Assessment of the capacity of Whatman filter papers as support to store stools for the molecular diagnostic testing of soil-transmitted helminthiasis.

Storage of stools for the detection of soil-transmitted helminths (STH) remains challenging for the molecular diagnostic testing of STH infections. This study aimed to overcome this challenge by assessing the capacity of Whatman filter papers to store stools for the molecular detection of STHs. Stool samples were collected from school-aged children of soil-transmitted helminthiasis endemic areas of Cameroon and then, analysed using Kato Katz technique. For this study, 128 and 40 stool samples respectively with and without STH eggs were analysed. From each sample, 10, 20, 40 and 80 mg of stool were weighted and spread on 6 grades of Whatman filter papers that were stored at room temperature from one to ten weeks. DNA was extracted from spread stool using CTAB based-method. The amount of stool to spread on filter papers and the grade of filter paper offering good storage were determined by amplifying specific DNA fragments of Ascaris lumbricoides. The capacity of filter papers to store stool samples for several weeks before the molecular detection of STH species was assessed by amplifying specific DNA fragments of different STHs. The amplification rates of A. lumbricoides were significantly higher (P < 0.0001) for 10 and 20 mg of stored stools. Stools spread on Whatman paper grade 2 yielded the highest amplification rate of 100% for A. lumbricoides, T. trichiura and hookworm. PCR revealed STH infections in all the 128 spread stools carrying STH eggs. It also revealed Necator americanus and Ancylostoma duodenale respectively in 10 and 13 of 15 spread stools contained hookworm eggs. PCR confirmed the co-infections of these hookworm species as well as that of A. lumbricoides and Trichuris trichiura in 7 spread stools. Out of 40 stools without STH eggs, PCR revealed that 5 (12.5%) and 9 (22.5%) had respectively A. lumbricoides and T. trichiura infections. The amplification rate of each STH species was 100% from one to 8 weeks and decreased to 86.7% after 10 weeks of storage. This study highlighted the capacity of filter papers to store stools for the molecular detection of STHs. Storing stools on these papers will enable to monitor and evaluate control programs and ensure post-elimination surveillance.

Trypanosome infections in animals from tsetse infected areas of Cameroon and their sensitivity and resistance molecular profiles for diminazene aceturate and isometamidium chloride.

Monitoring and assessment of control strategies for African trypanosomoses' elimination require not only updating data on trypanosome infections, but also to have an overview on the molecular profiles of trypanocides resistance in different epidemiological settings. This study was designed to determine, in animals from six tsetse-infested areas of Cameroon, the prevalence of trypanosome infections as well as the diminazene aceturate (DA) and isometamidium chloride (ISM) sensitivity/resistance molecular profiles of these trypanosomes. From 2016 to 2019, blood was collected in pigs, dogs, sheep, goats and cattle from six tsetse infested areas of Cameroon. DNA was extracted from blood and trypanosome species were identified by PCR. The sensitivity/resistance molecular profiles of trypanosomes to DA and ISM were investigated using PCR-RFLP. From 1343 blood samples collected, Trypanosoma vivax, Trypanosoma congolense forest and savannah, Trypanosoma theileri and trypanosomes of the sub-genus Trypanozoon were identified. The overall prevalence of trypanosome infections was 18.7%. These prevalence vary between trypanosome species, animal taxa, within and between sampling sites. Trypanosoma theileri was the predominant species with an infection rate of 12.1%. Trypanosomes showing resistant molecular profiles for ISM and DA were identified in animals from Tibati (2.7% for ISM and 65.6% for DA) and Kontcha (0.3% for ISM and 6.2% for DA). No trypanosome carrying resistant molecular profile for any of the two trypanocides was detected in animals from Fontem, Campo, Bipindi and Touboro. Mixed molecular profiles of sensitive/resistant trypanosomes were detected in animals from Tibati and Kontcha. Results of this study highlighted the presence of various trypanosome species as well as parasites carrying sensitive/resistant molecular profiles for DA and ISM in animals of tsetse infested areas of Cameroon. They indicate that the control strategies must be adapted according to epidemiological settings. The diversity of trypanosomes indicates that AAT remains a serious threat for animal breeding and animal health in these tsetse infested areas.

Assessment of cetyl-trimethyl-ammonium bromide (CTAB) based method for the extraction of soil-transmitted helminth DNAs from stools for molecular dagnostic of soil-transmitted helminthiasis.

