every 24 h post addition of an extract. The Worminator system was used to test the effects of the extracts on adult B. pahangi motility, and mean motility units were determined for each worm. Cytotoxicity of the active extracts on N27 cells was assessed using the MTS assay. RESULTS: Extracts from D. oliveri and P. febrifugum were effective against the adult roundworms O. ochengi and B. pahangi. Interestingly, extracts showing macrofilaricidal activities against O. ochengi also showed activity against O. ochengi mfs. The hexane stem bark extract of D. oliveri (DOBHEX) was more selective for adult O. ochengi than for mfs, with a half maximal and 100% inhibitory concentration (IC50 and IC100, respectively) against adult O. ochengi of 13.9 and 31.3 µg/ml, respectively. The in vitro cytotoxicity of all active extracts on N27 cells showed selective toxicity for parasites (selectivity index > 1). Bioassay-guided fractionation of the extracts yielded fractions with activity against adult B. pahangi, thus confirming the presence of bioactive principles in the plant extracts. CONCLUSIONS: Our study supports the use of D. oliveri and P. febrifugum in the traditional treatment of onchocerciasis and LF. The further purification of active extracts from these plants could yield lead compounds for filarial drug discovery and development.

Comparison of conventional and non-invasive diagnostic tools for detecting Plasmodium falciparum infection in southwestern Cameroon: a cross-sectional study.

BACKGROUND: Malaria remains a significant health challenge in sub-Saharan Africa, with early diagnosis critical to reducing its morbidity and mortality. Despite the increasing Plasmodium spp. diagnostic capabilities, access to testing is limited in some cases by the almost absolute requirement for blood from potentially infected subjects as the only sample source for all conventional methods. A rapid test on non-invasive specimen with comparable performance to microscopy for the screening or diagnosis of all participants is invaluable. This study sought to compare conventional and non-invasive diagnostic tools for detecting Plasmodium falciparum. METHODS: This was a cross-sectional study, carried out between March and August 2019 to evaluate and compare the diagnostic performance of a PfHRP2/pLDH-based malaria rapid diagnostic test (mRDT) on patients' blood, saliva and urine relative to conventional light microscopy and nested PCR at outpatient clinics in the Buea and Tiko health districts of Southwestern Cameroon. The significance of differences in proportions was explored using the Pearson's χ2 test whereas differences in group means were assessed using analyses of variance. RESULTS: A total of 359 individuals of both sexes, aged 1-92 years, were enrolled into the study. Of the 301 individuals tested by light microscopy and mRDTs on blood, saliva and urine, 84 (27.9%), 81 (26.9%), 87 (28.9%) and 107 (35.5%) respectively were positive. However, only 34.3%, 90.5%, 91.4%, 83.9% and 65.4% febrile, light microscopy and mRDT positives on blood, saliva and urine respectively had P. falciparum infection as confirmed by PCR. The sensitivity and specificity of presumptive diagnosis, light microscopy and mRDT on blood, saliva and urine were 86.9% and 19.7%, 77.8% and 96.1%, 75.8% and 96.6%, 74.5% and 93.1%, and 70.7% and 81.8%, respectively. The agreement between mRDT on saliva (k = 0.696) and microscopy (k = 0.766) compared to PCR was good. CONCLUSION: The study highlighted the low performance of presumptive diagnosis, reinforcing the need for parasitological tests prior to antimalarial therapy. The higher PfHRP2/pLDH mRDT parasite detection rates and sensitivity in saliva compared to urine suggests that the former is a practical adjunct to or alternative worth optimising for the routine diagnosis of malaria. Flow chart for diagnosis of P. falciparum infection by light microscopy, rapid diagnostic tests and nested PCR.

Occupational use of agrochemicals results in inhibited cholinesterase activity and altered reproductive hormone levels in male farmers from Buea, Cameroon.

