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المؤلفون: Pati Pyana, Veerle Lejon, Ipos Ngay Lukusa, Nick Van Reet, Erick Mwamba Miaka, Philippe Büscher, Felix Akwaso, Dieudonné Mumba Ngoyi, Wilfried Mutombo, Lewis Kaninda, Medard Ilunga, Justin Masumu, Vincent Kobo, Sandra Rembry, Antoine Tarral, Olaf Valverde Mordt, Digas Ngolo, Sylvain Mutanda, Dieudonné Mpoyi Muamba
المصدر: PLoS Neglected Tropical Diseases, Vol 15, Iss 9, p e0009739 (2021)
PLoS Neglected Tropical Diseasesمصطلحات موضوعية: Physiology, Trypanosoma brucei gambiense, RC955-962, Nervous System, law.invention, Cerebrospinal fluid, Medical Conditions, law, Zoonoses, Arctic medicine. Tropical medicine, Medicine and Health Sciences, African trypanosomiasis, Polymerase chain reaction, Cerebrospinal Fluid, Protozoans, Eukaryota, Body Fluids, Blood, Infectious Diseases, Democratic Republic of the Congo, RNA extraction, Lymph, Anatomy, Public aspects of medicine, RA1-1270, RNA, Protozoan, Research Article, Neglected Tropical Diseases, Trypanosoma, African Trypanosomiasis, Extraction techniques, Trypanosomiasis, medicine, Parasitic Diseases, Humans, Protozoan Infections, business.industry, Public Health, Environmental and Occupational Health, Organisms, RNA, Biology and Life Sciences, medicine.disease, Tropical Diseases, Virology, Reverse transcriptase, Parasitic Protozoans, Research and analysis methods, Trypanosomiasis, African, Parasitology, business
الوصف: Background Spliced Leader (SL) trypanosome RNA is detectable only in the presence of live trypanosomes, is abundant and the Trypanozoon subgenus has a unique sequence. As previously shown in blood from Guinean human African trypanosomiasis (HAT) patients, SL-RNA is an accurate target for diagnosis. Detection of SL-RNA in the cerebrospinal fluid (CSF) has never been attempted. In a large group of Congolese gambiense HAT patients, the present study aims i) to confirm the sensitivity of SL-RNA detection in the blood and; ii) to assess the diagnostic performance of SL-RNA compared to trypanosome detection in CSF. Methodology/Principal findings Blood and CSF from 97 confirmed gambiense HAT patients from the Democratic Republic of Congo were collected using PAXgene blood RNA Tubes. Before RNA extraction, specimens were supplemented with internal extraction control RNA to monitor the extraction, which was performed with a PAXgene Blood RNA Kit. SL-RNA qPCR was carried out with and without reverse transcriptase to monitor DNA contamination. In blood, 92/97 (94.8%) HAT patients tested SL-RNA positive, which was significantly more than combined trypanosome detection in lymph and blood (78/97 positive, 80.4%, p = 0.001). Of 96 CSF RNA specimens, 65 (67.7%) were SL-RNA positive, but there was no significant difference between sensitivity of SL-RNA and trypanosome detection in CSF. The contribution of DNA to the Cq values was negligible. In CSF with normal cell counts, a fraction of SL-RNA might have been lost during extraction as indicated by higher internal extraction control Cq values. Conclusions/Significance Detection of SL-RNA in blood and CSF allows sensitive demonstration of active gambiense HAT infection, even if trypanosomes remain undetectable in blood or lymph. As this condition often occurs in treatment failures, SL-RNA detection in blood and CSF for early detection of relapses after treatment deserves further investigation. Trial registration This study was an integral part of the diagnostic trial "New Diagnostic Tools for Elimination of Sleeping Sickness and Clinical Trials: Early tests of Cure" (DiTECT-HAT-WP4, ClinicalTrials.gov Identifier: NCT03112655).
