RESUMO
BACKGROUND: The prevalence of sexually transmitted infections (STIs) in sub-Saharan Africa is poorly described. We aimed to determine the prevalence of five treatable STIs (Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma genitalium, Treponema pallidum) in a sample of Gambian women from the general population. METHODS: Archived specimens from 420 women aged 15 - 69 years living in The Gambia enrolled in a clinical trial of human papilloma virus vaccine schedules were tested in this study. Urine samples were tested for C. trachomatis, N. gonorrhoeae, T. vaginalis and M. genitalium using a commercially available, open-platform multiplex PCR kit. A fragment of the ompA gene was amplified from C. trachomatis-positive samples and sequenced. Serum samples were tested for T. pallidum using the Chembio DPP Syphilis Screen and Confirm test. RESULTS: Overall, 41/420 (9.8%) women tested positive for at least one STI. 32 (7.6%), 9 (2.1%), 1 (0.2%), 1 (0.2%) and 0 (0.0%) tested positive for T. vaginalis, C. trachomatis, N gonorrhoeae, M. genitalium and T. pallidum, respectively. ompA gene sequence was available from five C. trachomatis infections: four were genovar D,one was genovar G and one was genovar F. CONCLUSIONS: STIs are endemic in The Gambia. Monitoring systems should be established.
Assuntos
Infecções por Chlamydia , Gonorreia , Infecções por Mycoplasma , Mycoplasma genitalium , Infecções Sexualmente Transmissíveis , Feminino , Humanos , Infecções por Chlamydia/epidemiologia , Chlamydia trachomatis/genética , Gâmbia/epidemiologia , Gonorreia/epidemiologia , Mycoplasma genitalium/genética , Infecções por Mycoplasma/epidemiologia , Neisseria gonorrhoeae/genética , Prevalência , Rios , Infecções Sexualmente Transmissíveis/epidemiologia , Adolescente , Adulto Jovem , Adulto , Pessoa de Meia-Idade , IdosoRESUMO
BACKGROUND: To eliminate trachoma as a public health problem, the World Health Organization recommends the SAFE (surgery, antibiotics, facial cleanliness, and environmental improvement) strategy. As part of the SAFE strategy in the Amhara Region, Ethiopia, the Trachoma Control Program distributed >124 million doses of antibiotics between 2007 and 2015. Despite this, trachoma remained hyperendemic in many districts and a considerable level of Chlamydia trachomatis (Ct) infection was evident. METHODS: We utilized residual material from Abbott m2000 Ct diagnostic tests to sequence 99 ocular Ct samples from Amhara and investigated the role of Ct genomic variation in continued transmission of Ct. RESULTS: Sequences were typical of ocular Ct at the whole-genome level and in tissue tropism-associated genes. There was no evidence of macrolide resistance in this population. Polymorphism around the ompA gene was associated with village-level trachomatous inflammation-follicular prevalence. Greater ompA diversity at the district level was associated with increased Ct infection prevalence. CONCLUSIONS: We found no evidence for Ct genomic variation contributing to continued transmission of Ct after treatment, adding to evidence that azithromycin does not drive acquisition of macrolide resistance in Ct. Increased Ct infection in areas with more ompA variants requires longitudinal investigation to understand what impact this may have on treatment success and host immunity.
Assuntos
Gonorreia , Doenças do Recém-Nascido , Tracoma , Antibacterianos/uso terapêutico , Azitromicina/uso terapêutico , Chlamydia trachomatis/genética , Farmacorresistência Bacteriana/genética , Etiópia/epidemiologia , Genômica , Gonorreia/tratamento farmacológico , Humanos , Lactente , Recém-Nascido , Macrolídeos/uso terapêutico , Prevalência , Tracoma/tratamento farmacológico , Tracoma/epidemiologia , Tracoma/prevenção & controleRESUMO
Cytokine-induced memory-like (CIML) NK cells generated in response to proinflammatory cytokines in vitro and in vivo can also be generated by vaccination, exhibiting heightened responses to cytokine stimulation months after their initial induction. Our previous study demonstrated that in vitro human NK cell responses to inactivated influenza virus were also indirectly augmented by very low doses of IL-15, which increased induction of myeloid cell-derived cytokine secretion. These findings led us to hypothesize that IL-15 stimulation could reveal a similar effect for active influenza vaccination and influence CIML NK cell effector functions. In this study, 51 healthy adults were vaccinated with seasonal influenza vaccine, and PBMC were collected before and up to 30 d after vaccination. Myeloid and lymphoid cell cytokine secretion was measured after in vitro PBMC restimulation with low-dose IL-15, alone or in combination with inactivated H3N2 virus; the associated NK cell response was assessed by flow cytometry. PBMC collected 30 d postvaccination showed heightened cytokine production in response to IL-15 compared with PBMC collected at baseline; these responses were further enhanced when IL-15 was combined with H3N2. NK cell activation in response to IL-15 alone (CD25) and H3N2 plus IL-15 (CD25 and IFN-γ) was enhanced postvaccination. We also observed proliferation of less-differentiated NK cells with downregulation of cytokine receptors as early as 3 d after vaccination, suggesting cytokine stimulation in vivo. We conclude that vaccination-induced "training" of accessory cells combines with the generation of CIML NK cells to enhance the overall NK cell response postvaccination.
