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1.
Front Cell Infect Microbiol ; 11: 646467, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34084754

RESUMO

HIV-1 infected individuals under antiretroviral therapy can control viremia but often develop non-AIDS diseases such as cardiovascular and metabolic disorders. Gut microbiome dysbiosis has been indicated to be associated with progression of these diseases. Analyses of gut/fecal microbiome in individual regions are important for our understanding of pathogenesis in HIV-1 infections. However, data on gut/fecal microbiome has not yet been accumulated in West Africa. In the present study, we examined fecal microbiome compositions in HIV-1 infected adults in Ghana, where approximately two-thirds of infected adults are females. In a cross-sectional case-control study, age- and gender-matched HIV-1 infected adults (HIV+; n = 55) and seronegative controls (HIV-; n = 55) were enrolled. Alpha diversity of fecal microbiome in HIV+ was significantly reduced compared to HIV- and associated with CD4 counts. HIV+ showed reduction in varieties of bacteria including Faecalibacterium, the most abundant in seronegative controls, but enrichment of Proteobacteria. Ghanaian HIV+ exhibited enrichment of Dorea and Blautia; bacteria groups whose depletion has been reported in HIV-1 infected individuals in several other cohorts. Furthermore, HIV+ in our cohort exhibited a depletion of Prevotella, a genus whose enrichment has recently been shown in men having sex with men (MSM) regardless of HIV-1 status. The present study revealed the characteristics of dysbiotic fecal microbiome in HIV-1 infected adults in Ghana, a representative of West African populations.


Assuntos
Infecções por HIV , HIV-1 , Microbiota , Adulto , Estudos de Casos e Controles , Estudos Transversais , Disbiose , Feminino , Gana , Humanos , Masculino
2.
Jpn J Infect Dis ; 74(1): 42-47, 2021 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-32611986

RESUMO

Recent studies have indicated an association between gut microbiome composition and various disorders, including infectious diseases. The composition of the microbiome differs among ethnicities and countries, possibly resulting in diversified interactions between host immunity and the gut microbiome. Characterization of baseline microbiome composition in healthy people is an essential step for better understanding of the biological interactions associated with individual populations. However, data on the gut/fecal microbiome have not been accumulated for individuals in West Africa. In the present study, we examined the fecal microbiome composition in healthy adults in Ghana. Toward this, 16S rRNA gene libraries were prepared using bacterial fractions derived from 55 Ghanaian adults, which were then subjected to next-generation sequencing. The fecal microbiome of the Ghanaian adults was dominated by Firmicutes (Faecalibacterium, Subdoligranulum, and Ruminococcaceae UCG-014), Proteobacteria (Escherichia-Shigella and Klebsiella), and Bacteroidetes (Prevotella 9 and Bacteroides), consistent with previous observations in African cohorts. Further, our analysis revealed differences in microbiome composition and a lower diversity of the fecal microbiome in the Ghanaian cohort compared with those reported in non-African countries. This is the first study to describe substantial fecal microbiome data obtained using high-throughput metagenomic tools on samples derived from a cohort in Ghana. The data may provide a valuable basis for determining the association between the fecal microbiome and progression of various diseases in West African populations.


Assuntos
Fezes/microbiologia , Microbioma Gastrointestinal/genética , Adulto , Bacteroidetes/genética , Estudos Transversais , Feminino , Firmicutes/genética , Gana , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Metagenômica , Microbiota , Pessoa de Meia-Idade , Proteobactérias/genética , RNA Bacteriano/isolamento & purificação , RNA Ribossômico 16S/genética
3.
J Hum Genet ; 65(1): 35-40, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31582773

RESUMO

Infectious disease is still a major threat in the world today. Five decades ago, it was considered soon to be eradicated, but the adaptation of pathogens to environmental pressure, such as antimicrobials, encouraged the emergence and reemergence of infectious disease. The fight with infectious disease starts with prevention, diagnosis, and treatment. Diagnosis can be upheld by observing the cause of disease under the microscope or detecting the presence of nucleic acid and proteins of the pathogens. The molecular techniques span from classical polymerase chain reaction (PCR) to sequencing the nucleic acid composition. Here, we are reviewing the works have been undertaken to utilize a portable sequencer, MinION, in various aspects of infectious disease management.