Author summary Human African trypanosomiasis is a parasitic infection occurring in sub-Saharan Africa, which is fatal if left untreated. Diagnosis relies on demonstration of trypanosomes, which may occur at such low concentrations that they remain microscopically undetectable. Nucleic acid detection offers an alternative, in particular RNA, which is unstable and a better marker for live organisms than DNA. Trypanosomal SL-RNA detection in blood by reverse transcriptase quantitative PCR has hitherto only been tested twice. Although in cerebrospinal fluid, trypanosome presence indicates brain infection, SL-RNA detection has never been attempted. We evaluated sensitivity of SL-RNA detection in blood and cerebrospinal fluid. For each specimen, 2 controls were included: presence of genomic DNA contamination and efficacy of RNA extraction. Sensitivity of SL-RNA detection in blood was higher than of combined blood and lymph microscopy. In cerebrospinal fluid, SL-RNA and trypanosome detection had similar sensitivity. In a few specimens, traces of DNA were amplified. In some cerebrospinal fluids, some RNA was lost during extraction. Performing both internal controls is crucial, to ensure that negative SL-RNA cerebrospinal fluid findings are not due to a failed extraction and, in particular when testing treated patients, to differentiate live parasite RNA from reminiscent DNA. -
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المؤلفون: Barkissa Mélika Traoré, Mathurin Koffi, Martial Kassi N'Djetchi, Dramane Kaba, Jacques Kaboré, Hamidou Ilboudo, Bernadin Ahouty Ahouty, Minayégninrin Koné, Bamoro Coulibaly, Thomas Konan, Adeline Segard, Lingué Kouakou, Thierry De Meeûs, Sophie Ravel, Philippe Solano, Jean-Mathieu Bart, Vincent Jamonneau
المصدر: PLoS Neglected Tropical Diseases
PLoS Neglected Tropical Diseases, Vol 15, Iss 12, p e0010036 (2021)مصطلحات موضوعية: Trypanosoma, Swine, Trypanosoma congolense, Physiology, Trypanosoma brucei gambiense, RC955-962, Artificial Gene Amplification and Extension, Research and Analysis Methods, Polymerase Chain Reaction, African Trypanosomiasis, Medical Conditions, Trypanosomiasis, Arctic medicine. Tropical medicine, Zoonoses, parasitic diseases, Medicine and Health Sciences, Parasitic Diseases, Trypanosoma Brucei, Animals, Humans, Molecular Biology Techniques, Molecular Biology, Disease Reservoirs, Swine Diseases, Mammals, Protozoans, Protozoan Infections, Organisms, Biology and Life Sciences, Eukaryota, Tropical Diseases, Parasitic Protozoans, Body Fluids, Cote d'Ivoire, Trypanosomiasis, African, Infectious Diseases, Blood, Animals, Domestic, Vertebrates, Amniotes, Parasitology, Public aspects of medicine, RA1-1270, Anatomy, Zoology, Research Article, Neglected Tropical Diseases
الوصف: Background The existence of an animal reservoir of Trypanosoma brucei gambiense (T. b. gambiense), the agent of human African trypanosomiasis (HAT), may compromise the interruption of transmission targeted by World Health Organization. The aim of this study was to investigate the presence of trypanosomes in pigs and people in the Vavoua HAT historical focus where cases were still diagnosed in the early 2010’s. Methods For the human survey, we used the CATT, mini-anion exchange centrifugation technique and immune trypanolysis tests. For the animal survey, the buffy coat technique was also used as well as the PCR using Trypanosoma species specific, including the T. b. gambiense TgsGP detection using single round and nested PCRs, performed from animal blood samples and from strains isolated from subjects positive for parasitological investigations. Results No HAT cases were detected among 345 people tested. A total of 167 pigs were investigated. Free-ranging pigs appeared significantly more infected than pigs in pen. Over 70% of free-ranging pigs were positive for CATT and parasitological investigations and 27–43% were positive to trypanolysis depending on the antigen used. T. brucei was the most prevalent species (57%) followed by T. congolense (24%). Blood sample extracted DNA of T. brucei positive subjects were negative to single round TgsGP PCR. However, 1/22 and 6/22 isolated strains were positive with single round and nested TgsGP PCRs, respectively. Discussion Free-ranging pigs were identified as a multi-reservoir of T. brucei and/or T. congolense with mixed infections of different strains. This trypanosome diversity hinders the easy and direct detection of T. b. gambiense. We highlight the lack of tools to prove or exclude with certainty the presence of T. b. gambiense. This study once more highlights the need of technical improvements to explore the role of animals in the epidemiology of HAT.