Assuntos
Citocinas/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Células Matadoras Naturais/imunologia , Células Mieloides/imunologia , Adulto , Idoso , Feminino , Humanos , Vírus da Influenza A Subtipo H3N2/imunologia , Interferon gama/imunologia , Interleucina-15/imunologia , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Vacinação/métodos , Adulto JovemRESUMO
Trachoma is initiated during childhood following repeated conjunctival infection with Chlamydia trachomatis, which causes a chronic inflammatory response in some individuals that leads to scarring and in-turning of the eyelids in later life. There is currently no treatment to halt the progression of scarring trachoma due to an incomplete understanding of disease pathogenesis. A cohort study was performed in northern Tanzania in 616 children aged 6 to 10 years at enrollment. Every 3 months for 4 years, children were examined for clinical signs of trachoma, and conjunctival swabs were collected for C. trachomatis detection and to analyze the expression of 46 immunofibrogenic genes. Data were analyzed in relation to progressive scarring status between baseline and the final time point. Genes that were significantly associated with scarring progression included those encoding proinflammatory chemokines (CXCL5, CCL20, CXCL13, and CCL18), cytokines (IL23A, IL19, and IL1B), matrix modifiers (MMP12 and SPARCL1), immune regulators (IDO1, SOCS3, and IL10), and a proinflammatory antimicrobial peptide (S100A7). In response to C. trachomatis infection, IL23A and PDGF were significantly upregulated in scarring progressors relative to in nonprogressors. Our findings highlight the importance of innate proinflammatory signals from the epithelium and implicate interleukin 23A (IL-23A)-responsive cells in driving trachomatous scarring, with potential key mechanistic roles for PDGFB, MMP12, and SPARCL1 in orchestrating fibrosis.
Assuntos
Cicatriz/patologia , Cicatriz/fisiopatologia , Túnica Conjuntiva/patologia , Imunidade Inata , Fatores Imunológicos/biossíntese , Tracoma/patologia , Tracoma/fisiopatologia , Criança , Chlamydia trachomatis/crescimento & desenvolvimento , Feminino , Perfilação da Expressão Gênica , Humanos , Fatores Imunológicos/genética , Estudos Longitudinais , Masculino , TanzâniaRESUMO
Chlamydia trachomatis is the most commonly diagnosed bacterial sexually transmitted infection and can lead to tubal factor infertility, a disease characterised by fibrosis of the fallopian tubes. Genetic polymorphisms in molecular pathways involving G protein-coupled receptor signalling, the Akt/PI3K cascade, the mitotic cell cycle, and immune response have been identified in association with the development of trachomatous scarring, an ocular form of chlamydia-related fibrotic pathology. In this case-control study, we performed genome-wide association and pathways-based analysis in a sample of 71 Dutch women who attended an STI clinic who were seropositive for Chlamydia trachomatis antibodies and 169 high-risk Dutch women who sought similar health services but who were seronegative. We identified two regions of within-gene SNP association with Chlamydia trachomatis serological response and found that GPCR signalling and cell cycle pathways were also associated with the trait. These pathway-level associations appear to be common to immunological sequelae of chlamydial infections in both ocular and urogenital tropisms. These pathways may be central mediators of human refractoriness to chlamydial diseases.