Although several protocols were developed to extract DNA for soil-transmitted helminthiasis diagnostic, amplifying these extracts remains challenging due to DNA polymerase inhibitors. This study aimed to assess a DNA extraction method for efficient detection of soil-transmitted helminth species by determining stool mass and the type of DNA polymerase that can be used for this extraction method. For this study, 141 stool samples harbouring soil-transmitted eggs and 50 samples without egg were obtained from school-aged children of Makenene in the Centre region of Cameroon. DNA was extracted from 10, 20, 40 and 80 mg of stool using commercial kit and/or cetyltrimethylammonium bromide (CTAB)-based method. The amount of stool for molecular diagnostic of soil-transmitted helminthiasis was determined by amplifying Ascaris lumbricoides DNA. The performances of three DNA polymerases and CTAB-based method were assessed by amplifying DNA of different soil-transmitted helminth species. For this study, 94 stools with A. lumbricoides eggs, 39 with Trichuris trichuria and 15 with hookworm were analyzed. DNA of A. lumbricoides, T. trichuria, Necator americanus and Ancylostoma duodenale were detected in 97.9% of extracts from stools harbouring soil-transmitted helminth eggs. Soil-transmitted helminth DNAs were significantly (X2 = 17.66; df = 3; p ã€ˆ00001) more amplified in extracts from 10 and 20 mg than those from 40 and 80 mg. The amplification rate with "Q5 high fidelity DNA polymerase" was significantly (X2 = 30.54; df = 2; p < 0.00001) higher than that of other DNA polymerases. Multiplex-PCR confirmed co-infections of A. lumbricoides with either T. trichuria or N. americanus. The extraction cost for the CTAB-based method was $1.45. This method appearedis reliable and 3 times cost effective than commercial kit. Its combination with the "Q5 high fidelity DNA polymerase" may improve soil-transmitted helminthiasis diagnostic.

Fine mapping of Ascaris lumbricoides, Trichuris trichiura and hookworm infections in sub-districts of Makenene in Centre Region of Cameroun.

Preventive chemotherapy (PC) that remains the main control strategy recommended by the World Health Organization to achieve the elimination of soil-transmitted helminth (STH) infections as a public health problem must be strengthened by identifying the remaining transmission hot-spots for the deployment of appropriate control measures. This study was designed to assess the prevalence and infections intensities of soil-transmitted helminths and perform micro scale mapping in order to identify transmission hot-spots for targeted control operations. Stool samples were collected from 1775 children in ten primary schools of eight sub-districts of Makenene in Cameroon. Kato Katz technique was used to process and examine stool samples to detect the eggs of soil-transmitted nematodes. The prevalence of soil-transmitted helminth species as well as the infection intensities was compared. Data visualizations in forms of maps were made using Quantum geographic information system (QGIS) software. The overall prevalence of soil-transmitted helminth infections was 4.8% with a 95% confidence interval (CI) of 3.8-5.9%: 3.0% (95% CI 2.2-3.9) for Ascaris lumbricoides, 1.4% (95% CI 0.9-2.0) for Trichuris trichiura and 0.8% (95% CI 0.5-1.4) for hookworms. The prevalence of soil-transmitted helminth species differ significantly between schools and sub-districts. The intensity of infections was light (2.4%, 1.1% and 0.8%), moderate (0.4%, 0.1% and 0.1%) and heavy (0.2%, 0.2% and 0%) for A. lumbricoides, T. trichiura and hookworm respectively. The mean intensity of infections was 7255 EPG for A. lumbricoides, 2900 EPG for T. trichiura and 298 EPG for hookworm. Between schools, significant difference was recorded in the means of infection intensities of T. Trichiura and hookworms but not for A. lumbricoides. This difference was also significant for T. Trichiura when comparison were between sex. No significant difference were recorded when the comparison were between age. Fine mapping revealed that children harbouring heavy infections were clustered in the same sub-districts; highlighting the presence of high endemicity sub-districts and hot-spots for the transmission of different soil-transmitted helminth species. This study showed a diversity in the prevalence and transmission of different soil-transmitted helminth species. It also hightlighted the need for micro scale mapping to enable the localisation of high endemicity sub-districts and transmission hot-spot sites where targeted control operations must be deployed to achieve STH elimination.

Association between IL1 gene polymorphism and human African trypanosomiasis in populations of sleeping sickness foci of southern Cameroon.

