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Expression and Evaluation of Wb-SXP-1 and Wb-123 Recombinant Antigens as Potential Diagnostic Biomarkers for Lymphatic Filariasis

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American Journal of Molecular Biology 2023年第2期13卷 95-112页

作者： Sharlene Kerubo Mageto rebecca wanjiku waihenya Anne Wanjiru Mwangi Peter Kipkemboi Rotich Matthew Mutinda Munyao Tonny Teya Robinson Mugasiali Irekwa Joanne Jepkemei Yego Caroline Wangui Njoroge Grace Ng’endo Kanyita Nicole Sian Tanchu Dawala Koromtili Oumar Primrose Muthoni Ndungu Samson Muuo Nzou Department of Molecular Biology and Biotechnology Pan-African University Institute of Basic Sciences Technology and Innovation Nairobi Kenya Zoology Department Jomo Kenyatta University for Agriculture and Technology Nairobi Kenya Nagasaki University Institute of Tropical Medicine Kenya Medical Research Institute (NUITM-KEMRI) Nairobi Kenya Production Department Kenya Medical Research Institute Nairobi Kenya Centre for Microbiology Research Kenya Medical Research Institute Nairobi Kenya Department of Biochemistry Microbiology and Biotechnology Kenyatta University Nairobi Kenya Department of Pharmacology Clinical Pharmacy and Pharmacy Practice University of Nairobi Nairobi Kenya

Lymphatic filariasis (LF) remains a public health concern as it can cause permanent morbidity and disability to those infected. While the global elimination of LF in these endemic areas is ongoing through mass drug administration, there is the need to develop diagnostic tools that would be utilized to track the progress of total global eradication as well as perform surveillance for the recurrence of lymphatic filariasis transmission. Currently, approved LF diagnosis tools are faced with lack of specificity, low sensitivity, and periodicity dependence. Recombinant filarial antigen-based assays can address these drawbacks and offer practical instruments for LF diagnosis and surveillance. This present study, evaluated rWb-SXP-1 and rWb-123 antigens as potential diagnostic biomarker tools for Wuchereria banchrofti in human sera using microspheres-based multiplex serological assay. Based on statistical analysis using XLSTAT 2019 (Addinsoft) on data generated from multiplex technology assay, generated ROC curves for both rWb-SXP-1 and rWb-123 demonstrated 87.1% sensitivity to Wuchereria banchrofti human sera with rWb-SXP-1 antigens having the highest specificity of 96%. Indication that rWb-SXP-1 and rWb-123 antigens are capable of detecting immunoglobulin G4 (IgG4) antibodies in human sera synthesized specifically against W. banchrofti infections. Therefore, rWb-SXP-1 and rWb-123 antigens can be utilized to detect W. banchrofti infections by antibody profiling with excellent diagnostic sensitivity and specificity using microsphere-based multiplex serological tests. This method can be particularly practical for screening a large number of sera samples and/or for quick, extensive field-testing due to the high-throughput and quick formats applied.

关键词： Lymphatic Filariasis Recombinant Antigens Diagnosis

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Investigation of Plasmodium falciparum Resistance Biomarkers among Primary School Children in Western Kenya

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American Journal of Molecular Biology 2022年第2期12卷 43-53页

作者： Otilmoi Poul Stephen Tonny Teya Nyandwaro Robinson Mugasiali Irekwa rebecca wanjiku waihenya Matthew Mutinda Munyao Peter Kipkemboi Rotich Caroline Wangui Njoroge Anne Wanjiru Mwangi Joanne Jepkemei Yego Nicole Sian Tanchu Dawala Koromtili Oumar Grace Ngendo Kanyita Primrose Muthoni Ndungu Samson Muuo Nzou Department of Molecular Biology and Biotechnology Pan-African University Institute of Basic Sciences Technology and Innovation Nairobi Kenya Innovation Technology Transfer Department Kenya Medical Research Institute (ITTD-KEMRI) Nairobi Kenya Zoology Department School of Biological Sciences College of Pure and Applied Sciences Jomo Kenyatta University of Agriculture and Technology Nairobi Kenya Production Department KEMRI Nairobi Kenya Centre for Microbiology Research Kenya Medical Research Institute Nairobi Kenya Nagasaki University Institute of Tropical Medicine Kenya Medical Research Institute (NUITM-KEMRI) Nairobi Kenya Department of Biochemistry Microbiology and Biotechnology Kenyatta University Nairobi Kenya Department of Pharmacology and Pharmacognosy University of Nairobi Nairobi Kenya