Author summary Significant efforts to control human African trypanosomiasis (HAT) since the 1990’s have drastically reduced the prevalence of the disease. Its elimination as a public health problem is being achieved. World Health Organization now targets the interruption of transmission for 2030. However, potential animal reservoirs of Trypanosoma brucei gambiense (T. b. gambiense), the main agent of HAT, may compromise this ambitious objective. It is the case in the Vavoua historical focus in Côte d’Ivoire where HAT cases were still diagnosed in the early 2010’s. During a study conducted in this area, we scrutinized the trypanosomes circulating in pigs and people sharing the same environment using serological, immunological, parasitological and molecular tools. No HAT cases were detected. We showed that T. brucei s.l. and T. congolense actively circulated in free-ranging pigs. Even if no tools were sensitive and specific enough to unambiguously identify T. b. gambiense directly from biological samples, six isolated strains from pigs positive for trypanosomes were amplified for TgsGP, the only currently accepted T. b. gambiense specific molecular marker. The apparent discrepancies between the presence of T. b. gambiense in pigs despite the absence of human cases is discussed. These results stress the need for an efficient “molecular toolbox” to easily detect and identify T. b. gambiense in any animal it may infect. -
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المؤلفون: Isabel Saldanha, Lucas J. Cunningham, Emily R. Adams, Stephen J. Torr, Gala Garrod, Jessica K. Lingley
المصدر: PLoS Neglected Tropical Diseases
PLoS Neglected Tropical Diseases, Vol 14, Iss 11, p e0008308 (2020)مصطلحات موضوعية: Trypanosoma brucei rhodesiense, Epidemiology, Trypanosoma brucei gambiense, RC955-962, Artificial Gene Amplification and Extension, Disease Vectors, Nucleic Acid Denaturation, Polymerase Chain Reaction, law.invention, Medical Conditions, law, Limit of Detection, Arctic medicine. Tropical medicine, Zoonoses, Medicine and Health Sciences, Mass Screening, Multiplex, African trypanosomiasis, Polymerase chain reaction, Protozoans, education.field_of_study, biology, Eukaryota, Insects, Infectious Diseases, Public aspects of medicine, RA1-1270, Research Article, Neglected Tropical Diseases, Trypanosoma, Glossina, Arthropoda, Infectious Disease Control, Tsetse Flies, Tsetse Fly, Population, Trypanosoma brucei, Research and Analysis Methods, Real-Time Polymerase Chain Reaction, Proof of Concept Study, African Trypanosomiasis, Trypanosomiasis, parasitic diseases, medicine, Trypanosoma Brucei, Parasitic Diseases, Animals, Humans, education, Molecular Biology Techniques, Molecular Biology, DNA Primers, Protozoan Infections, Public Health, Environmental and Occupational Health, Organisms, Tsetse fly, Biology and Life Sciences, DNA, Protozoan, biology.organism_classification, medicine.disease, Tropical Diseases, Virology, Invertebrates, Parasitic Protozoans, Insect Vectors, Species Interactions, Trypanosomiasis, African, Medical Risk Factors, Zoology, Entomology
الوصف: Human African Trypanosomiasis (HAT) is a potentially fatal parasitic infection caused by the trypanosome sub-species Trypanosoma brucei gambiense and T. b. rhodesiense transmitted by tsetse flies. Currently, global HAT case numbers are reaching less than 1 case per 10,000 people in many disease foci. As such, there is a need for simple screening tools and strategies to replace active screening of the human population which can be maintained post-elimination for Gambian HAT and long-term for Rhodesian HAT. Here, we describe the proof of principle application of a novel high-resolution melt assay for the xenomonitoring of Trypanosoma brucei gambiense and T. b. rhodesiense in tsetse. Both novel and previously described primers which target species-specific single copy genes were used as part of a multiplex qPCR. An additional primer set was included in the multiplex to determine if samples had sufficient genomic material for detecting genes present in low copy number. The assay was evaluated on 96 wild-caught tsetse previously identified to be positive for T. brucei s. l. of which two were known to be positive for T. b. rhodesiense. The assay was found to be highly specific with no cross-reactivity with non-target trypanosome species and the assay limit of detection was 104 tryps/mL. The qPCR successfully identified three T. b. rhodesiense positive flies, in agreement with the reference species-specific PCRs. This assay provides an alternative to running multiple PCRs when screening for pathogenic sub-species of T. brucei s. l. and produces results in less than 2 hours, avoiding gel electrophoresis and subjective analysis. This method could provide a component of a simple and efficient method of screening large numbers of tsetse flies in known HAT foci or in areas at risk of recrudescence or threatened by the changing distribution of both forms of HAT.