BACKGROUND: Human African Trypanosomiasis (HAT) is a neglected tropical disease caused by infections due to Trypanosoma brucei subspecies. In addition to the well-established environmental and behavioural risks of becoming infected, there is evidence for a genetic component to the response to trypanosome infection. We undertook a candidate gene case-control study to investigate genetic associations further. METHODOLOGY: We genotyped one polymorphism in each of seven genes (IL1A, IL1RN, IL4RN, IL6, HP, HPR, and HLA-G) in 73 cases and 250 controls collected from 19 ethno-linguistic subgroups stratified into three major ethno-linguistic groups, 2 pooled ethno-linguistic groups and 11 ethno-linguistic subgroups from three Cameroonian HAT foci. The seven polymorphic loci tested consisted of three SNPs, three variable numbers of tandem repeat (VNTR) and one INDEL. RESULTS: We found that the genotype (TT) and minor allele (T) of IL1A gene as well as the genotype 1A3A of IL1RN were associated with an increased risk of getting Trypanosoma brucei gambiense and develop HAT when all data were analysed together and also when stratified by the three major ethno-linguistic groups, 2 pooled ethno-linguistic subgroups and 11 ethno-linguistic subgroups. CONCLUSION: This study revealed that one SNP rs1800794 of IL1A and one VNTR rs2234663 of IL1RN were associated with the increased risk to be infected by Trypanosoma brucei gambiense and develop sleeping sickness in southern Cameroon. The minor allele T and the genotype TT of SNP rs1800794 in IL1A as well as the genotype 1A3A of IL1RN rs2234663 VNTR seem to increase the risk of getting Trypanosoma brucei gambiense infections and develop sleeping sickness in southern Cameroon.

Molecular characterization of lower vaginal swabs for Human papilloma virus in association with Chlamydia trachomatis infection in Cameroonian Women.

Human papilloma virus (HPV) infection is an etiological factor for cervical cancer development and Chlamydia trachomatis (Ct) is considered as a cofactor. Understanding the dynamics of HPV and Ct infection could help to explain the incidence of early onset of cervical cancer (CC) observed in Cameroon. Lower vaginal swabs and sera from sexually active women were analyzed for HPV and Ct infection in association with risk factors. Questionnaires were used to document patients' lifestyle and risk factors. A total of 206 women participated in the study average 28.1±8years (16-50 years). HPV prevalence was 23.3% with subtypes 16 and 18 at respectively 2.9% and 1%. Ct infection totalised 40.8%, of which 23.8% were HPV- Ct co-infections. HPV infection was inversely associated with age (p=0.028). We found a positive association between Ct infection and the number of sex partners (p=0.012) and a negative association with parity (p=0.032). There was no significant association between HPV and Ct infections. High rates of HPV and Ct infections could be an indicator of cervical cancer risk in the near future. There is therefore an urgent need for sensitization as well as implementation of appropriate preventive measures.

Genetic structure of Trypanosoma congolense "forest type" circulating in domestic animals and tsetse flies in the South-West region of Cameroon.

Despite the economic impact of trypanosome infections, few investigations have been undertaken on the population genetics and transmission dynamics of animal trypanosomes. In this study, microsatellite markers were used to investigate the population genetics of Trypanosoma congolense "forest type", with the ultimate goal of understanding its transmission dynamics between tsetse flies and domestic animals. Blood samples were collected from pigs, sheep, goats and dogs in five villages in Fontem, South-West region of Cameroon. In these villages, tsetse were captured, dissected and their mid-guts collected. DNA was extracted from blood and tsetse mid-guts and specific primers were used to identify T. congolense "forest type". All positive samples were genetically characterized with seven microsatellite markers. Genetic analyses were performed on samples showing single infections of T. congolense "forest type". Of the 299 blood samples, 137 (46%) were infected by T. congolense "forest type". About 3% (54/1596) of tsetse fly mid-guts were infected by T. congolense "forest type". Of 182 samples with T. congolense "forest type", 52 were excluded from the genetic analysis. The genetic analysis on the 130 remaining samples revealed polymorphism within and between subpopulations of the target trypanosome. The dendrogram of genetic similarities was subdivided into two clusters and three sub-clusters, indicating one major and several minor genotypes of T. congolense "forest type" in tsetse and domestic animals. The low FSTvalues suggest low genetic differentiation and no sub-structuration within subpopulations. The same T. congolense genotypes appear to circulate in tsetse and domestic animals.

A polymorphism in the haptoglobin, haptoglobin related protein locus is associated with risk of human sleeping sickness within Cameroonian populations.