The efficiency of agro pesticides and fertilizers in eliminating pests and scaling up crop yield has motivated farmers to increase their use. Unfortunately, health hazards caused on farmers by these agrochemicals are of growing concern, though not well elucidated. In order to evaluate the effects of occupational exposure to agrochemicals on some key parameters of male farmers' health in Buea Subdivision, Cameroon, a total of 101 men, including 62 farmers using the agrochemicals and a reference population of 39 men not involved in occupational utilization of the agrochemicals, were interviewed on use of protective equipment, exposure symptoms and reproductive health status. Thereafter, serum cholinesterase [acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)] activities, total antioxidant capacity and reproductive hormones [follicle-stimulating hormone (FSH), luteinizing hormone and testosterone] were assessed. Results revealed that farmers mainly used insecticides followed by fungicides, herbicides and fertilizers, but with inadequate protective measures. The use of agrochemicals resulted in several exposure symptoms including weakness, itches, burning sensation, headache, sneezing, coughing and vomiting, as well as decrease in serum AChE activity when compared to the reference population. The agrochemicals impacted negatively on the farmers' reproductive health as evidenced by increased FSH levels. Taken altogether, these results suggested that exposure to agrochemicals adversely affects farmers' health. Therefore, there is a need to further sensitize the farmers on the use of protective equipment to mitigate the exposure and resulting health hazards.

An Integrated Approach to Identify New Anti-Filarial Leads to Treat River Blindness, a Neglected Tropical Disease.

Filarial worms cause multiple debilitating diseases in millions of people worldwide, including river blindness. Currently available drugs reduce transmission by killing larvae (microfilariae), but there are no effective cures targeting the adult parasites (macrofilaricides) which survive and reproduce in the host for very long periods. To identify effective macrofilaricides, we carried out phenotypic screening of a library of 2121 approved drugs for clinical use against adult Brugia pahangi and prioritized the hits for further studies by integrating those results with a computational prioritization of drugs and associated targets. This resulted in the identification of 18 hits with anti-macrofilaricidal activity, of which two classes, azoles and aspartic protease inhibitors, were further expanded upon. Follow up screening against Onchocerca spp. (adult Onchocerca ochengi and pre-adult O. volvulus) confirmed activity for 13 drugs (the majority having IC50 < 10 µM), and a counter screen of a subset against L. loa microfilariae showed the potential to identify selective drugs that prevent adverse events when co-infected individuals are treated. Stage specific activity was also observed. Many of these drugs are amenable to structural optimization, and also have known canonical targets, making them promising candidates for further optimization that can lead to identifying and characterizing novel anti-macrofilarial drugs.

Alkaloids with Anti-Onchocercal Activity from Voacanga africana Stapf (Apocynaceae): Identification and Molecular Modeling.

A new iboga-vobasine-type isomeric bisindole alkaloid named voacamine A (1), along with eight known compounds-voacangine (2), voacristine (3), coronaridine (4), tabernanthine (5), iboxygaine (6), voacamine (7), voacorine (8) and conoduramine (9)-were isolated from the stem bark of Voacangaafricana. The structures of the compounds were determined by comprehensive spectroscopic analyses. Compounds 1, 2, 3, 4, 6, 7 and 8 were found to inhibit the motility of both the microfilariae (Mf) and adult male worms of Onchocerca ochengi, in a dose-dependent manner, but were only moderately active on the adult female worms upon biochemical assessment at 30 µM drug concentrations. The IC50 values of the isolates are 2.49-5.49 µM for microfilariae and 3.45-17.87 µM for adult males. Homology modeling was used to generate a 3D model of the O. ochengi thioredoxin reductase target and docking simulation, followed by molecular dynamics and binding free energy calculations attempted to offer an explanation of the anti-onchocercal structure-activity relationship (SAR) of the isolated compounds. These alkaloids are new potential leads for the development of antifilarial drugs. The results of this study validate the traditional use of V. africana in the treatment of human onchocerciasis.

Antischistosomal, antionchocercal and antitrypanosomal potentials of some Ghanaian traditional medicines and their constituents.