Background: A marked decrease in malaria-related deaths worldwide has been attributed to the administration of effective antimalarials against Plasmodium falciparum. However, the continuous spread of P. falciparum resistance to anti-malarial drugs is raising a serious problem in controlling Malaria to the vulnerable children’s immune system. In recent studies, Plasmodium falciparum Kelch 13 propeller gene (Pfk13) has been reported to develop resistance to artemisinin in South Asia. In this study, we checked Plasmodium falciparum chloroquine resistance transporter gene (Pfcrt) involved in chloroquine (CQ) resistance. Method: In this study, archived 280 samples were collected from Alupe primary school children in Busia, Western Kenya from May, 2016 to November, 2016. Genomic DNA was extracted using the MightyPrep reagent. The samples were investigated for P. falciparum positivity out of which 67 of them tested positive giving a prevalence rate of 24%. The sixty-seven were subjected to PCR amplification for the molecular marker resistance to Pfcrt. After PCR amplification, the amplicons were purified and sequenced using Sanger Sequencing. The sequence data were analyzed using BioEdit software to identify point mutations. Results: 14 samples sequences were analyzed on Bioedit software giving the following amino acid changes F76C, Y66H, L70A, Y58C, T59V, V65I, P67L, T81L, Y60S, Y66S, P67T and I71F). New mutations have been reported at position 76 leading to an amino acid change, one of Pfcrt gold standard biomarkers. However, amino acid changes Y66H, L70A, Y58C, T59V, V65I, P67L, T81L, Y60S, Y66S, P67T and I71F are newly reported giving an increase in Pfcrt prevalence of concern from zero to 5.0%. A phylogenetic evolutionary relationship was constructed as shown below. Generally, the results showed a continuous resistance of *** to Pfcrt which calls for robust continuous monitoring and surveillance. Conclusion: Due to the increase of the resistant Pfcrt gene pre

关键词： P. falciparum Chloroquine Drug Resistance Malaria P. falciparum Chloroquine Resistance Transporter Gene Mutations

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Use of Polyclonal Antibody for the Diagnosis of Human African Trypanosomiasis

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American Journal of Molecular Biology 2023年第2期13卷 127-139页

作者： Dawala Koromtili Oumar Matthew Mutinda Munyao Anne Wanjiru Mwangi rebecca wanjiku waihenya Peter Kipkemboi Rotich Robinson Mugasiali Irekwa Tonny Teya Nyandwaro Caroline Wangui Njoroge Joanne Jepkemei Yego Primrose Muthoni Ndungu Sharlene Kerubo Mageto Damaris Mutethya Kilei Otilmoi Poul Stephen Nicole Sian Tanchu Grace Ngendo Kanyita Shingo Inoue Lucy Gitau Samson Muuo Nzou Department of Molecular Biology and Biotechnology Pan-African University Institute of Basic Sciences Technology and Innovation Nairobi Kenya Centre for Microbiology Research Kenya Medical Research Institute Nairobi Kenya Nagasaki University Institute of Tropical Medicine-Kenya Medical Research Institute Project Nairobi Kenya Production Department Kenya Medical Research Institute Nairobi Kenya Zoology Department Jomo Kenyatta University for Agriculture and Technology Nairobi Kenya Department of Pharmacology and Pharmacognosy University of Nairobi Nairobi Kenya Department of Microbiology Jomo Kenyatta University for Agriculture and Technology Nairobi Kenya Department of Biochemistry Microbiology and Biotechnology Kenyatta University Nairobi Kenya Center for Biotechnology Research and Development Kenya Medical Research Institute Nairobi Kenya

Human African trypanosomiasis (HAT), commonly known as sleeping sickness is one of the neglected tropical diseases (NTDs), which is fatal if left untreated. Its diagnosis is a challenge since the signs and symptoms of the primary phase are not specific, the existing diagnostic methods have low sensitivity and specificity, and the available drugs have some toxicity. New, robust, and cost-effective techniques are needed for the early identification of parasites. This study aimed to assess the sensitivity and specificity of two different types of polyclonal antibodies against T. b. gambiense using antigen detection ELISA. Polyclonal antibodies against the expressed proteins Tbg I2 and Tbg I17 were produced using New Zealand white rabbits. The antibody titer measured was greater than 32 g/L after the 3rd immunization for the expressed protein Tbg I2. For the expressed protein Tbg I17, the antibody titer measured was greater than 32 g/L after the 4th immunization. The sensitivity and specificity of the Tbg I2 polyclonal antibody confirmed with Polymerase Chain Reaction (PCR) as gold standard were respectively 89.5% and 80.6%, while for the Tbg I17 polyclonal antibody, the sensitivity and specificity were respectively 92.1% and 88.9%. The area under the curve for the Tbg I2 polyclonal antibody was 0.90 ± 0.032, while for the Tbg I17 polyclonal antibody, the area under the curve was 0.92 ± 0.0. The Tbg I17 polyclonal antibody produced in New Zealand white rabbits has good sensitivity and good specificity;it can be successfully used in the diagnosis of HAT.

