Molecular identification of Crimean-Congo haemorrhagic fever virus in Hyalomma rufipes and Amblyomma variegatum in the Upper East Region of Ghana.

Sampled ticks were screened for Crimean-Congo haemorrhagic fever virus (CCHFV) using an assay that targets the nucleoprotein gene region of the S segment, a conserved region of the CCHFV genome. Minimum infection rates of 0.34% and 0.10% were obtained when testing pools of Hyalomma rufipes and Amblyomma variegatum, respectively. Next-generation sequencing and phylogenetic analysis showed that the S and L segments of the CCHFV isolate clustered with those of similar isolates of genotype III. However, analysis of the M segment showed that reassortment had occurred, causing this segment to cluster with those of isolates of genotype I, providing the first evidence of such an occurrence in Ghana.

Klebsiella pneumoniae ST147 harboring blaNDM-1, multidrug resistance and hypervirulence plasmids.

The spread of hypervirulent (hv) and carbapenem-/multidrug-resistant Klebsiella pneumoniae is an emerging problem in healthcare settings. The New Delhi metallo-ß-lactamase-1 (blaNDM-1) is found in Enterobacteriaceae including K. pneumoniae. The blaNDM-1 is capable of hydrolyzing ß-lactam antibiotics which are used for treatment of severe infections caused by multidrug-resistant Gram-negative bacteria. This is associated with the unacceptably high mortality rate in immunocompromised burn injury patients. This study reports on the characterization of blaNDM-1 gene and virulence factors in hv carbapenem-/multidrug-resistant K. pneumoniae ST147 in the burns unit of a tertiary teaching hospital during routine surveillance. Two K. pneumoniae strains were obtained from wounds of burn-infected patients from May 2020 to July 2021. The hypervirulence genes and genetic context of the blaNDM-1 gene and mobile genetic elements potentially involved in the transposition of the gene were analyzed. We identified a conserved genetic background and an IS26 and open reading frame flanking the blaNDM-1 gene that could suggest its involvement in the mobilization of the gene. The plasmid harbored additional antibiotic resistance predicted regions that were responsible for resistance to almost all the routinely used antibiotics. To ensure the identification of potential outbreak strains during routine surveillance, investigations on resistance genes and their environment in relation to evolution are necessary for molecular epidemiology.IMPORTANCEData obtained from this study will aid in the prompt identification of disease outbreaks including evolving resistance and virulence of the outbreak bacteria. This will help establish and implement antimicrobial stewardship programs and infection prevention protocols in fragile health systems in countries with limited resources. Integration of molecular surveillance and translation of whole-genome sequencing in routine diagnosis will provide valuable data for control of infection. This study reports for the first time a high-risk clone K. pneumoniae ST147 with hypervirulence and multidrug-resistance features in Ghana.

First Molecular Identification of Rickettsia aeschlimannii and Rickettsia africae in Ticks from Ghana.

The threats from vector-borne pathogens transmitted by ticks place people (including deployed troops) at increased risk for infection, frequently contributing to undifferentiated febrile illness syndromes. Wild and domesticated animals are critical to the transmission cycle of many tick-borne diseases. Livestock can be infected by ticks, and serve as hosts to tick-borne diseases such as rickettsiosis. Thus, it is necessary to identify the tick species and determine their potential to transmit pathogens. A total of 1,493 adult ticks from three genera-Amblyomma, Hyalomma, and Rhipicephalus-were identified using available morphological keys and were pooled (n = 541) by sex and species. Rickettsia species were detected in 308 of 541 (56.9%) pools by genus-specific quantitative polymerase chain reaction assay (Rick17b). Furthermore, sequencing of the outer membrane protein A and B genes (ompA and ompB) of random samples of Rickettsia-positive samples led to the identification of Rickettsia aeschlimannii and Rickettsia africae with most R. africae DNA (80.2%) detected in pools of Amblyomma variegatum. We report the first molecular detection and identification of the rickettsial pathogens R. africae and R. aeschlimannii in ticks from Ghana. Our findings suggest there is a need to use control measures to prevent infections from occurring among human populations in endemic areas in Ghana. This study underscores the importance of determining which vector-borne pathogens are in circulation in Ghana. Further clinical and prevalence studies are needed to understand more comprehensively the clinical impact of these rickettsial pathogens contributing to human disease and morbidity in Ghana.

Whole genome sequencing of outbreak strains from 2017 to 2018 reveals an endemic clade of dengue 1 virus in Cameroon.