BACKGROUND: Ghana is endemic for some neglected tropical diseases (NTDs) including schistosomiasis, onchocerciasis and lymphatic filariasis. The major intervention for these diseases is mass drug administration of a few repeatedly recycled drugs which is a cause for major concern due to reduced efficacy of the drugs and the emergence of drug resistance. Evidently, new treatments are needed urgently. Medicinal plants, on the other hand, have a reputable history as important sources of potent therapeutic agents in the treatment of various diseases among African populations, Ghana inclusively, and provide very useful starting points for the discovery of much-needed new or alternative drugs. METHODOLOGY/PRINCIPAL FINDINGS: In this study, extracts of fifteen traditional medicines used for treating various NTDs in local communities were screened in vitro for efficacy against schistosomiasis, onchocerciasis and African trypanosomiasis. Two extracts, NTD-B4-DCM and NTD-B7-DCM, prepared from traditional medicines used to treat schistosomiasis, displayed the highest activity (IC50 = 30.5 µg/mL and 30.8 µg/mL, respectively) against Schistosoma mansoni adult worms. NTD-B2-DCM, also obtained from an antischistosomal remedy, was the most active against female and male adult Onchocera ochengi worms (IC50 = 76.2 µg/mL and 76.7 µg/mL, respectively). Antitrypanosomal assay of the extracts against Trypanosoma brucei brucei gave the most promising results (IC50 = 5.63 µg/mL to 18.71 µg/mL). Incidentally, NTD-B4-DCM and NTD-B2-DCM, also exhibited the greatest antitrypanosomal activities (IC50 = 5.63 µg/mL and 7.12 µg/mL, respectively). Following the favourable outcome of the antitrypanosomal screening, this assay was selected for bioactivity-guided fractionation. NTD-B4-DCM, the most active extract, was fractionated and subsequent isolation of bioactive constituents led to an eupatoriochromene-rich oil (42.6%) which was 1.3-fold (IC50 <0.0977 µg/mL) more active than the standard antitrypanosomal drug, diminazene aceturate (IC50 = 0.13 µg/mL). CONCLUSION/SIGNIFICANCE: These findings justify the use of traditional medicines and demonstrate their prospects towards NTDs drug discovery.

Repurposing Auranofin and Evaluation of a New Gold(I) Compound for the Search of Treatment of Human and Cattle Parasitic Diseases: From Protozoa to Helminth Infections.

Neglected parasitic diseases remain a major public health issue worldwide, especially in tropical and subtropical areas. Human parasite diversity is very large, ranging from protozoa to worms. In most cases, more effective and new drugs are urgently needed. Previous studies indicated that the gold(I) drug auranofin (Ridaura®) is effective against several parasites. Among new gold(I) complexes, the phosphole-containing gold(I) complex {1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl (abbreviated as GoPI) is an irreversible inhibitor of both purified human glutathione and thioredoxin reductases. GoPI-sugar is a novel 1-thio-ß-d-glucopyranose 2,3,4,6-tetraacetato-S-derivative that is a chimera of the structures of GoPI and auranofin, designed to improve stability and bioavailability of GoPI. These metal-ligand complexes are of particular interest because of their combined abilities to irreversibly target the essential dithiol/selenol catalytic pair of selenium-dependent thioredoxin reductase activity, and to kill cells from breast and brain tumors. In this work, screening of various parasites-protozoans, trematodes, and nematodes-was undertaken to determine the in vitro killing activity of GoPI-sugar compared to auranofin. GoPI-sugar was found to efficiently kill intramacrophagic Leishmania donovani amastigotes and adult filarial and trematode worms.

Evaluation of the performance of an in-house duplex PCR assay targeting the IS6110 and rpoB genes for tuberculosis diagnosis in Cameroon.

BACKGROUND: Tuberculosis (TB) remains a major public health concern in many low-income countries accounting for approximately two-thirds of deaths in people living with human immunodeficiency virus (HIV) infection. With prompt, accurate and appropriate treatment, almost all TB disease can be cured. The present study was to evaluate the diagnostic performance of an in-house duplex PCR (D-PCR) using IS1610 and rpoB specific primers in sputum samples from TB suspected patients. METHODS: A hospital-based cross-sectional study was conducted at the Limbe and Buea Regional Hospitals of the South West Region of Cameroon from June 2016 to April 2017. Sputum samples, decontaminated with hypertonic saline/sodium hydroxide solution were centrifuged and pellets processed for smear microscopy, culture and DNA extraction. Suspected inhibition was resolved by serial dilution of genomic DNA. Results were compared to culture as gold standard as well as a Composite Reference Standard (CRS). RESULTS: A total of 129 participants aged between 5 to 82 years were enrolled in to the study. The median age of the participants was 37 years (interquartile range, IQR: 27-50 years), with 54.3% being male. Forty-seven samples (36.4%) were positive by direct sputum microscopy, 49 (38%) by microscopy after concentration, 51 (39.5%) by culture and 62 (40.1%) by D-PCR. PCR inhibition was resolved in 85.7% (18/21) of the samples that had inhibition. The overall sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios and area under the curve AUC) of the D-PCR was 93.5, 94, 94, 94%, 15.6, 0.005 and 89.0% respectively using CRS as reference. The sensitivities of D-PCR observed among different sample categories were 95.7, 87.5 and 87.5% for smear-and culture-positives, smear-negative/culture-positive, and clinically diagnosed cases respectively. CONCLUSION: IS1610 and rpoB duplex PCR using relatively cheap decontamination and DNA extraction methods in addition to simple serial dilutions to resolve PCR inhibition shows high sensitivity in the diagnosis of paucibacillary tuberculosis.

