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Neisseria gonorrhoeae antimicrobial resistance (AMR) surveillance is essential for tracking the emergence and spread of AMR strains in local, national and international populations. This is crucial for developing or refining treatment guidelines. N. gonorrhoeae multiantigen sequence typing (NG-MAST) is beneficial for describing the molecular epidemiology of gonococci at national and international levels. Elucidation of AMR determinants to β-lactam drugs, is a means of monitoring the development of resistance. In Ghana, little is known about the current gonococcal AMR prevalence and no characterization of gonococcal isolates has been previously performed. In this study, gonococcal isolates (n = 44) collected from five health facilities in Ghana from 2012 to 2015, were examined using AMR testing, NG-MAST and sequencing of penA. High rates of resistance were identified to tetracycline (100%), benzylpenicillin (90.9%), and ciprofloxacin (81.8%). One isolate had a high cefixime MIC (0.75 μg/ml). Twenty-eight NG-MAST sequence types (STs) were identified, seventeen of which were novel. The isolate with the high cefixime MIC contained a mosaic penA-34 allele and belonged to NG-MAST ST1407, an internationally spreading multidrug-resistant clone that has accounted for most cefixime resistance in many countries. In conclusion, AMR testing, NG-MAST, and sequencing of the AMR determinant penA, revealed high rates of resistance to tetracycline, benzylpenicillin, and ciprofloxacin; as well as a highly diverse population of N. gonorrhoeae in Ghana. It is imperative to continue with enhanced AMR surveillance and to understand the molecular epidemiology of gonococcal strains circulating in Ghana and other African countries.

A three-antigen DNA-prime/chimpanzee adenovirus 63 (ChAd63) boost vaccine containing pre-erythrocytic Plasmodium falciparum (Pf) circumsporozoite protein (CSP), Pf apical membrane antigen-1 (AMA1) and malaria multiple epitopes (ME) fused to Pf thrombospondin-related adhesion protein (ME-TRAP) elicited higher vaccine efficacy (VE) in an open label, randomized Phase 1 trial against controlled human malaria infection (CHMI) than the two-antigen vaccine DNA/Human Adenovirus 5 (HuAd5) containing CSP and AMA1. The objective of this follow-up study was to determine whether responses to CSP, AMA1 or TRAP MHC Class I-restricted epitopes were associated with VE. Protected (n = 6) and non-protected participants (n = 26) were screened in FluoroSpot interferon gamma (IFN-γ) and Granzyme B (GzB) assays using antigen-specific 15mer peptide subpools spanning CSP (n = 9 subpools), AMA1 (n = 12 subpools), and TRAP (n = 11 subpools). Individual antigen-specific 15mers in the subpools with strong responses were then deconvoluted, evaluated for activities, and MHC Class I-restricted epitopes within the active 15mers were predicted using NetMHCpan algorithms. The predicted epitopes were synthesized and evaluated in the FluoroSpot IFN-γ and GzB assays. Protected and some non-protected participants had similar responses to individual antigen-specific peptide subpools, which did not distinguish only protected participants. However, deconvoluted antigen-specific positive subpools with high magnitudes of responses revealed individual 15mer peptides containing specific and/or predicted MHC Class I (HLA) epitopes. Responses to epitopes were either IFN-γ-only, IFN-γ and GzB, or GzB-only. Due to limitation of cells, most of the analysis concentrated on the identification of protection associated AMA1 epitopes, since most of the predominant pool specific responses were generated against AMA1 15mer subpools. Furthermore, we previously identified protection associated HLA class I-restricted epitopes in a previous gene-based vaccine trial. Seven predicted minimal epitopes in AMA1 were synthesized and upon testing, five recalled responses from protected participants confirming their possible contribution and association with protection, and two recalled responses from non-protected participants. Two protection-associated epitopes were promiscuous and may have also contributed to protection by recognition of different HLA alleles. In addition, strongly positive antigen-specific 15mers identified within active antigen-specific subpools contained 39 predicted but not tested epitopes were identified in CSP, AMA1 and TRAP. Finally, some non-protected individuals recognized HLA-matched protection-associated minimal epitopes and we discuss possible reasons. Other factors such as HLA allele fine specificity or interaction between other HLA alleles in same individual may also influence protective efficacy. This integrated approach using immunoassays and bioinformatics identified and confirmed AMA1-MHC Class I-restricted epitopes and a list of predicted additional epitopes which could be evaluated in future studies to assess possible association with protection against CHMI in the Phase 1 trial participants. The results suggest that identification of protection-associated epitopes within malaria antigens is feasible and can help design potent next generation multi-antigen, multi-epitope malaria vaccines for a genetically diverse population and to develop robust assays to measure protective cellular immunity against pre-erythrocytic stages of malaria. This approach can be used to develop vaccines for other novel emerging infectious disease pathogens.