Assuntos
Doenças Transmissíveis/microbiologia , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Análise de Sequência de DNA/instrumentação , Análise de Sequência de RNA/instrumentação , Doenças Transmissíveis/virologia , Epigenômica/instrumentação , Epigenômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodos
4.
Malar J ; 17(1): 217, 2018 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-29843734

RESUMO

BACKGROUND: The recent spread of artemisinin (ART)-resistant Plasmodium falciparum represents an emerging global threat to public health. In Southeast Asia, the C580Y mutation of kelch13 (k13) is the dominant mutation of ART-resistant P. falciparum. Therefore, a simple method for the detection of C580Y mutation is urgently needed to enable widespread routine surveillance in the field. The aim of this study is to develop a new diagnostic procedure for the C580Y mutation using loop-mediated isothermal amplification (LAMP) combined with the MinION nanopore sequencer. RESULTS: A LAMP assay for the k13 gene of P. falciparum to detect the C580Y mutation was successfully developed. The detection limit of this procedure was 10 copies of the reference plasmid harboring the k13 gene within 60 min. Thereafter, amplicon sequencing of the LAMP products using the MinION nanopore sequencer was performed to clarify the nucleotide sequences of the gene. The C580Y mutation was identified based on the sequence data collected from MinION reads 30 min after the start of sequencing. Further, clinical evaluation of the LAMP assay in 34 human blood samples collected from patients with P. falciparum malaria in Indonesia revealed a positive detection rate of 100%. All LAMP amplicons of up to 12 specimens were simultaneously sequenced using MinION. The results of sequencing were consistent with those of the conventional PCR and Sanger sequencing protocol. All procedures from DNA extraction to variant calling were completed within 3 h. The C580Y mutation was not found among these 34 P. falciparum isolates in Indonesia. CONCLUSIONS: An innovative method combining LAMP and MinION will enable simple, rapid, and high-sensitivity detection of the C580Y mutation of P. falciparum, even in resource-limited situations in developing countries.


Assuntos
Malária Falciparum/classificação , Mutação , Técnicas de Amplificação de Ácido Nucleico/métodos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Humanos , Indonésia , Malária Falciparum/parasitologia , Nanoporos , Plasmodium falciparum/isolamento & purificação
5.
BMC Infect Dis ; 17(1): 621, 2017 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-28903726

RESUMO

BACKGROUND: A simple and accurate molecular diagnostic method for malaria is urgently needed due to the limitations of conventional microscopic examination. In this study, we demonstrate a new diagnostic procedure for human malaria using loop mediated isothermal amplification (LAMP) and the MinION™ nanopore sequencer. METHODS: We generated specific LAMP primers targeting the 18S-rRNA gene of all five human Plasmodium species including two P. ovale subspecies (P. falciparum, P. vivax, P. ovale wallikeri, P. ovale curtisi, P. knowlesi and P. malariae) and examined human blood samples collected from 63 malaria patients in Indonesia. Additionally, we performed amplicon sequencing of our LAMP products using MinION™ nanopore sequencer to identify each Plasmodium species. RESULTS: Our LAMP method allowed amplification of all targeted 18S-rRNA genes of the reference plasmids with detection limits of 10-100 copies per reaction. Among the 63 clinical samples, 54 and 55 samples were positive by nested PCR and our LAMP method, respectively. Identification of the Plasmodium species by LAMP amplicon sequencing analysis using the MinION™ was consistent with the reference plasmid sequences and the results of nested PCR. CONCLUSIONS: Our diagnostic method combined with LAMP and MinION™ could become a simple and accurate tool for the identification of human Plasmodium species, even in resource-limited situations.


Assuntos
Malária/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Primers do DNA , Humanos , Indonésia , Limite de Detecção , Malária Falciparum/diagnóstico , Malária Vivax/diagnóstico , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos , Nanoporos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Plasmodium/genética , Reação em Cadeia da Polimerase , RNA Ribossômico 18S
6.
Antiviral Res ; 146: 1-11, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28818572

RESUMO

Zika fever, a mosquito-borne infectious disease caused by Zika virus (ZIKV), is an epidemic disease for which no effective therapy has been established. The recent outbreaks of ZIKV in Brazil and French Polynesia have been linked to a considerable increase in the incidence of fetal microcephaly and other diseases such as Guillain-Barre syndrome. Because there is currently no specific therapy or vaccine, the early exploitation of a method to prevent expansion of ZIKV is a high priority. To validate commonly used antiviral drugs, we evaluated the effect of ribavirin, a drug used to treat hepatitis C with interferon-ß (IFN-ß), on ZIKV replication. In mammalian cells, we observed an inhibitory effect of ribavirin on ZIKV replication and ZIKV-induced cell death without cytotoxic effect. Furthermore, we found that STAT1-deficient mice, which lack type I IFN signaling, were highly sensitive to ZIKV infection and exhibited lethal outcome. Ribavirin abrogated viremia in ZIKV-infected STAT-1-deficient mice. These data suggest that the inhibition of viral RNA-dependent RNA polymerases may be effective for treatment of ZIKV infection. Our data provide a new insight into the mechanisms for inhibition of ZIKV replication and prevention of Zika fever.