Author summary With global cases of Human African Trypanosomiasis (HAT) reaching pre-elimination numbers, identifying areas of trypanosome transmission becomes increasingly important. With decreasing numbers of cases, the cost of identifying each positive case increases. Screening for trypanosomes in tsetse flies instead of in the human host allows for a faster, more cost-effective and efficient method of detecting areas in which parasite transmission is on-going. Molecular tools such as PCR play a critical role in molecular identification of parasites in vectors. However, at present, multiple PCRs are required to confirm the presence or absence of human infective trypanosomes. Here we present a novel assay which allows for the screening of both species of trypanosomes responsible for HAT in one assay. This method eliminates the need for multiple assays per sample, produces results which are easier to interpret, and reduces the risk of contamination of samples. We found the assay to be as sensitive as the current gold-standard PCR when evaluated on a subset of wild tsetse flies. With further field evaluation, this method could provide an alternative monitoring method for HAT, which can be used leading up to elimination and maintained once elimination has been achieved. -
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المؤلفون: Inaki Tirados, Lucas J. Cunningham, Johan Esterhuizen, Mercy A. Opiyo, Michael J. Lehane, Jessica K. Lingley, Clement T. N. Mangwiro, Stephen J. Torr
المصدر: PLoS Neglected Tropical Diseases, Vol 14, Iss 4, p e0007737 (2020)
PLoS Neglected Tropical Diseasesمصطلحات موضوعية: 0301 basic medicine, Disease reservoir, Veterinary medicine, Swine, Trypanosoma brucei gambiense, RC955-962, Artificial Gene Amplification and Extension, Polymerase Chain Reaction, 0302 clinical medicine, Zoonoses, Arctic medicine. Tropical medicine, Prevalence, Medicine and Health Sciences, African trypanosomiasis, Uganda, Protozoans, Mammals, education.field_of_study, Microscopy, Eukaryota, Ruminants, Blood, Infectious Diseases, Animals, Domestic, Vertebrates, Livestock, Topography, Medical, Public aspects of medicine, RA1-1270, Research Article, Neglected Tropical Diseases, Trypanosoma, Veterinary parasitology, Tsetse Flies, 030231 tropical medicine, Population, Biology, Trypanosoma brucei, Research and Analysis Methods, African Trypanosomiasis, 03 medical and health sciences, Trypanosomiasis, Bovines, parasitic diseases, medicine, Trypanosoma Brucei, Parasitic Diseases, Animals, Humans, education, Molecular Biology Techniques, Molecular Biology, Disease Reservoirs, Protozoan Infections, business.industry, Public Health, Environmental and Occupational Health, Organisms, Biology and Life Sciences, medicine.disease, biology.organism_classification, Tropical Diseases, Veterinary Parasitology, Parasitic Protozoans, 030104 developmental biology, Trypanosomiasis, African, Amniotes, Cattle, Parasitology, Veterinary Science, business
الوصف: Background Large-scale control of sleeping sickness has led to a decline in the number of cases of Gambian human African trypanosomiasis (g-HAT) to
Author summary The decline of annual cases of West-African sleeping sickness in Uganda raises the prospect that elimination of the disease is achievable for the country. However, with the decrease in incidence and the likely subsequent change in priorities there is a need to confirm that the disease is truly eliminated. One unanswered question is the role that domestic animals play in maintaining transmission of the disease. The potential of cryptic-animal reservoirs is a serious threat to successful and sustained elimination of the disease. It is with the intent of resolving this question that we have carried out this study whereby we examined 2088 cattle, 400 pigs and 2184 tsetse for Trypanosoma brucei gambiense, the parasite responsible for the disease. Our study found T. brucei s.l. in local cattle, pigs and tsetse flies, with their respective prevalences as follows, 1.9%, 6.3% and 1.8%. Further analysis to establish identity of these positives to the sub-species level found that no cattle, pigs or tsetse were carrying the pathogen responsible for Gambian sleeping sickness. Our work highlights the difficulty of establishing the absence of a disease, especially in an extremely low endemic setting, and the limitations of some of the most commonly used methods. -