A DNA-prime/human adenovirus serotype 5 (HuAd5) boost vaccine encoding Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP) and Pf apical membrane antigen-1 (PfAMA1), elicited protection in 4/15 (27%) of subjects against controlled human malaria infection (CHMI) that was statistically associated with CD8+ T cell responses. Subjects with high level pre-existing immunity to HuAd5 were not protected, suggesting an adverse effect on vaccine efficacy (VE). We replaced HuAd5 with chimpanzee adenovirus 63 (ChAd63), and repeated the study, assessing both the two-antigen (CSP, AMA1 = CA) vaccine, and a novel three-antigen (CSP, AMA1, ME-TRAP = CAT) vaccine that included a third pre-erythrocytic stage antigen [malaria multiple epitopes (ME) fused to the Pf thrombospondin-related adhesive protein (TRAP)] to potentially enhance protection. This was an open label, randomized Phase 1 trial, assessing safety, tolerability, and VE against CHMI in healthy, malaria naïve adults. Forty subjects (20 each group) were to receive three monthly CA or CAT DNA priming immunizations, followed by corresponding ChAd63 boost four months later. Four weeks after the boost, immunized subjects and 12 infectivity controls underwent CHMI by mosquito bite using the Pf3D7 strain. VE was assessed by determining the differences in time to parasitemia as detected by thick blood smears up to 28-days post CHMI and utilizing the log rank test, and by calculating the risk ratio of each treatment group and subtracting from 1, with significance calculated by the Cochran-Mantel-Haenszel method. In both groups, systemic adverse events (AEs) were significantly higher after the ChAd63 boost than DNA immunizations. Eleven of 12 infectivity controls developed parasitemia (mean 11.7 days). In the CA group, 15 of 16 (93.8%) immunized subjects developed parasitemia (mean 12.0 days). In the CAT group, 11 of 16 (63.8%) immunized subjects developed parasitemia (mean 13.0 days), indicating significant protection by log rank test compared to infectivity controls (p = 0.0406) and the CA group (p = 0.0229). VE (1 minus the risk ratio) in the CAT group was 25% compared to -2% in the CA group. The CA and CAT vaccines induced robust humoral (ELISA antibodies against CSP, AMA1 and TRAP, and IFA responses against sporozoites and Pf3D7 blood stages), and cellular responses (IFN-[gamma] FluoroSpot responses to CSP, AMA1 and TRAP) that were not associated with protection. This study demonstrated that the ChAd63 CAT vaccine exhibited significant protective efficacy, and confirmed protection was afforded by adding a third antigen (T) to a two-antigen (CA) formulation to achieve increased VE. Although the ChAd63-CAT vaccine was associated with increased frequencies of systemic AEs compared to the CA vaccine and, historically, compared to the HuAd5 vectored malaria vaccine encoding CSP and AMA1, they were transient and associated with increased vector dosing.