Assuntos
Infecções por Chlamydia/genética , Estudo de Associação Genômica Ampla/métodos , Polimorfismo de Nucleotídeo Único/genética , Adolescente , Adulto , Estudos de Casos e Controles , Chlamydia trachomatis/patogenicidade , Feminino , Genótipo , Humanos , Técnicas In Vitro , Receptores Acoplados a Proteínas G/genética , Fatores de Risco , Adulto JovemRESUMO
BACKGROUND: Mass drug administration (MDA) with azithromycin is a corner-stone of trachoma control however it may drive the emergence of antimicrobial resistance. In a cluster-randomized trial (Clinical trial gov NCT00792922), we compared the reduction in the prevalence of active trachoma in communities that received three annual rounds of MDA to that in communities that received a single treatment round. We used the framework of this trial to carry out an opportunistic study to investigate if the increased rounds of treatment resulted in increased prevalence of nasopharyngeal carriage of macrolide-resistant Staphylococcus aureus. Three cross-sectional surveys were conducted in two villages receiving three annual rounds of MDA (3 × treatment arm). Surveys were conducted immediately before the third round of MDA (CSS-1) and at one (CSS-2) and six (CSS-3) months after MDA. The final survey also included six villages that had received only one round of MDA 30 months previously (1 × treatment arm). RESULTS: In the 3 × treatment arm, a short-term increase in prevalence of S. aureus carriage was seen following MDA from 24.6% at CSS-1 to 38.6% at CSS-2 (p < 0.001). Prevalence fell to 8.8% at CSS-3 (p < 0.001). A transient increase was also seen in prevalence of carriage of azithromycin resistant (AzmR) strains from 8.9% at CSS-1 to 34.1% (p < 0.001) in CSS-2 and down to 7.3% (p = 0.417) in CSS-3. A similar trend was observed for prevalence of carriage of macrolide-inducible-clindamycin resistant (iMLSB) strains. In CSS-3, prevalence of carriage of resistant strains was higher in the 3 × treatment arm than in the 1 × treatment (AzmR 7.3% vs. 1.6%, p = 0.010; iMLSB 5.8% vs. 0.8%, p < 0.001). Macrolide resistance was attributed to the presence of msr and erm genes. CONCLUSIONS: Three annual rounds of MDA with azithromycin were associated with a short-term increase in both the prevalence of nasopharyngeal carriage of S. aureus and prevalence of carriage of AzmR and iMLSB S. aureus. TRIAL REGISTRATION: This study was ancillary to the Partnership for the Rapid Elimination of Trachoma, ClinicalTrials.gov NCT00792922 , registration date November 17, 2008.
Assuntos
Azitromicina/administração & dosagem , Azitromicina/uso terapêutico , Macrolídeos/uso terapêutico , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Nasofaringe/microbiologia , Prevalência , Tracoma/tratamento farmacológico , Administração Oral , Adolescente , Antibacterianos/uso terapêutico , Portador Sadio/epidemiologia , Portador Sadio/microbiologia , Criança , Estudos Transversais , Farmacorresistência Bacteriana , Feminino , Gâmbia/epidemiologia , Humanos , Programas de Imunização , Masculino , Testes de Sensibilidade Microbiana , Nasofaringite/tratamento farmacológico , Nasofaringite/microbiologia , Fatores de Risco , Manejo de Espécimes/métodos , Infecções Estafilocócicas/complicações , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/epidemiologia , Streptococcus pneumoniae/efeitos dos fármacos , Tracoma/complicaçõesRESUMO
BACKGROUND: Trachoma, caused by ocular infection with Chlamydia trachomatis, is the leading infectious cause of blindness worldwide. We conducted the first population-based trachoma prevalence survey in the Casamance region of Senegal to enable the Senegalese National Eye Care Programme (NECP) to plan its trachoma control activities. The World Health Organization (WHO) guidelines state that any individual with trachomatous trichiasis (TT) should be offered surgery, but that surgery should be prioritised where the prevalence is >0.1%, and that districts and communities with a trachomatous inflammation, follicular (TF) prevalence of ≥10% in 1-9 year-olds should receive mass antibiotic treatment annually for a minimum of three years, along with hygiene promotion and environmental improvement, before re-assessing the prevalence to determine whether treatment can be discontinued (when TF prevalence in 1-9 year-olds falls <5%). METHODS: Local healthcare workers conducted a population-based household survey in four districts of the Bignona Department of Casamance region to estimate the prevalence of TF in 1-9 year-olds, and TT in ≥15 year-olds. Children's facial cleanliness (ocular and/or nasal discharge, dirt on the face, flies on the face) was measured at time of examination. Risk factor questionnaires were completed at the household level. RESULTS: Sixty communities participated with a total censused population of 5580 individuals. The cluster-, age- and sex-adjusted estimated prevalence of TF in 1-9 year-olds was 2.5% (95% Confidence Interval (CI) 1.8-3.6) (38/1425) at the regional level and <5% in all districts, although the upper 95%CI exceeded 5% in all but one district. The prevalence of TT in those aged ≥15 years was estimated to be 1.4% (95%CI 1.0-1.9) (40/2744) at the regional level and >1% in all districts. CONCLUSION: With a prevalence <5%, TF does not appear to be a significant public health problem in this region. However, TF monitoring and surveillance at sub-district level will be required to ensure that elimination targets are sustained and that TF does not re-emerge as a public health problem. TT surgery remains the priority for trachoma elimination efforts in the region, with an estimated 1819 TT surgeries to conduct.