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المؤلفون: Sylvain Biéler, Anne J. N. Kazibwe, Enock Matovu, Crispin Lumbala, Pascal Lutumba, Hakim Sendagire, Simon Kayembe, Joseph Mathu Ndung'u, Hypolite Muhindo Mavoko, Jean-Pierre Van Geertruyden, Joris Losimba Likwela
المصدر: PLoS Neglected Tropical Diseases, Vol 14, Iss 4, p e0008168 (2020)
PLoS neglected tropical diseases
PLoS Neglected Tropical Diseasesمصطلحات موضوعية: Male, 0301 basic medicine, Physiology, Trypanosoma brucei gambiense, RC955-962, Protozoan Proteins, Artificial Gene Amplification and Extension, Polymerase Chain Reaction, Geographical Locations, 0302 clinical medicine, Zoonoses, Arctic medicine. Tropical medicine, Medicine and Health Sciences, Uganda, African trypanosomiasis, Prospective Studies, Protozoans, Rapid diagnostic test, Plasma samples, biology, Eukaryota, Body Fluids, Blood, Infectious Diseases, Democratic Republic of the Congo, Engineering and Technology, Female, Anatomy, Public aspects of medicine, RA1-1270, Research Article, Neglected Tropical Diseases, Adult, Trypanosoma, medicine.medical_specialty, Adolescent, Plasmodium falciparum, 030231 tropical medicine, Antigens, Protozoan, Research and Analysis Methods, Sensitivity and Specificity, Blood Plasma, African Trypanosomiasis, Young Adult, 03 medical and health sciences, Trypanosomiasis, Internal medicine, parasitic diseases, Parasitic Diseases, medicine, Humans, Prototypes, Molecular Biology Techniques, Molecular Biology, Protozoan Infections, Diagnostic Tests, Routine, business.industry, Organisms, Public Health, Environmental and Occupational Health, Biology and Life Sciences, Tropical Diseases, medicine.disease, biology.organism_classification, equipment and supplies, Parasitic Protozoans, Malaria, Trypanosomiasis, African, Technology Development, 030104 developmental biology, People and Places, Africa, Combined test, Human medicine, business
الوصف: Background Malaria is endemic in all regions where gambiense or rhodesiense human African trypanosomiasis (HAT) is reported, and both diseases have similarities in their symptomatology. A combined test could be useful for both diseases and would facilitate integration of the screening for gambiense HAT (gHAT) and malaria diagnosis. This study aimed to evaluate a combined prototype rapid diagnostic test (RDT) for gHAT and malaria. Methods Blood samples were collected in the Democratic Republic of the Congo and in Uganda to evaluate the performance of a prototype HAT/Malaria Combined RDT in comparison to an individual malaria RDT based on Plasmodium falciparum (P.f.) Histidine Rich Protein II (HRP-II or HRP2) antigen (SD BIOLINE Malaria Ag P.f. RDT) for malaria detection and an individual gHAT RDT based on recombinant antigens, the SD BIOLINE HAT 2.0 RDT for HAT screening. Due to the current low prevalence of gHAT in endemic regions, the set of blood samples that were collected was used to evaluate the specificity of the RDTs for gHAT, and additional archived plasma samples were used to complete the evaluation of the HAT/Malaria Combined RDT in comparison to the HAT 2.0 RDT. Results Frozen whole blood samples from a total of 486 malaria cases and 239 non-malaria controls, as well as archived plasma samples from 246 gHAT positive and 246 gHAT negative individuals were tested. For malaria, the sensitivity and specificity of the malaria band in the HAT/Malaria Combined RDT were 96.9% (95% CI: 95.0–98.3) and 97.1% (95% CI: 94.1–98.8) respectively. The sensitivity and specificity of the SD BIOLINE malaria Ag P.f. RDT were 97.3% (95% CI: 95.5–98.6) and 97.1% (95% CI: 94.1–98.8) respectively. For gHAT, using archived plasma samples, the sensitivity and specificity were respectively 89% (95% CI: 84.4–92.6) and 93.5% (95% CI: 89.7–96.2) with the HAT/Malaria Combined RDT, and 88.2% (95% CI: 83.5–92) and 94.7% (95% CI: 91.1–97.2) with the HAT 2.0 RDT. Using the whole blood samples that were collected during the study, the specificity of the HAT/Malaria Combined RDT for gHAT was 95.8% (95% CI: 94.3–97.0). Conclusion The HAT/Malaria Combined prototype RDT was as accurate as the individual malaria or gHAT RDTs. The HAT/Malaria Combined prototype RDT is therefore suitable for both malaria diagnosis and gHAT screening. However, there is a need to assess its accuracy using fresh samples in prospective clinical trials.