关键词： Human African Trypanosomiasis Polyclonal Antibody Tbg I2 Expressed Protein Tbg I17 Expressed Protein Sensitivity Specificity

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Epidemiology andinteractions of Human Immunodeficiency Virus - 1 and Schistosoma mansoni in sub-SaharanAfrica

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Infectious Diseases of Poverty 2013年第1期2卷 6-16页

作者： Humphrey D Mazigo Fred Nuwaha Shona Wilson Safari M Kinunghi Domenica Morona rebecca waihenya Jorg Heukelbach David W Dunne Department of Medical Parasitology and Entomology School of MedicineCathollc University of Health and Aled SclencesP.O.Box 1464MwanzaTanzanla Department of Environmental Health and Communicable Disease Control School of Public HealthCollege of Health SclencesMakerere UniversiyP.O.Box 7072KampalaUganda National Institute for Medical.Research Mwanza Research CentreP.Q.Box 1462MwanzaTanzanla Department of Pathology University of CambidgeTennis Court RoadCambridge CB21OPUK Department of Zoology Faculty of SclenceJomo Kenyatta University of Agriculture and TechnologyP.O.Box ***NairobiKenya Department of Community Health School of MedicineFederal University of CearaCearaBail

Human Immunodeficiency Virus-1/AIDS and Schis tosoma mansoni are widespread in sub Saharan Africa and co-infection occurs *** the early 1990s,it has been suggested that the two infections may interact and potentiate the effects of each other within co-infected human ***,S mansoni infection has been suggested to be a risk factor for HIV transmission and progression in *** so,it would follow that mass deworming could have beneficial effects on HIV-1 transmission *** epidemiology of HIV in African countries is changing,shifting from urban to rural areas where the prevalence of Schistosoma mansoni is high and public health services are *** the other side,the consequent pathogenesis of HIV-1/*** co-infection remains *** we give an account of the epidemiology of HIV-1 and ***,discuss co-infection and possible biological causal relationships between the two infections,and the potential impact of praziquantel treatment on HIV-1 viral loads,CD4^(+) counts and CD4^(+)/CD8^(+) *** review of the available literature indicates that there is evidence to support the hypothesis that *** infections can influence the replication of the HIV-1,cel-to-cell transmission,as well as increase HIV progression as measured by reduced CD4^(+) T lymphocytes *** so,then deworming of HIV positive individuals living in endemic areas may impact on HV-1 viral loads and CD4^(+) T lymphocyte counts.

关键词： Schitosoma mansoni HIV-1 Co-infections Immunological interactions Deworming

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Perforin Expression in <i>Theileria parva</i>Specific Cytotoxic T Cells Correlates with Cytotoxicity

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Open Journal of Immunology 2014年第3期4卷 117-127页

作者： Jerome Wendoh rebecca waihenya Rosemary Saya Elias Awino Vishvannath Nene Lucilla Steinaa International Livestock Research Institute Nairobi Kenya Jomo Kenyatta University of Agriculture and Technology Nairobi Kenya Kenya Medical Research Institute Nairobi Kenya

Theileria parva is a protozoan parasite that causes the disease East Coast fever in cattle which results in major economic losses in Eastern, Central and Southern Africa. Efforts to generate vaccines involve measurements of cytotoxic activity since CD8 cells are believed to be responsible for protection of the animals. CTL assays are time consuming, and may require use of radioactive material and they also impose a considerable amount of in vitro work, which may skew the response compared to ex vivo assays. Hence it would be beneficial to identify other markers that correlate with the cytotoxicity. In this in vitro study we examined if the number of tetramer positive CD8 cells and the staining intensity of these correlated with lysis of the target cells. Furthermore, we investigated if the expression of the activation marker perforin correlated with the cytotoxicity. Perforin is involved in permeabilization of the cell membrane of the target cell. Bulk CTL lines and purified CD8 cell lines generated from cattle of the A18 BoLA (MHC) type were analysed for the Theileria parva specific immune responses using a peptide-MHC tetramer and antibodies for perforinin FACS analysis. Thelysis of target cells was determined by a chromium release assay. Results demonstrate that the percentage of tetramer positive cells, in six cell lines generated against the whole parasite, correlate with killing of PBMC pulsed with the peptide. The product of the percentages of perforin and tetramer double positive cells and the net MFI of perforin showed a perfect correlation with the cytotoxicity of the peptide pulsed PBMC. Likewise, the product of percentage perforin positive cells and the staining intensity had the best significant correlation with killing of the pulsed PBMC. The present results suggest that perforin could be a possible biomarker for the cytotoxicity to Theileria parva infections/immunizations.