Assuntos
Tracoma/epidemiologia , Triquíase/epidemiologia , Adolescente , Antibacterianos/uso terapêutico , Criança , Pré-Escolar , Estudos Transversais , Esquema de Medicação , Feminino , Promoção da Saúde , Humanos , Lactente , Recém-Nascido , Masculino , Prevalência , Saúde Pública , Fatores de Risco , Senegal/epidemiologia , Tracoma/tratamento farmacológico , Triquíase/terapiaRESUMO
NKG2C is an activating receptor that is preferentially expressed on natural killer (NK) cells. The gene encoding NKG2C (killer cell lectin-like receptor C2, KLRC2) is present at different copy numbers in the genomes of different individuals. Deletion at the NKG2C locus was investigated in a case-control study of 1522 individuals indigenous to East- and West-Africa and the association with the ocular Chlamydia trachomatis infection and its sequelae was explored. The frequency of homozygous KLRC2 deletion was 13.7 % in Gambians and 4.7 % in Tanzanians. A significantly higher frequency of the deletion allele was found in West-Africans from the Gambia and Guinea-Bissau (36.2 % p = 2.105 × 10(-8), 26.8 % p = 0.050; respectively) in comparison to East-African Tanzanians where the frequency of the deletion is comparable to other human populations (20.9 %). We found no evidence for an association between the numbers of KLRC2 gene copies and the clinical manifestations of trachoma (follicular trachoma or conjunctival scarring). A new method for imputation of KLRC2 genotypes from single nucleotide polymorphism (SNP) data in 2621 individuals from the Gambia further confirmed these results. Our data suggest that NKG2C does not play a major role in trachomatous disease. We found that the deletion allele is present at different frequencies in different populations but the reason behind these differences is currently not understood. The new method offers the potential to use SNP arrays from genome wide association studies to study the frequency of KLRC2 deletion in other populations and its association with other diseases.
Assuntos
Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Subfamília C de Receptores Semelhantes a Lectina de Células NK/genética , Tracoma/genética , Adolescente , Adulto , África Ocidental , Idoso , Idoso de 80 Anos ou mais , Alelos , Criança , Pré-Escolar , Feminino , Genótipo , Homozigoto , Humanos , Lactente , Recém-Nascido , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Deleção de Sequência/genética , Tracoma/epidemiologia , Tracoma/patologiaRESUMO
BACKGROUND: Trachoma, a preventable blinding eye disease, is initiated by ocular infection with Chlamydia trachomatis (Ct). We previously showed that microRNAs (miR) -147b and miR-1285 were up-regulated in inflammatory trachomatous scarring. During the initial stage of disease, follicular trachoma with current Ct infection, the differential expression of miR has not yet been investigated. METHODS: Conjunctival samples were collected from 163 children aged 1-9 years old living in a trachoma-endemic region of Guinea Bissau, West Africa. Small RNA sequencing (RNAseq) was carried out on samples from five children with follicular trachoma and current Ct infection and five children with healthy conjunctivae and no Ct infection. Small RNAseq was also carried out on human epithelial cell lines infected with ocular Ct strains A2497 and isogenic plasmid-free A2497 in vitro. Results were validated by quantitative PCR (qPCR) in 163 clinical samples. RESULTS: Differential expression of RNAseq data identified 12 miR with changes in relative expression during follicular trachoma, of which 9 were confirmed as differentially expressed by qPCR (miR-155, miR-150, miR-142, miR-181b, miR-181a, miR-342, miR-132, miR-4728 and miR-184). MiR-155 and miR-184 expression had a direct relationship with the degree of clinical inflammation. MiR-155 was up-regulated (OR = 2.533 ((95 % CI = 1.291-4.971); P = 0.0069) and miR-184 was down-regulated (OR = 0.416 ((95 % CI = 0.300-0.578); P = 1.61*10(-7)) as the severity of clinical inflammation increased. Differential miR expression was not detected in HEp-2 or HCjE epithelial cells 48 h post infection with Ct in vitro. HCjE cells, a conjunctival epithelial cell line, had a markedly different miR background expression compared to HEp-2 cells. CONCLUSIONS: In follicular trachoma, expression of miR-155 and miR-184 is correlated with the severity of inflammation. This likely reflects host regulation of the immune response and a prolonged period of wound healing following the clearance of Ct. Prolonged healing may be associated with subsequent development of scarring trachoma.