Author summary The annual number of reported cases of human African trypanosomiasis (HAT), also known as sleeping sickness (SS), is currently below 1,000 cases worldwide. The Democratic Republic of the Congo (DRC), the most affected country, and Uganda, which shares a border with DRC, are both endemic for gambiense HAT (gHAT). The main strategy to control gHAT is screening of at-risk individuals, followed by diagnosis and treatment of confirmed cases. However, this strategy and even the passive screening as currently implemented become less efficient with declining incidence, justifying innovative strategies to efficiently detect the remaining cases. All areas where gHAT occurs are also endemic for malaria, presenting an opportunity to integrate gHAT screening activities within malaria control activities. This integration is warranted by the fact that in early disease stage, gHAT patients present with signs and symptoms strikingly similar to those of malaria. In order to use malaria diagnosis as an entry point to screen for gHAT, Standard Diagnostics (SD), Republic of Korea (now Abbott Diagnostics, Korea Inc–ADK) made a Combined prototype RDT for both malaria and gHAT, expected to be as accurate as the individual gHAT and malaria RDTs. In this study, we evaluated the accuracy of the Combined prototype RDT using whole blood samples collected in Uganda and DRC, and archived plasma samples collected in DRC, Angola and Central African Republic. We found that the Combined prototype performs just as well as individual RDTs. -
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المؤلفون: Elvis Ofon, Harry Noyes, Vincent Ebo'o Eyanga, Flobert Njiokou, Mathurin Koffi, Pythagore Fogue, Christiane Hertz-Fowler, Annette MacLeod, Enock Matovu, Gustave Simo, TrypanoGEN Research Group, as members of The H3Africa Consortium
المصدر: PLoS Neglected Tropical Diseases, Vol 13, Iss 3, p e0007283 (2019)
PLoS Neglected Tropical Diseases
PLOS NEGLECTED TROPICAL DISEASESمصطلحات موضوعية: 0301 basic medicine, Male, Heredity, Trypanosoma brucei gambiense, Artificial Gene Amplification and Extension, Polymerase Chain Reaction, Geographical Locations, 0302 clinical medicine, Zoonoses, Genotype, Medicine and Health Sciences, African trypanosomiasis, Cameroon, Child, Genetics, Aged, 80 and over, Protozoans, biology, lcsh:Public aspects of medicine, Neglected Diseases, Eukaryota, Middle Aged, 3. Good health, Genetic Mapping, Infectious Diseases, Female, Research Article, Neglected Tropical Diseases, Adult, Risk, Trypanosoma, lcsh:Arctic medicine. Tropical medicine, Adolescent, lcsh:RC955-962, 030231 tropical medicine, Single-nucleotide polymorphism, Variant Genotypes, Trypanosoma brucei, Research and Analysis Methods, Polymorphism, Single Nucleotide, African Trypanosomiasis, Molecular Genetics, 03 medical and health sciences, Young Adult, Trypanosomiasis, parasitic diseases, medicine, Parasitic Diseases, Humans, Genetic Predisposition to Disease, Molecular Biology Techniques, Molecular Biology, Alleles, Genetic Association Studies, Aged, Protozoan Infections, Public Health, Environmental and Occupational Health, Organisms, Biology and Life Sciences, lcsh:RA1-1270, medicine.disease, biology.organism_classification, Tropical Diseases, Parasitic Protozoans, Minor allele frequency, 030104 developmental biology, Trypanosomiasis, African, Genetic Loci, Case-Control Studies, People and Places, Africa, Gene polymorphism
الوصف: 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.