Human challenge models for enterotoxigenic Escherichia coli (ETEC) facilitate vaccine down-selection. The B7A (O148:H28 CS6+LT+ST+) strain is important for vaccine development. We sought to refine the B7A model by identifying a dose and fasting regimen consistently inducing moderate-severe diarrhea. An initial cohort of 28 subjects was randomized (1:1:1:1) to receive B7A following an overnight fast at doses of 108 or 109 colony forming units (cfu) or a 90-minute fast at doses of 109 or 1010 cfu. A second cohort included naïve and rechallenged subjects who had moderate-severe diarrhea and were given the target regimen. Immune responses to important ETEC antigens were assessed. Among subjects receiving 108 cfu of B7A, overnight fast, or 109 cfu, 90-minute fast, 42.9% (3/7) had moderate-severe diarrhea. Higher attack rates (71.4%; 5/7) occurred in subjects receiving 109 cfu, overnight fast, or 1010 cfu, 90-minute fast. Upon rechallenge with 109 cfu of B7A, overnight fast, 5/11 (45.5%) had moderate-severe diarrhea; the attack rate among concurrently challenge naïve subjects was 57.9% (11/19). Anti-CS6, O148 LPS and LT responses were modest across all groups. An overnight fast enabled a reduction in the B7A inoculum dose; however, the attack rate was inconsistent and protection upon rechallenge was minimal.

Melioidosis is a severe infectious disease caused by the gram-negative soil bacterium Burkholderia pseudomallei. Melioidosis is well known to be a major cause of morbidity and mortality in Southeast Asia, particularly in Thailand. However, melioidosis remains underreported in surrounding areas such as Cambodia. We report a case series of melioidosis in seven patients from Takeo Province, Cambodia. The patients, aged 24-65 years, were enrolled from May 2014 to May 2015 during a one year prospective study of sepsis at Takeo Provincial Hospital. They presented with fever, rigors, dyspnea, fatigue, diaphoresis, productive cough, and skin abscesses. Six of the seven patients were also hyponatremic. B. pseudomallei was cultured from the blood of six patients and the sputum of one patient. In this manuscript, we provide a detailed description of the clinical presentation, case management and laboratory confirmation of B. pseudomallei, as well as discuss the difficulties of identifying and treating melioidosis in low resource settings.

Background Travelers’ diarrhea is a common malady afflicting up to 50% of travelers after a 2-week travel period. An appreciable percentage of these cases will become persistent or chronic. We summarized the published literature reporting persistent/chronic diarrhea in travelers elucidating current understanding of disease incidence, etiology and regional variability. Methods We searched electronic databases (Medline, Embase, and Cochrane database of clinical trials) from 1990 to 2015 using the following terms: “chronic or persistent diarrh* and (returning) travel* or enteropathogen, GeoSentinel, and travel-associated infection. Included studies published in the English language on adult returning travelers (duration < 3-months) reporting denominator data. Point estimates and standard 95% confidence intervals were calculated for incidence using a random-effects model. Study incidence heterogeneity rates were assessed using x 2 heterogeneity statistics, graphically represented with Forest plots. Results We identified 19 studies meeting the inclusion criteria (all published after 1999). 18 studies reported upon the incidence of persistent/chronic diarrhea as a syndromic diagnosis in returning travelers; one study reported adequate denominator data from which to assess pathogen specific etiology. Giardiasis comprise an appreicaible percentage of infectious mediated persistent/chronic diarrhea in returning travelers. The overall estimate of persistent/chronic diarrhea incidence was 6% (0.05–0.07) in 321,454, travelers; with significant heterogeniety observed across regions. The total number of regional travelers, and point estimates for incidence (95% CI) for Latin American, African, and Asian travelers were [15816 (0.09 [0.07–0.11]), 42290 (0.06 [0.05–0.07]), and 27433 (0.07 [0.06–0.09])] respectively. We identified lower published rates of chronic diarrhea from Sub-Saharan Africa relative to [North Africa, South Central Asia, and Central America]. Persistent/chronic diarrhea ranked fourth as a syndromic diagnosis in all regions. Conclusions Persistent/Chronic diarrhea is a leading syndromic diagnosis in returning travelers across all regions. The 6% incidence [proportionate morbidity (PM) of 60] observed in over >300,000 global travelers is comparable to prior estimates. We identified lower published rates of chronic diarrhea from Sub-Saharan Africa relative to [North Africa, South Central Asia, and Central America]. Giardiasis comprises an appreciabile percentatge of travel-associated infectious mediated persistent/chronic diarrhea. There’s a dearth of published data characterizing the incidence of specific enteropathogenic etiologies for persistent/chronic diarrhea in returning travelers.