Assuntos
Chlamydia trachomatis/imunologia , Túnica Conjuntiva/imunologia , Regulação da Expressão Gênica , MicroRNAs/genética , Tracoma/imunologia , Cegueira , Criança , Pré-Escolar , Túnica Conjuntiva/parasitologia , Regulação para Baixo , Feminino , Guiné-Bissau , Humanos , Lactente , Inflamação , Masculino , Tracoma/parasitologia , Regulação para CimaRESUMO
Trachoma is a blinding disease usually caused by infection with Chlamydia trachomatis (Ct) serovars A, B, and C in the upper tarsal conjunctiva. Individuals in endemic regions are repeatedly infected with Ct throughout childhood. A proportion of individuals experience prolonged or severe inflammatory episodes that are known to be significant risk factors for ocular scarring in later life. Continued scarring often leads to trichiasis and in-turning of the eyelashes, which causes pain and can eventually cause blindness. The mechanisms driving the chronic immunopathology in the conjunctiva, which largely progresses in the absence of detectable Ct infection in adults, are likely to be multifactorial. Socioeconomic status, education, and behavior have been identified as contributing to the risk of scarring and inflammation. We focus on the contribution of host and pathogen genetic variation, bacterial ecology of the conjunctiva, and host epigenetic imprinting including small RNA regulation by both host and pathogen in the development of ocular pathology. Each of these factors or processes contributes to pathogenic outcomes in other inflammatory diseases and we outline their potential role in trachoma.
Assuntos
Chlamydia trachomatis/patogenicidade , Tracoma/genética , Animais , Epigenômica/métodos , Infecções Oculares/genética , Infecções Oculares/microbiologia , Genômica/métodos , HumanosRESUMO
The Chlamydia trachomatis plasmid is a virulence factor. Plasmid copy number, C. trachomatis load and disease severity were assessed in a treatment-naive population where trachoma is hyperendemic. By using droplet digital PCR, plasmid copy number was found to be stable (median, 5.34 [range, 1 to 18]) and there were no associations with C. trachomatis load or disease severity.
Assuntos
Chlamydia trachomatis/genética , Dosagem de Genes , Plasmídeos , Tracoma/microbiologia , Tracoma/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carga Bacteriana , Criança , Pré-Escolar , Chlamydia trachomatis/patogenicidade , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Virulência , Adulto JovemRESUMO
OBJECTIVE: To evaluate the effect of repeated mass drug administration (MDA) of azithromycin in the Gambia on the nasopharyngeal carriage of Streptococcus pneumoniae and on the emergence of antibiotic-resistant strains. METHODS: This study involved villages that participated in a cluster randomized trial comparing the effect of one versus three azithromycin MDA rounds on the prevalence of trachoma. Only villages in which most children received 7-valent pneumococcal conjugate vaccine were included. Three cross-sectional surveys were performed in two villages that received three annual MDA rounds: the first immediately before the third MDA round and the second and third, 1 and 6 months, respectively, after the third MDA round. The third survey also covered six villages that had received one MDA round 30 months previously. Pneumococcal carriage was assessed using nasopharyngeal swabs and azithromycin resistance was detected using the Etest. FINDINGS: The prevalence of pneumococcal carriage decreased from 43.4% to 19.2% between the first and second surveys (P < 0.001) but rebounded by the third survey (45.8%; P = 0.591). Being a carrier at the first survey was a risk factor for being a carrier at the second (odds ratio: 3.71; P < 0.001). At the third survey, the prevalence of carriage was similar after one and three MDA rounds (50.3% versus 45.8%, respectively; P = 0.170), as was the prevalence of azithromycin resistance (0.3% versus 0.9%, respectively; P = 0.340). CONCLUSION: Three azithromycin MDA rounds did not increase the prevalence of nasopharyngeal carriage of azithromycin-resistant S. pneumoniae strains compared with one round.