A 6.5-kb intergenic structural variation enhances P450-mediated resistance to pyrethroids in malaria vectors lowering bed net efficacy.

Elucidating the complex evolutionary armory that mosquitoes deploy against insecticides is crucial to maintain the effectiveness of insecticide-based interventions. Here, we deciphered the role of a 6.5-kb structural variation (SV) in driving cytochrome P450-mediated pyrethroid resistance in the malaria vector, Anopheles funestus. Whole-genome pooled sequencing detected an intergenic 6.5-kb SV between duplicated CYP6P9a/b P450s in pyrethroid-resistant mosquitoes through a translocation event. Promoter analysis revealed a 17.5-fold higher activity (p < .0001) for the SV- carrying fragment than the SV- free one. Quantitative real-time PCR expression profiling of CYP6P9a/b for each SV genotype supported its role as an enhancer because SV+/SV+ homozygote mosquitoes had a significantly greater expression for both genes than heterozygotes SV+/SV- (1.7- to 2-fold) and homozygotes SV-/SV- (4-to 5-fold). Designing a PCR assay revealed a strong association between this SV and pyrethroid resistance (SV+/SV+ vs. SV-/SV-; odds ratio [OR] = 2,079.4, p < .001). The 6.5-kb SV is present at high frequency in southern Africa (80%-100%) but absent in East/Central/West Africa. Experimental hut trials revealed that homozygote SV mosquitoes had a significantly greater chance to survive exposure to pyrethroid-treated nets (OR 27.7; p < .0001) and to blood feed than susceptible mosquitoes. Furthermore, mosquitoes homozygote-resistant at the three loci (SV+/CYP6P9a_R/CYP6P9b_R) exhibited a higher resistance level, leading to a far superior ability to survive exposure to nets than those homozygotes susceptible at the three loci, revealing a strong additive effect. This study highlights the important role of structural variations in the development of insecticide resistance in malaria vectors and their detrimental impact on the effectiveness of pyrethroid-based nets.

Evaluation of the Effects of Agro Pesticides Use on Liver and Kidney Function in Farmers from Buea, Cameroon.

Agro pesticides are increasingly used worldwide to increase crop production. However, health hazards resulting from human exposure to these chemicals, especially from agricultural areas of developing countries have been a growing concern. The objective of this study was to evaluate the impact of occupational exposure to agro pesticides on the health of farmers in the Buea subdivision, which is one of the major agrarian areas in Cameroon. The study was transversal and involved 90 participants including 58 farmers using pesticides and a reference population of 32 men not involved in occupational use of agro pesticides. The participants were interviewed on agro pesticide use and their health status. Thereafter, blood samples were collected from the participants and used for the assessment of biochemical markers of the liver (alanine aminotransferase and aspartate aminotransferase) and the kidney (creatinine and uric acid) function. Results revealed that farmers frequently used insecticides, fungicides, and herbicides in their farming activities. Farmers reported several acute health symptoms related to pesticides use with the common ones being skin rash, eye irritation, and face burn. When compared to the reference population, the farmers showed significantly elevated (p < 0.01) alanine aminotransferase activity. However, other parameters investigated were not affected significantly. These results suggested that farmers were exposed to 3 different classes of agro pesticides, which induced eye and skin affections. Pesticides exposure resulted in alterations of the liver function hence the increased serum alanine aminotransferase activity. Therefore, there is a need to sensitize the farmers on toxicity and liver alteration potential of agro pesticides and the importance of appropriate protective equipment that may minimize exposure.