Dengue fever is expanding as a global public health threat including countries within Africa. For the past few decades, Cameroon has experienced sporadic cases of arboviral infections including dengue fever. Here, we conducted genomic analyses to investigate the origin and phylogenetic profile of Cameroon DENV-1 outbreak strains and predict the impact of emerging therapeutics on these strains. Bayesian and maximum-likelihood phylogenetic inference approaches were employed in virus evolutionary analyses. An in silico analysis was performed to assess the divergence in immunotherapeutic and vaccine targets in the new genomes. Six complete DENV-1 genomes were generated from 50 samples that met a clinical definition for DENV infection. Phylogenetic analyses revealed that the strains from the current study belong to a sub-lineage of DENV-1 genotype V and form a monophyletic taxon with a 2012 strain from Gabon. The most recent common ancestor (TMRCA) of the Cameroon and Gabon strains was estimated to have existed around 2008. Comparing our sequences to the vaccine strains, 19 and 15 amino acid (aa) substitutions were observed in the immuno-protective prM-E protein segments of the Dengvaxia® and TetraVax-DV-TV003 vaccines, respectively. Epitope mapping revealed mismatches in aa residues at positions E155 and E161 located in the epitope of the human anti-DENV-1 monoclonal antibody HMAb 1F4. The new DENV strains constitute a conserved genomic pool of viruses endemic to the Central African region that needs prospective monitoring to track local viral evolution. Further work is needed to ascertain the performance of emerging therapeutics in DENV strains from the African region.

Detection of Leishmania DNA in Phlebotomine Sand Flies in Tsatee, a Community in the Volta Region, Ghana.

Leishmania parasites, which are spread by infected female sand flies, are the cause of the disease leishmaniasis. Although cutaneous leishmaniasis has been found to occur in the Volta Region, there is limited data on vector species and reservoirs. This study focused on the Tsatee community, in the South Dayi District of the Volta Region, and is aimed at identifying the sand fly fauna and detecting the presence of Leishmania DNA by the use of primers that target the conserved region of Leishmania spp. minicircle DNA of the parasite kinetoplast. The miniature light traps and hand aspirators provided by the Centers for Disease Control and Prevention (CDC) were used to collect outdoor and indoor sand flies for five months in a guinea woodland and semideciduous forest area. From the collections, 4,580 phlebotomine sand flies were obtained and identified, and females were examined for Leishmania DNA using PCR. The male flies were 1,202 (26.24%), non-blood-fed females were 3,321 (72.51%), and 57 (1.25%) were blood-fed females. It was observed that Sergentomyia species constituted 99.91% of the total collected sand flies with S. africana (76.77%) as the predominant species. Phlebotomus rodhaini (0.09%) was the only Phlebotomus species identified from the study area. From 283 non-blood-fed sand fly pools and 57 individual blood-fed species screened, Leishmania DNA was detected in 12 (4.24%) pools and 8 (14.04%) individuals, respectively. It was observed that Leishmania DNA was detected in all the sand fly species identified except S. collarti. This study reports the first detection of Leishmania DNA in P. rodhaini in Ghana, with an infection rate of 33.33% (95% CI, 1.23-88.32). The findings suggest that the role of Phlebotomus in disease transmission in the study area cannot be discounted. Future studies should include continuous surveillance, blood meal preferences, and vector competence of the various infected phlebotomine sand flies to create effective control measures.

Molecular Characterization of Circulating Yellow Fever Viruses from Outbreak in Ghana, 2021-2022.

Yellow fever virus, transmitted by infected Aedes spp. mosquitoes, causes an acute viral hemorrhagic disease. During October 2021-February 2022, a yellow fever outbreak in some communities in Ghana resulted in 70 confirmed cases with 35 deaths (case-fatality rate 50%). The outbreak started in a predominantly unvaccinated nomadic community in the Savannah region, from which 65% of the cases came. The molecular amplification methods we used for diagnosis produced full-length DNA sequences from 3 confirmed cases. Phylogenetic analysis characterized the 3 sequences within West Africa genotype II; strains shared a close homology with sequences from Cote d'Ivoire and Senegal. We deployed more sensitive advanced molecular diagnostic techniques, which enabled earlier detection, helped control spread, and improved case management. We urge increased efforts from health authorities to vaccinate vulnerable groups in difficult-to-access areas and to educate the population about potential risks for yellow fever infections.

Whole genome analysis and antimicrobial resistance of Neisseria gonorrhoeae isolates from Ghana.