Assuntos
Antibacterianos/administração & dosagem , Azitromicina/administração & dosagem , Nasofaringe/microbiologia , Streptococcus pneumoniae/efeitos dos fármacos , Tracoma/tratamento farmacológico , Portador Sadio , Criança , Pré-Escolar , Estudos Transversais , Farmacorresistência Bacteriana , Feminino , Gâmbia/epidemiologia , Humanos , Programas de Imunização , Lactente , Recém-Nascido , Masculino , Vacinas Pneumocócicas/administração & dosagem , Prevalência , Fatores de Risco , População Rural , Tracoma/epidemiologiaRESUMO
OBJECTIVE: To assess the effect of azithromycin mass drug administration regimens on spleen rates in children aged 0-5 years. METHODS: Clinical assessment of spleen size was carried out during a cluster-randomised trial of azithromycin mass treatment for trachoma elimination in The Gambia. Twenty-four communities received three annual mass treatments with azithromycin, and 24 communities received treatment at baseline only. RESULTS: At the 30-month follow-up, 3646 children aged 0-5 years had spleen examination and measurement. Palpable splenomegaly was significantly lower in annually treated vs. baseline-only treatment communities and in treated vs. untreated children at 24 months in the annual treatment arm. CONCLUSION: The results suggest an effect of azithromycin on spleen rates at the individual level and are most plausibly due to the antimalarial effects of azithromycin.
Assuntos
Antibacterianos/uso terapêutico , Azitromicina/uso terapêutico , Baço/efeitos dos fármacos , Esplenomegalia/prevenção & controle , Tracoma/tratamento farmacológico , Antibacterianos/farmacologia , Azitromicina/farmacologia , Pré-Escolar , Chlamydia trachomatis , Seguimentos , Gâmbia , Humanos , Lactente , Baço/patologia , Tracoma/patologia , Resultado do TratamentoRESUMO
BACKGROUND: Chlamydia trachomatis is a pathogen of worldwide importance, causing more than 100 million cases of sexually transmitted infections annually. Whole-genome sequencing is a powerful high resolution tool that can be used to generate accurate data on bacterial population structure, phylogeography and mutations associated with antimicrobial resistance. The objective of this study was to perform whole-genome enrichment and sequencing of C. trachomatis directly from clinical samples. METHODS: C. trachomatis positive samples comprising seven vaginal swabs and three urine samples were sequenced without prior in vitro culture in addition to nine cultured C. trachomatis samples, representing different serovars. A custom capture RNA bait set, that captures all known diversity amongst C. trachomatis genomes, was used in a whole-genome enrichment step during library preparation to enrich for C. trachomatis DNA. All samples were sequenced on the MiSeq platform. RESULTS: Full length C. trachomatis genomes (>95-100% coverage of a reference genome) were successfully generated for eight of ten clinical samples and for all cultured samples. The proportion of reads mapping to C. trachomatis and the mean read depth across each genome were strongly linked to the number of bacterial copies within the original sample. Phylogenetic analysis confirmed the known population structure and the data showed potential for identification of minority variants and mutations associated with antimicrobial resistance. The sensitivity of the method was >10-fold higher than other reported methodologies. CONCLUSIONS: The combination of whole-genome enrichment and deep sequencing has proven to be a non-mutagenic approach, capturing all known variation found within C. trachomatis genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of C. trachomatis directly from clinical samples and has the potential to be adapted to other pathogens with a similar clonal nature.
Assuntos
Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/genética , Sequência de Bases , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Filogenia , Análise de Sequência de DNARESUMO
BACKGROUND: Mass drug administration (MDA) with azithromycin, carried out for the control of blinding trachoma, has been linked to reduced mortality in children. While the mechanism behind this reduction is unclear, it may be due, in part, to improved nutritional status via a potential reduction in the community burden of infectious disease. To determine whether MDA with azithromycin improves anthropometric indices at the community level, we measured the heights and weights of children aged 1 to 4 years in communities where one (single MDA arm) or three annual rounds (annual MDA arm) of azithromycin had been distributed. METHODS: Data collection took place three years after treatment in the single MDA arm and one year after the final round of treatment in the annual MDA arm. Mean height-for-age, weight-for-age and weight-for-height z scores were compared between treatment arms. RESULTS: No significant differences in mean height-for-age, weight-for-age or weight-for-height z scores were found between the annual MDA and single MDA arms, nor was there a significant reduction in prevalence of stunting, wasting or underweight between arms. CONCLUSIONS: Our data do not provide evidence that community MDA with azithromycin improved anthropometric outcomes of children in The Gambia. This may suggest reductions in mortality associated with azithromycin MDA are due to a mechanism other than improved nutritional status.
Assuntos
Antropometria , Antibacterianos/administração & dosagem , Azitromicina/administração & dosagem , Tracoma/prevenção & controle , Serviços de Saúde da Criança , Pré-Escolar , Serviços de Saúde Comunitária , Feminino , Gâmbia , Humanos , Lactente , Masculino , Estado Nutricional , PrevalênciaRESUMO
The human pathogen Chlamydia trachomatis has multiple serovariants that have distinct organotropisms. We recently revised genomic sequence data linked to ocular reference strain, B/HAR36. Now linked to its correct genomic data in the European Nucleotide Archive, we describe its genomic features.