Assuntos
Antivirais/farmacologia , Ribavirina/farmacologia , Fator de Transcrição STAT1/deficiência , Infecção por Zika virus/tratamento farmacológico , Zika virus/efeitos dos fármacos , Animais , Antivirais/administração & dosagem , Antivirais/uso terapêutico , Chlorocebus aethiops , Replicação do DNA/efeitos dos fármacos , Camundongos , Ribavirina/administração & dosagem , Ribavirina/uso terapêutico , Fator de Transcrição STAT1/genética , Células Vero , Viremia/tratamento farmacológico , Viremia/virologia , Replicação Viral/efeitos dos fármacos , Zika virus/fisiologia , Infecção por Zika virus/virologia
7.
BMC Res Notes ; 10(1): 147, 2017 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-28376874

RESUMO

BACKGROUND: Malaria still poses one of the major threats to human health. Development of effective antimalarial drugs has decreased this threat; however, the emergence of drug-resistant Plasmodium falciparum, a cause of Malaria, is disconcerting. The antimalarial drug chloroquine has been effectively used, but resistant parasites have spread worldwide. Interestingly, the withdrawal of the drug reportedly leads to an increased population of susceptible parasites in some cases. We examined the prevalence of genomic polymorphisms in a malaria parasite P. falciparum, associated with resistance to an antimalarial drug chloroquine, after the withdrawal of the drug from Indonesia. RESULTS: Blood samples were collected from 95 malaria patients in North Sulawesi, Indonesia, in 2010. Parasite DNA was extracted and analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for pfcrt and pfmdr1. In parallel, multiplex amplicon sequencing for the same genes was carried out with Illumina MiSeq. Of the 59 cases diagnosed as P. falciparum infection by microscopy, PCR-RFLP analysis clearly identified the genotype 76T in pfcrt in 44 cases. Sequencing analysis validated the identified genotypes in the 44 cases and demonstrated that the haplotype in the surrounding genomic region was exclusively SVMNT. Results of pfmdr1 were successfully obtained for 51 samples, where the genotyping results obtained by the two methods were completely consistent. In pfmdr1, the 86Y mutant genotype was observed in 45 cases (88.2%). CONCLUSIONS: Our results suggest that the prevalence of the mutated genotypes remained dominant even 6 years after the withdrawal of chloroquine from this region. Diversified haplotype of the resistance-related locus, potentially involved in fitness costs, unauthorized usage of chloroquine, and/or a short post-withdrawal period may account for the observed high persistence of prevalence.


Assuntos
Cloroquina/uso terapêutico , Resistência a Medicamentos/genética , Malária Falciparum/tratamento farmacológico , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Polimorfismo Genético , Proteínas de Protozoários/genética , Antimaláricos/uso terapêutico , DNA de Protozoário/química , DNA de Protozoário/genética , Resistência a Múltiplos Medicamentos/genética , Frequência do Gene , Genótipo , Geografia , Haplótipos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Indonésia , Malária Falciparum/parasitologia , Mutação , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição
8.
Parasit Vectors ; 7: 143, 2014 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-24685121

RESUMO

BACKGROUND: Dengue virus infection manifests in three distinct forms in humans: dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Infection with the virus is a fatal disease; no vaccine is available and prevention depends on interruption of the chain of transmission. The study of dengue viral transmission by mosquitoes is hindered due to the lack of an affordable animal model. In general, immuno-competent mice are used as a simple and inexpensive animal model, but mice are not susceptible to dengue virus infection and therefore viremia will not occur following the inoculation of the virus in such mice. Here, we report a method for creating artificial viremia in immuno-competent mice, and further demonstrate the use of viremic mice to simultaneously infect a large number of Aedes aegypti. METHODS: We infected K562 cells with DENV-2 in the presence of an antibody against DENV-4. We then incubated the cells for 2 d before injecting the infected cells into C3H mice. After 5 h incubation, we allowed 100-150 female Aedes aegypti to feed on blood from the mice directly. We collected blood samples from the mice and from randomly selected Ae. aegypti at 2, 6, 12, and 24 h post-blood meal and screened the samples for DENV-2 genome as well as for virus concentration. RESULTS: Our procedure provided high virus concentrations in the mice for at least 7 h after viral inoculation. We found that 13 out of 14 randomly picked mosquitoes were infected with DENV-2. High concentrations of virus were detected in the mosquitoes until at least 12 h post-infection. CONCLUSIONS: Using the viremic immuno-competent mouse, we show that mass infection of Ae. aegypti is achievable. Compared to other infection techniques using direct inoculation, membrane-feeding, or immuno-deficient/humanized mice, we are confident that this method will provide a simpler and more efficient infection technique.


Assuntos
Aedes/fisiologia , Vírus da Dengue/fisiologia , Dengue/transmissão , Viremia , Aedes/virologia , Animais , Linhagem Celular , Dengue/virologia , Feminino , Camundongos , Camundongos Endogâmicos C3H
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