Genetic diversity and antiretroviral resistance-associated mutation profile of treated and naive HIV-1 infected patients from the Northwest and Southwest regions of Cameroon.

BACKGROUND: Antiretroviral therapy (ART) has improved the survival of HIV infected persons. However, rapid scale-up of ART and the high HIV-1 genetic variability, has greatly influenced the emergence of drug-resistant strains. This constitutes a potential threat to achieving the UNAIDS' 90-90-90 goals by 2020. We investigated the prevalent HIV-1 genotypes, drug resistance-associated mutations and assessed some predictors of the occurrence of these mutations. METHODS: This was a hospital-based cross-sectional study conducted between October 2010 and June 2012. Participants were consecutively enrolled from selected HIV treatment centers of the Southwest and Northwest regions of Cameroon. Viral load was determined with the automated Abbott Real-time HIV-1 m2000rt System. HIV genotyping and antiretroviral resistance mutations analysis were performed using Bayer's HIV-1 TRUGENE™ Genotyping Kit and OpenGene DNA Sequencing system. The drug resistance mutation was interpreted with the Stanford HIV database. Epidemiological data were obtained using pre-tested semi-structured questionnaires. RESULTS: Of the 387 participants, 239 were successfully genotyped. The median age of these participants was 33 years (interquartile range, IQR: 28-40 years), and a majority (65.7%) were female. A total of 29.3% of the participants were receiving ART. The median duration of ART was 10.5 months (IQR: 4-17.25 months). The median CD4 count and log10 viral load of study participants were 353.5 cells/ml (IQR:145-471) and 4.89 copies/ml (IQR: 3.91-5.55) respectively. CRF02 (A/G) (69%) was the most prevalent subtype followed by G (8.2%) and F (6.7%). Overall, resistance mutations were present in 37.1% of ART-experienced and 10.7% of ART-naive patients. Nucleoside reverse transcriptase inhibitors (NRTI) mutations occurred in 30% of ART-experienced and 2.4% of ART-naïve patients, while non-nucleoside reverse transcriptase inhibitors (NNRTI) mutations occurred in 34.2% of ART-experienced and 10.1% of -naïve patients. M184V (8.4%, 20/239) and K103N (5.4%, 13/239) were the most prevalent mutations. Major protease inhibitor mutations occurred in 3 (1.3%) out of the 239 sequences. The duration of ART independently predicted the occurrence of resistance mutation among ART-experienced patients. CONCLUSION: The high resistance to NNRTIs, which are the main support to the backbone (NRTIs) first-line antiretroviral regimen in Cameroon, has prompted the need to rollout an integrase strand transfer inhibitor regimen (containing Dolutegravir) with a higher genetic barrier to resistance as the preferred first line regimen.

Development and validation of an Onchocerca ochengi adult male worm gerbil model for macrofilaricidal drug screening.

BACKGROUND: Onchocerciasis currently afflicts an estimated 15 million people and is the second leading infectious cause of blindness world-wide. The development of a macrofilaricide to cure the disease has been hindered by the lack of appropriate small laboratory animal models. This study therefore, was aimed at developing and validating the Mongolian gerbil, as an Onchocerca ochengi (the closest in phylogeny to O. volvulus) adult male worm model. METHODOLOGY/PRINCIPAL FINDINGS: Mongolian gerbils (Meriones unguiculatus) were each implanted with 20 O. ochengi male worms (collected from infected cattle), in the peritoneum. Following drug or placebo treatments, the implanted worms were recovered from the animals and analyzed for burden, motility and viability. Worm recovery in control gerbils was on average 35%, with 89% of the worms being 100% motile. Treatment of the gerbils implanted with male worms with flubendazole (FBZ) resulted in a significant reduction (p = 0.0021) in worm burden (6.0% versus 27.8% in the control animals); all recovered worms from the treated group had 0% worm motility versus 91.1% motility in control animals. FBZ treatment had similar results even after four different experiments. Using this model, we tested a related drug, oxfendazole (OFZ), and found it to also significantly (p = 0.0097) affect worm motility (22.7% versus 95.0% in the control group). CONCLUSIONS/SIGNIFICANCE: We have developed and validated a novel gerbil O. ochengi adult male worm model for testing new macrofilaricidal drugs in vivo. It was also used to determine the efficacy of oxfendazole in vivo.