RESUMO
Trachoma, a neglected tropical disease caused by Chlamydia trachomatis (Ct) serovars A-C, is the leading infectious cause of blindness worldwide. Africa bears the highest burden, accounting for over 86â% of global trachoma cases. We investigated Ct serovar A (SvA) and B (SvB) whole genome sequences prior to the induction of mass antibiotic drug administration in The Gambia. Here, we explore the factors contributing to Ct strain diversification and the implications for Ct evolution within the context of ocular infection. A cohort study in 2002-2003 collected ocular swabs across nine Gambian villages during a 6 month follow-up study. To explore the genetic diversity of Ct within and between individuals, we conducted whole-genome sequencing (WGS) on a limited number (n=43) of Ct-positive samples with an omcB load ≥10 from four villages. WGS was performed using target enrichment with SureSelect and Illumina paired-end sequencing. Out of 43 WGS samples, 41 provided sufficient quality for further analysis. ompA analysis revealed that 11 samples had highest identity to ompA from strain A/HAR13 (NC_007429) and 30 had highest identity to ompA from strain B/Jali20 (NC_012686). While SvB genome sequences formed two distinct village-driven subclades, the heterogeneity of SvA sequences led to the formation of many individual branches within the Gambian SvA subclade. Comparing the Gambian SvA and SvB sequences with their reference strains, Ct A/HAR13 and Ct B/Jali20, indicated an single nucleotide polymorphism accumulation rate of 2.4×10-5 per site per year for the Gambian SvA and 1.3×10-5 per site per year for SvB variants (P<0.0001). Variant calling resulted in a total of 1371 single nucleotide variants (SNVs) with a frequency >25â% in SvA sequences, and 438 SNVs in SvB sequences. Of note, in SvA variants, highest evolutionary pressure was recorded on genes responsible for host cell modulation and intracellular survival mechanisms, whereas in SvB variants this pressure was mainly on genes essential for DNA replication/repair mechanisms and protein synthesis. A comparison of the sequences between observed separate infection events (4-20 weeks between infections) suggested that the majority of the variations accumulated in genes responsible for host-pathogen interaction such as CTA_0166 (phospholipase D-like protein), CTA_0498 (TarP) and CTA_0948 (deubiquitinase). This comparison of Ct SvA and SvB variants within a trachoma endemic population focused on their local evolutionary adaptation. We found a different variation accumulation pattern in the Gambian SvA chromosomal genes compared with SvB, hinting at the potential of Ct serovar-specific variation in diversification and evolutionary fitness. These findings may have implications for optimizing trachoma control and prevention strategies.
Assuntos
Tracoma , Humanos , Tracoma/epidemiologia , Tracoma/genética , Chlamydia trachomatis/genética , Gâmbia/epidemiologia , Estudos de Coortes , Seguimentos , GenômicaRESUMO
BACKGROUND: Trachoma is a leading cause of infection-related blindness worldwide. This disease is caused by recurrent Chlamydia trachomatis (Ct) infections of the conjunctiva and develops in two phases: i) active (acute trachoma, characterized by follicular conjunctivitis), then long-term: ii) scarring (chronic trachoma, characterized by conjunctival fibrosis, corneal opacification and eyelid malposition). Scarring trachoma is driven by the number and severity of reinfections. The immune system plays a pivotal role in trachoma including exacerbation of the disease. Hence the immune system may also be key to developing a trachoma vaccine. Therefore, we characterized clinical and local immune response kinetics in a non-human primate model of acute conjunctival Ct infection and disease. METHODOLOGY/PRINCIPAL FINDINGS: The conjunctiva of non-human primate (NHP, Cynomolgus monkeys-Macaca fascicularis-) were inoculated with Ct (B/Tunis-864 strain, B serovar). Clinical ocular monitoring was performed using a standardized photographic grading system, and local immune responses were assessed using multi-parameter flow cytometry of conjunctival cells, tear fluid cytokines, immunoglobulins, and Ct quantification. Clinical findings were similar to those observed during acute trachoma in humans, with the development of typical follicular conjunctivitis from the 4th week post-exposure to the 11th week. Immunologic analysis indicated an early phase influx of T cells in the conjunctiva and elevated interleukins 4, 8, and 5, followed by a late phase monocytic influx accompanied with a decrease in other immune cells, and tear fluid cytokines returning to initial levels. CONCLUSION/SIGNIFICANCE: Our NHP model accurately reproduces the clinical signs of acute trachoma, allowing for an accurate assessment of the local immune responses in infected eyes. A progressive immune response occurred for weeks after exposure to Ct, which subsided into a persistent innate immune response. An understanding of these local responses is the first step towards using the model to assess new vaccine and therapeutic strategies for disease prevention.