A three-antigen DNA-prime/chimpanzee adenovirus 63 (ChAd63) boost vaccine containing pre-erythrocytic Plasmodium falciparum (Pf) circumsporozoite protein (CSP), Pf apical membrane antigen-1 (AMA1) and malaria multiple epitopes (ME) fused to Pf thrombospondin-related adhesion protein (ME-TRAP) elicited higher vaccine efficacy (VE) in an open label, randomized Phase 1 trial against controlled human malaria infection (CHMI) than the two-antigen vaccine DNA/Human Adenovirus 5 (HuAd5) containing CSP and AMA1. The objective of this follow-up study was to determine whether responses to CSP, AMA1 or TRAP MHC Class I-restricted epitopes were associated with VE. Protected (n = 6) and non-protected participants (n = 26) were screened in FluoroSpot interferon gamma (IFN-[gamma]) and Granzyme B (GzB) assays using antigen-specific 15mer peptide subpools spanning CSP (n = 9 subpools), AMA1 (n = 12 subpools), and TRAP (n = 11 subpools). Individual antigen-specific 15mers in the subpools with strong responses were then deconvoluted, evaluated for activities, and MHC Class I-restricted epitopes within the active 15mers were predicted using NetMHCpan algorithms. The predicted epitopes were synthesized and evaluated in the FluoroSpot IFN-[gamma] and GzB assays. Protected and some non-protected participants had similar responses to individual antigen-specific peptide subpools, which did not distinguish only protected participants. However, deconvoluted antigen-specific positive subpools with high magnitudes of responses revealed individual 15mer peptides containing specific and/or predicted MHC Class I (HLA) epitopes. Responses to epitopes were either IFN-[gamma]-only, IFN-[gamma] and GzB, or GzB-only. Due to limitation of cells, most of the analysis concentrated on the identification of protection associated AMA1 epitopes, since most of the predominant pool specific responses were generated against AMA1 15mer subpools. Furthermore, we previously identified protection associated HLA class I-restricted epitopes in a previous gene-based vaccine trial. Seven predicted minimal epitopes in AMA1 were synthesized and upon testing, five recalled responses from protected participants confirming their possible contribution and association with protection, and two recalled responses from non-protected participants. Two protection-associated epitopes were promiscuous and may have also contributed to protection by recognition of different HLA alleles. In addition, strongly positive antigen-specific 15mers identified within active antigen-specific subpools contained 39 predicted but not tested epitopes were identified in CSP, AMA1 and TRAP. Finally, some non-protected individuals recognized HLA-matched protection-associated minimal epitopes and we discuss possible reasons. Other factors such as HLA allele fine specificity or interaction between other HLA alleles in same individual may also influence protective efficacy. This integrated approach using immunoassays and bioinformatics identified and confirmed AMA1-MHC Class I-restricted epitopes and a list of predicted additional epitopes which could be evaluated in future studies to assess possible association with protection against CHMI in the Phase 1 trial participants. The results suggest that identification of protection-associated epitopes within malaria antigens is feasible and can help design potent next generation multi-antigen, multi-epitope malaria vaccines for a genetically diverse population and to develop robust assays to measure protective cellular immunity against pre-erythrocytic stages of malaria. This approach can be used to develop vaccines for other novel emerging infectious disease pathogens.