Author summary Human African Trypanosomiasis (HAT), or sleeping sickness, is a parasitic disease caused by flagellated parasites of the genus Trypanosoma. This disease has been included into the WHO roadmap for neglected tropical diseases with elimination as a public health problem targeted for 2020 and the interruption of transmission to humans for 2030. To achieve these elimination and interruption goals, it is important to identify and understand the factors that may hamper these goals. Understanding the contribution of human genetics to the response of trypanosome infections is important for the development of new control strategies. In this study, polymorphism in seven genes was investigated between controls and sleeping sickness patients of three sleeping sickness foci of Southern Cameroon in order to see if there is any association with the development of disease. Results of this study have shown that the genotype (TT) and minor allele (T) of IL1A gene and the genotype 1A3A VNTR of IL1RN are associated with an increased risk of getting T. b. gambiense infections and develop sleeping sickness in major ethno-linguistic groups of the Cameroonian population. They suggest that the association between host genetic determinants and the susceptibility to T. b. gambiense infections could vary according to the population studied. These results will improve our knowledge on the role of human genetics determinants and the risk to be infected by T. b. gambiense and develop sleeping sickness. They could thus lead to the identification of novel biomarkers which could open a frame work for the development of new diagnostics, treatments and intervention strategies.وصف الملف: application/pdf
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المؤلفون: Kyoko Hayashida, Junya Yamagishi, Peter Nambala, Naoko Kawai, Chihiro Sugimoto, Mable Mwale Mutengo, Janelisa Musaya, Philippe Büscher, Nick Van Reet, Boniface Namangala
المصدر: PLoS Neglected Tropical Diseases
PLoS Neglected Tropical Diseases, Vol 14, Iss 10, p e0008753 (2020)مصطلحات موضوعية: 0301 basic medicine, Trypanosoma brucei rhodesiense, Veterinary medicine, Malawi, Physiology, Trypanosoma brucei gambiense, RC955-962, Diagnostic accuracy, Artificial Gene Amplification and Extension, Disaccharides, Polymerase Chain Reaction, law.invention, 0302 clinical medicine, Medical Conditions, law, Arctic medicine. Tropical medicine, Zoonoses, Medicine and Health Sciences, African trypanosomiasis, DNA extraction, Polymerase chain reaction, Protozoans, Organic Compounds, Eukaryota, Body Fluids, Chemistry, Infectious Diseases, Blood, Molecular Diagnostic Techniques, Physical Sciences, Public aspects of medicine, RA1-1270, Anatomy, Nucleic Acid Amplification Techniques, Research Article, Neglected Tropical Diseases, Trypanosoma, Point-of-Care Systems, 030231 tropical medicine, Loop-mediated isothermal amplification, Carbohydrates, Research and Analysis Methods, Sensitivity and Specificity, African Trypanosomiasis, 03 medical and health sciences, Extraction techniques, Trypanosomiasis, Diagnostic Medicine, parasitic diseases, medicine, Parasitic Diseases, Animals, Humans, Molecular Biology Techniques, Molecular Biology, Protozoan Infections, business.industry, Disease progression, Organic Chemistry, Public Health, Environmental and Occupational Health, Organisms, Chemical Compounds, Biology and Life Sciences, Trehalose, DNA, Protozoan, medicine.disease, Tropical Diseases, Parasitic Protozoans, 030104 developmental biology, Trypanosomiasis, African, business
الوصف: Human African trypanosomiasis (HAT) is one of the neglected tropical diseases in sub-Saharan Africa. Early diagnosis and treatment prior to disease progression are crucial for the survival of HAT patients. We had previously established a loop-mediated isothermal amplification (LAMP) method for HAT diagnosis in which the reagents were dried for field-use purposes. In this study, we used a semi-automated process to produce the test tubes using a bio-inkjet printer to achieve an accurate production. The performance of the inkjet printer-produced dried LAMP test (CZC-LAMP) was found to be stable after storage for up to 180 days at 30 °C. The diagnostic accuracy of CZC-LAMP HAT was evaluated using DNA samples that were extracted from 116 Trypanosoma brucei gambiense patients and 66 T. b. rhodesiense patients. The sensitivity was 72% for T. b. gambiense (95%CI: 63%–80%) and 80% for T. b. rhodesiense (95%CI: 69%–89%). The specificity determined using DNA from 116 endemic control DNA samples was 95% (95%CI: 89%–98%). The performance of the CZC-LAMP HAT and CZC-LAMP rHAT were also evaluated using 14 crude blood lysate samples obtained from T. b. rhodesiense patients and endemic control samples collected from Rumphi District in Malawi. The sensitivity and specificity were both 100% (95%CI: 77%–100%). As the developed CZC-LAMP test does not require a cold chain or a sophisticated laboratory, it holds promise for use as a routine simple molecular tool for point-of-care HAT diagnosis in endemic areas.