Assuntos
Chlamydia trachomatis , Túnica Conjuntiva , Modelos Animais de Doenças , Macaca fascicularis , Tracoma , Animais , Tracoma/imunologia , Tracoma/microbiologia , Túnica Conjuntiva/imunologia , Túnica Conjuntiva/patologia , Túnica Conjuntiva/microbiologia , Chlamydia trachomatis/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Masculino , FemininoRESUMO
Trachoma is the leading infectious cause of blindness worldwide and is now largely confined to around 40 low- and middle-income countries. It is caused by Chlamydia trachomatis (Ct), a contagious intracellular bacterium. The World Health Organization recommends mass drug administration (MDA) with azithromycin for treatment and control of ocular Ct infections, alongside improving facial cleanliness and environmental conditions to reduce transmission. To understand the molecular epidemiology of trachoma, especially in the context of MDA and transmission dynamics, the identification of Ct genotypes could be useful. While many studies have used the Ct major outer membrane protein gene (ompA) for genotyping, it has limitations. Our study applies a typing system novel to trachoma, Multiple Loci Variable Number Tandem Repeat Analysis combined with ompA (MLVA-ompA). Ocular swabs were collected post-MDA from four trachoma-endemic zones in Ethiopia between 2011-2017. DNA from 300 children with high Ct polymerase chain reaction (PCR) loads was typed using MLVA-ompA, utilizing 3 variable number tandem repeat (VNTR) loci within the Ct genome. Results show that MLVA-ompA exhibited high discriminatory power (0.981) surpassing the recommended threshold for epidemiological studies. We identified 87 MLVA-ompA variants across 26 districts. No significant associations were found between variants and clinical signs or chlamydial load. Notably, overall Ct diversity significantly decreased after additional MDA rounds, with a higher proportion of serovar A post-MDA. Despite challenges in sequencing one VNTR locus (CT1299), MLVA-ompA demonstrated cost-effectiveness and efficiency relative to whole genome sequencing, providing valuable information for trachoma control programs on local epidemiology. The findings suggest the potential of MLVA-ompA as a reliable tool for typing ocular Ct and understanding transmission dynamics, aiding in the development of targeted interventions for trachoma control.
Assuntos
Proteínas da Membrana Bacteriana Externa , Chlamydia trachomatis , Genótipo , Repetições Minissatélites , Tracoma , Chlamydia trachomatis/genética , Chlamydia trachomatis/isolamento & purificação , Chlamydia trachomatis/classificação , Tracoma/epidemiologia , Tracoma/microbiologia , Tracoma/tratamento farmacológico , Humanos , Etiópia/epidemiologia , Repetições Minissatélites/genética , Proteínas da Membrana Bacteriana Externa/genética , Feminino , Masculino , Pré-Escolar , Tipagem Molecular/métodos , Azitromicina/uso terapêutico , Variação Genética , Lactente , Criança , Antibacterianos/farmacologia , DNA Bacteriano/genéticaRESUMO
Droplet digital PCR (ddPCR) is an emulsion PCR process that performs absolute quantitation of nucleic acids. We developed a ddPCR assay for Chlamydia trachomatis infections and found it to be accurate and precise. Using PCR mixtures containing plasmids engineered to include the PCR target sequences, we were able to quantify with a dynamic range between 0.07 and 3,160 targets/µl (r(2) = 0.9927) with >95% confidence. Using 1,509 clinical conjunctival swab samples from a population in which trachoma is endemic in Guinea Bissau, we evaluated the specificity and sensitivity of the quantitative ddPCR assay in diagnosing ocular C. trachomatis infections by comparing the performances of ddPCR and the Roche Amplicor CT/NG test. We defined ddPCR tests as positive when we had ≥95% confidence in a nonzero estimate of target load. The sensitivity of ddPCR against Amplicor was 73.3% (95% confidence interval [CI], 67.9 to 78.7%), and specificity was 99.1% (95% CI, 98.6 to 99.6%). Negative and positive predictive values were 94.6% (95% CI, 93.4 to 95.8%) and 94.5% (95% CI, 91.3 to 97.7%), respectively. Based on Amplicor CT/NG testing, the estimated population prevalence of C. trachomatis ocular infection was â¼17.5%. Receiver-operator curve analysis was used to select critical cutoff values for use in clinical settings in which a balance between higher sensitivity and specificity is required. We concluded that ddPCR is an effective diagnostic technology suitable for both research and clinical use in diagnosing ocular C. trachomatis infections.