BACKGROUND: Human African Trypanosomiasis (HAT) is a neglected disease targeted for elimination as a public health problem by 2020. Elimination requires a better understanding of the epidemiology and clinical evolution of HAT. In addition to the classical clinical evolution of HAT, asymptomatic carriers and spontaneous cure have been reported in West Africa. A genetic component to human susceptibility to HAT has been suggested to explain these newly observed responses to infection. In order to test for genetic associations with infection response, genetic polymorphism in 17 genes were tested (APOL1, IL1B, IL4, IL4R, IL6, IL8, IL12B, IL12RB1, IL10, TNFA, INFG, MIF, HLA-G, HLA-A, HP, HPR and CFH). METHODOLOGY: A case-control study was performed on 180 blood samples collected from 56 cases and 124 controls from Cameroon. DNA was extracted from blood samples. After quality control, 25 samples (24 controls and 1 case) were eliminated. The genotyping undertaken on 155 individuals including 55 cases and 100 controls were investigated at 96 loci (88 SNPs and 8 indels) located on 17 genes. Associations between these loci and HAT were estimated via a case-control association test. RESULTS: Analyses of 64 SNPs and 4 indels out of 96 identified in the selected genes reveal that the minor allele (T) of rs8062041 in haptoglobin (HP) appeared to be protective against HAT (p = 0.0002395, OR 0.359 (CI95 [0.204-0.6319])); indicating higher frequency in cases compared to controls. This minor allele with adjusted p value of 0.0163 is associated with a lower risk (protective effect) of developing sleeping sickness. CONCLUSION: The haptoglobin related protein HPR and HP are tightly linked and both are duplicated in some people and may lead to higher activity. This increased production could be responsible of the protection associated with rs8062041 even though this SNP is within HP.

Population genetics of forest type of Trypanosoma congolense circulating in Glossina palpalis palpalis of Fontem in the South-West region of Cameroon.

BACKGROUND: Genetic variation of microsatellite loci is a widely used method for the analysis of population genetic structure of several organisms. To improve our knowledge on the population genetics of trypanosomes, Trypanosoma congolense forest and savannah types were identified in the mid-guts of Glossina palpalis palpalis caught in five villages of Fontem in the South-West region of Cameroon. From the positive samples of Trypanosoma congolense forest, the genetic diversity and the population genetic structure of these parasites were evaluated. METHOD: For this study, pyramidal traps were set up during three entomological surveys and 3347 tsetse flies were collected, dissected and 1903 midguts collected. DNA was extracted from midguts and specific primers were used to identify Trypanosoma congolense forest and savannah. All Trypanosoma congolense forest positive samples were characterized with seven microsatellite markers. RESULTS: Microscopic examination revealed 25 (1.31%) mid-gut infections with trypanosomes while the PCR method identified 120 (6.3%) infections due to Trypanosoma congolense: 94 (78.33%) Trypanosoma congolense forest and 28 (21.77%) Trypanosoma congolense savannah. The trypanosome infection rates varied significantly between villages and years of capture. Menji recorded the highest infection rate (15.11%); and samples captured in 2009 were more infected (14.33%). The microsatellite markers revealed a genetic variability between Trypanosoma congolense forest populations of Fontem villages and 6.38% of mixed infections due to different genotypes of T. congolense "forest type". CONCLUSION: Our data on the population genetics play in favor of a clonal reproduction of this parasite. The microsatellite markers used here showed a low genetic differentiation and an absence of sub-structuration (FST ≤ 0.0003) between Trypanosoma congolense forest populations of Fontem villages. However, the high FST value (FST ≥ 0.3911) between samples of the Democratic Republic of Congo and those of Fontem villages indicates low migration rates between trypanosomes of these subpopulations.

Substituted 2-aminothiophenes: antifungal activities and effect on Microsporum gypseum protein profile.

The increasing recognition and importance of fungal infections, the difficulties encountered in their treatment and the increase in resistance to antifungal agents have stimulated the search for therapeutic alternatives. The objective of this study was to evaluate the antifungal activities of three substituted 2-aminothiophenes (1, 2 and 3) against some fungal species. The synthesis of substituted 2-aminothiophenes was carried out through the most versatile synthetic method developed by Gewald et al. The antifungal activity was performed against yeast, dermatophytes and Aspergillus species using the broth microdilution method. The effect of these aminothiophenes was examined on the protein content and profile. Compound 2 was the most active (MIC varying from 2.00 to 128 µg ml(-1) ). All the three substituted 2-aminothiophenes had a relatively important dose-dependent effect on Microsporum gypseum protein profile and content. These compounds affected the structure and dye fixation of macroconidia of this fungus. The overall results indicate that the tested substituted 2-aminothiophenes can be used as precursors for new antifungal drugs development.