Author summary Nucleic acid amplification methods allow for a sensitive detection and specific identification of a number of pathogens, including Trypanosoma parasites that cause human African trypanosomiasis (HAT). However, conventional molecular tools such as the polymerase chain reaction (PCR) are highly resource-demanding and time-consuming and are seldom used in HAT-endemic countries. The loop-mediated isothermal amplification method (LAMP) provides a promising alternative to the conventional PCR, as this technique can amplify nucleic acids under isothermal conditions using minimal resources within one hour. In this study, we established a protocol to produce a dried-format LAMP kit for the diagnosis of HAT, that used a bio-ink-jet printer machine. The produced test kit (CZC-LAMP HAT and CZC-LAMP rHAT) was determined to be stable for up to 180 days at room temperature. The test provided a good performance for the detection of the trypanosome parasites using both DNA and crude blood samples, and the test results were comparable to those obtained from PCR. The findings from this study suggest that the dried LAMP test can be useful for the diagnosis of HAT, particularly in areas where laboratory resources are limited. -
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المؤلفون: Lauren Gummery, Alexandra G. Raftery, D. G. M. Sutton, Euan Bennet, Jean Rodgers, Saloum Jallow
المصدر: PLoS ONE
Gummery, L, Jallow, Raftery, Bennet, E D S, Rodgers & Sutton 2020, ' Comparison of loop-mediated isothermal amplification (LAMP) and PCR for the diagnosis of infection withTrypanosoma bruceissp. in equids in The Gambia ', PLoS ONE, vol. 15, no. 8, e0237187 . https://doi.org/10.1371/journal.pone.0237187
PLoS ONE, Vol 15, Iss 8, p e0237187 (2020)مصطلحات موضوعية: Male, 0301 basic medicine, Veterinary medicine, Physiology, Surfactants, Artificial Gene Amplification and Extension, Polymerase Chain Reaction, law.invention, Medical Conditions, 0302 clinical medicine, law, Zoonoses, Medicine and Health Sciences, Prospective Studies, DNA extraction, Materials, Polymerase chain reaction, Whole blood, Protozoans, Multidisciplinary, Eukaryota, Meningoencephalitis, Body Fluids, Blood, Infectious Diseases, Molecular Diagnostic Techniques, Physical Sciences, Medicine, Female, Gambia, Anatomy, Nucleic Acid Amplification Techniques, Research Article, Trypanosoma, Science, Trypanosoma brucei brucei, Materials Science, Detergents, 030231 tropical medicine, Loop-mediated isothermal amplification, Context (language use), Biology, Trypanosoma brucei, Sensitivity and Specificity, 03 medical and health sciences, Extraction techniques, Trypanosomiasis, Trypanosoma Brucei, Parasitic Diseases, medicine, Animals, Horses, Molecular Biology Techniques, Molecular Biology, Protozoan Infections, Organisms, Biology and Life Sciences, Gold standard (test), DNA, Protozoan, medicine.disease, biology.organism_classification, Parasitic Protozoans, Research and analysis methods, Cross-Sectional Studies, Trypanosomiasis, African, 030104 developmental biology, Horse Diseases, Trypanosoma Brucei Gambiense
الوصف: Introduction:\ud Infection of equids with Trypanosoma brucei (T. brucei) ssp. is of socioeconomic importance across sub-Saharan Africa as the disease often progresses to cause fatal meningoencephalitis. Loop-mediated isothermal amplification (LAMP) has been developed as a cost-effective molecular diagnostic test and is potentially applicable for use in field-based laboratories.\ud \ud Part I:\ud Threshold levels for T. brucei ssp. detection by LAMP were determined using whole equine blood specimens spiked with known concentrations of parasites. Results were compared to OIE antemortem gold standard of T. brucei-PCR (TBR-PCR).\ud \ud Results I:\ud Threshold for detection of T. brucei ssp. on extracted DNA from whole blood was 1 parasite/ml blood for LAMP and TBR-PCR, and there was excellent agreement (14/15) between tests at high (1 x 103/ml) concentrations of parasites. Detection threshold was 100 parasites/ml using LAMP on whole blood (LWB). Threshold for LWB improved to 10 parasites/ml with detergent included. Performance was excellent for LAMP at high (1 x 103/ml) concentrations of parasites (15/15, 100%) but was variable at lower concentrations. Agreement between tests was weak to moderate, with the highest for TBR-PCR and LAMP on DNA extracted from whole blood (Cohen’s kappa 0.95, 95% CI 0.64–1.00).\ud \ud Part II:\ud A prospective cross-sectional study of working equids meeting clinical criteria for trypanosomiasis was undertaken in The Gambia. LAMP was evaluated against subsequent TBR-PCR.\ud \ud Results II:\ud Whole blood samples from 321 equids in The Gambia were processed under field conditions. There was weak agreement between LWB and TBR-PCR (Cohen’s kappa 0.34, 95% CI 0.19–0.49) but excellent agreement when testing CSF (100% agreement on 6 samples).\ud \ud Conclusions:\ud Findings support that LAMP is comparable to PCR when used on CSF samples in the field, an important tool for clinical decision making. Results suggest repeatability is low in animals with low parasitaemia. Negative samples should be interpreted in the context of clinical presentation.
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