Abstract Background The relationship between postvaccination symptoms and strength of antibody responses is unclear. The goal of this study was to determine whether adverse effects caused by vaccination with the Pfizer/BioNTech BNT162b2 vaccine are associated with the magnitude of vaccine-induced antibody levels. Methods We conducted a single-center, observational cohort study consisting of generally healthy adult participants that were not severely immunocompromised, had no history of coronavirus disease 2019, and were seronegative for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein before vaccination. Severity of vaccine-associated symptoms was obtained through participant-completed questionnaires. Testing for immunoglobulin G antibodies against SARS-CoV-2 spike protein and receptor-binding domain was conducted using microsphere-based multiplex immunoassays performed on serum samples collected at monthly visits. Neutralizing antibody titers were determined by microneutralization assays. Results Two hundred six participants were evaluated (69.4% female, median age 41.5 years old). We found no correlation between vaccine-associated symptom severity scores and vaccine-induced antibody titers 1 month after vaccination. We also observed that (1) postvaccination symptoms were inversely correlated with age and weight and more common in women, (2) systemic symptoms were more frequent after the second vaccination, (3) high symptom scores after first vaccination were predictive of high symptom scores after second vaccination, and (4) older age was associated with lower titers. Conclusions Lack of postvaccination symptoms after receipt of the BNT162b2 vaccine does not equate to lack of vaccine-induced antibodies 1 month after vaccination. We found no correlation between BNT162b2-associated symptom severity and vaccine-induced antibody titers 1 month after vaccination. Adverse effects inversely correlated with age and weight, whereas symptom severity after first vaccination was predictive of that after second vaccination.

Neisseria gonorrhoeae antimicrobial resistance (AMR) surveillance is essential for tracking the emergence and spread of AMR strains in local, national and international populations. This is crucial for developing or refining treatment guidelines. N. gonorrhoeae multiantigen sequence typing (NG-MAST) is beneficial for describing the molecular epidemiology of gonococci at national and international levels. Elucidation of AMR determinants to [beta]-lactam drugs, is a means of monitoring the development of resistance. In Ghana, little is known about the current gonococcal AMR prevalence and no characterization of gonococcal isolates has been previously performed. In this study, gonococcal isolates (n = 44) collected from five health facilities in Ghana from 2012 to 2015, were examined using AMR testing, NG-MAST and sequencing of penA. High rates of resistance were identified to tetracycline (100%), benzylpenicillin (90.9%), and ciprofloxacin (81.8%). One isolate had a high cefixime MIC (0.75 [mu]g/ml). Twenty-eight NG-MAST sequence types (STs) were identified, seventeen of which were novel. The isolate with the high cefixime MIC contained a mosaic penA-34 allele and belonged to NG-MAST ST1407, an internationally spreading multidrug-resistant clone that has accounted for most cefixime resistance in many countries. In conclusion, AMR testing, NG-MAST, and sequencing of the AMR determinant penA, revealed high rates of resistance to tetracycline, benzylpenicillin, and ciprofloxacin; as well as a highly diverse population of N. gonorrhoeae in Ghana. It is imperative to continue with enhanced AMR surveillance and to understand the molecular epidemiology of gonococcal strains circulating in Ghana and other African countries.

We report a case of successful treatment of chronic osteomyelitis (emanating from contaminated soil exposure) caused by Clostridium sphenoides, an organism infrequently identified as a cause of human infection and more saliently osteomyelitis (only 1 reported case in the literature). Additional impetus for reporting this case resides in the insights gained regarding pathogen identification exploiting sophisticated molecular platforms coupled to traditional microbial culture-based methods. The fastidious nature of cultivating anaerobic organisms required initial attempts at 16S rRNA sequencing to identify a Clostridium species (Clostridium celerecrescens). However, on exploiting matrix-assisted laser desorption ionization time of flight (MALDI TOF) technology, C. sphenoides was identified, and confirmed on whole genome sequencing. The discrepancies noted in the varying platforms require vigilance to seek complementary testing for conflicting results. Although highly accurate, the MALDI TOF and 16S rRNA sequencing platforms are not immune to false identification particularly in differentiating closely related organisms. More germane, whole genome sequencing should be entertained when conflicting results are obtained from MALDI TOF and 16S rRNA sequencing. Precise species and/or strain level identification can be clinically relevant as antimicrobial sensitivity profiles may be discrepant between closely related species influencing clinical outcomes. Thus, it is incumbent on us to strive to acquire the correct species characterization when resources allow to dictate optimal treatment.