关键词： Cytotoxic T Cells Biomarkers Perforin Theileria parva

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HIV-1 Drug Resistance Mutations in Patients Failing 1st Line Therapy in a Comprehensive Care Center in Nairobi, Kenya

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World Journal of AIDS 2015年第2期5卷 83-89页

作者： Elizabeth Luvai rebecca waihenya James Munyao Lucy Sanguli Christina Mwachari Samoel Khamadi Institute of Tropical Medicine and Infectious Diseases (ITROMID) Jomo Kenyatta University of Agriculture and Technology Juja Kenya Jomo Kenyatta University of Agriculture and Technology Juja Kenya Kenya Medical Research Institute Nairobi Kenya

Background: HIV-1 drug resistance is an emerging challenge for HIV-1 infected clients who are on antiretroviral therapy (ART). In Kenya, as in many other developing countries, ART is now accesible to clients who need it. However, they must be done a CD4 test first and if the count is <300, then ART is commenced. With the initiation of ART comes the challenge of adherence to medication, a factor that is impacted greatly by the understanding of the client of the importance?of adherence and the financial ability to keep their appointments, especially if the clients come from a distant location. Objective: To identify HIV-1 drug resistance mutations inclientsfailing1st line antiretroviral therapy in Nairobi, Kenya. Methodology: A cross sectional study was carried out where whole blood samples were collected from clients attending a HIV care and treatment clinic in Nairobi. Clients who had been on ART for more than 6 months and had a viral load greater than 1000 were enrolled in the study. A total of 52 client samples were successfully sequenced in the reverse transcriptase region and analyzed. Results: After analysis of the generated sequences, it was seen that 43 (82.6%) of the clients had HIV-1 drug resistance mutations conferring resistance to one or more nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse-transcriptase inhibitors (NNRTIs). Majority of the clients (46%) were infected with HIV-1 subtype A viruses. Conclusion: The findings of the study showed that a significant proportion of the clients on ART had developed resistance mutations to one or more drugs that are used as 1st line therapy in Kenya. There is need for continuous education of the population on importance of adherence to medication. There is also need for clinicians to be trained on using viral load and HIV drug resistance testing, where available, as methods of monitoring treatment failure so that clients can be switched to alternative medication immediately the need a

关键词： HIV-1 Treatment Failure Antiretroviral Therapy Drug Resistance Subtype

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Evaluation of the TbgI_(2) and TbgI_(17) Tandem Repeat Antigens as Potential Antigens for the Diagnosis of Trypanosoma brucei rhodesiense

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Open Journal of Clinical Diagnostics 2019年第4期9卷 152-163页

作者： Vanessa Irumva rebecca waihenya Anne Wanjiru Mwangi Peter Kipkemboi Satoshi Kaneko Tonny Teya Joanne Jepkemei Caroline Njoroge Joseph Wambugu Gitari Matthew Mutinda Munyao Robinson MIrekwa Murima Ng’ang’a Samson Muuo Nzou Department of Molecular Biology and Biotechnology Pan-African University Institute of Basic SciencesTechnology and InnovationNairobiKenya Zoology Department Jomo Kenyatta University for Agriculture and TechnologyNairobiKenya Nagasaki University Institute of Tropical Medicine-Kenya Medical Research Institute Project NairobiKenya Production Department-Kenya Medical Research Institute NairobiKenya Institute of Tropical Medicine Eco-Epidemiology DepartmentNagasaki UniversityNagasakiJapan Division of Vector Borne and Neglected Tropical Diseases.Ministry of Health NairobiKenya Centre for Microbiology Research Kenya Medical Research InstituteBusiaKenya

Human African trypanosomiasis (HAT) affects up to half a million people every year in sub-Saharan Africa. Interruption of transmission of the disease by early diagnosis and treatment is core to the control and eventual elimination of HAT. The routine diagnostic method for HAT is light microscopy of blood samples. The present study sought to evaluate the potential of TbgI2 and TbgI17 tandem repeat antigens as candidates for the diagnosis of Trypanosoma brucei rhodesiense. The expressed proteins were purified and the antigenic reactivity evaluation was done using multiplex assay using sera obtained from HAT patients. Receiver operating characteristic analysis showed that recombinant antigen, TbgI2 had high sensitivity for sera from patients infected with T. b. rhodesiense with the area under the curve being 0.577 and a sensitivity of 0.641 and specificity 0.650. The results suggest that TbgI2 is a potential biomarker for T. b. rhodesiense HAT serodiagnostic tests.

关键词： Human African Trypanosomiasis TbgI2 TbgI17 Antigens Diagnosis Trypanosoma brucei rhodesiense

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