Introduction: Gonorrhoea is a major public health concern. With the global emergence and spread of resistance to last-line antibiotic treatment options, gonorrhoea threatens to be untreatable in the future. Therefore, this study performed whole genome characterization of Neisseria gonorrhoeae collected in Ghana to identify lineages of circulating strains as well as their phenotypic and genotypic antimicrobial resistance (AMR) profiles. Methods: Whole genome sequencing (WGS) was performed on 56 isolates using both the Oxford Nanopore MinION and Illumina MiSeq sequencing platforms. The Comprehensive Antimicrobial Resistance Database (CARD) and PUBMLST.org/neisseria databases were used to catalogue chromosomal and plasmid genes implicated in AMR. The core genome multi-locus sequence typing (cgMLST) approach was used for comparative genomics analysis. Results and Discussion: In vitro resistance measured by the E-test method revealed 100%, 91.0% and 85.7% resistance to tetracycline, penicillin and ciprofloxacin, respectively. A total of 22 sequence types (STs) were identified by multilocus sequence typing (MLST), with ST-14422 (n = 10), ST-1927 (n = 8) and ST-11210 (n = 7) being the most prevalent. Six novel STs were also identified (ST-15634, 15636-15639 and 15641). All isolates harboured chromosomal AMR determinants that confer resistance to beta-lactam antimicrobials and tetracycline. A single cefixime-resistant strain, that belongs to N. gonorrhoeae multiantigen sequence type (NG-MAST) ST1407, a type associated with widespread cephalosporin resistance was identified. Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR), identified 29 unique sequence types, with ST-464 (n = 8) and the novel ST-3366 (n = 8) being the most prevalent. Notably, 20 of the 29 STs were novel, indicative of the unique nature of molecular AMR determinants in the Ghanaian strains. Plasmids were highly prevalent: pTetM and pblaTEM were found in 96% and 92% of isolates, respectively. The TEM-135 allele, which is an amino acid change away from producing a stable extended-spectrum ß-lactamase that could result in complete cephalosporin resistance, was identified in 28.5% of the isolates. Using WGS, we characterized N. gonorrhoeae strains from Ghana, giving a snapshot of the current state of gonococcal AMR in the country and highlighting the need for constant genomic surveillance.

SARS-CoV-2 Molecular Evolutionary Dynamics in the Greater Accra Region, Ghana.

To assess dynamics of SARS-CoV-2 in Greater Accra Region, Ghana, we analyzed SARS-CoV-2 genomic sequences from persons in the community and returning from international travel. The Accra Metropolitan District was a major origin of virus spread to other districts and should be a primary focus for interventions against future infectious disease outbreaks.

A Preliminary Study to Compare Recombinase Polymerase Amplification-Lateral Flow and Quantitative PCR in the Detection of Cutaneous Leishmania in Communities from the Volta Region of Ghana.

Background: Leishmaniasis is a parasitic disease that mostly affects populations in tropical and subtropical countries. In Ghana, cutaneous leishmaniasis (CL) is the most common form of the disease affecting communities of the Volta Region. Conventional parasitological method (microscopy) is the commonly used test for CL diagnosis in many endemic countries, but has low sensitivity in chronic cases. Therefore, there is a clear need for a sensitive and easy-to-use point-of-care diagnostic method like an isothermal recombinase polymerase amplification-lateral flow (RPA-LF) test, suitable for use in austere and low-resource settings for the identification of CL cases. This study compared the efficacy of RPA-LF test with quantitative PCR (qPCR) in detecting Leishmania in suspected CL cases from the Volta Region. Methods: Twenty-five participants between 5 and 14 years were enrolled in the study from whom a total of 26 samples were obtained. Lesion samples were collected using FTA® filter papers applied to ulcerated lesions for molecular diagnosis. DNA isolated from filter papers was used for both the RPA-LF test and qPCR. Results: Twenty-two participants (88%) presented with one or two ulcerated active lesions per individual, while the rest of them had plaques or dried lesions. Among the 26 samples, 19/26 (73%) had concordant results when comparing the two diagnostic methods. Conclusion: Data from this study suggest that the RPA-LF test can be used in addition to a conventional parasitological diagnostic test (microscopy) to detect CL cases in communities of the Volta Region.

Occurrence of influenza and bacterial infections in cancer patients receiving radiotherapy in Ghana.

BACKGROUND: Influenza co-infection with bacteria is a leading cause of influenza-related deaths and severe respiratory infections, especially among high-risk groups like cancer patients undergoing treatment. However, acute respiratory infection (ARI)-like symptoms developed by upper-torso cancer (UTC) patients receiving radiotherapy are considered as side-effects of the radiation. Hence influenza and bacterial pathogens implicated in ARI are not investigated. METHODS: This prospective cohort study examined 85 in-patients with upper-torso cancers undergoing radiotherapy at the National Radiotherapy, Oncology and Nuclear Medicine Centre (NRONMC) of Korle-Bu Teaching Hospital (KBTH) in Accra, Ghana. Eligible patients who consented were recruited into the study from September 2018 to April 2019. Influenza viruses A and B in addition to the following bacteria species Streptococcus pneumonia, Haemophilus influenzae, Neisseria meningitidis and Staphylococcus aureus were detected from oropharyngeal and nasopharyngeal swab specimens collected at three different time points. Presence of respiratory pathogens were investigated by influenza virus isolation in cell culture, bacterial culture, polymerase chain reaction (PCR) and next generation sequencing (NGS) assays. RESULTS: Of the 85 eligible participants enrolled into the study, 87% were females. Participants were 17 to 77 years old, with a median age of 49 years. Most of the participants (88%) enrolled had at least one pathogen present. The most prevalent pathogen was N. meningitidis (63.4%), followed by H. influenzae (48.8%), Influenza viruses A and B (32.9%), S. pneumoniae (32.9%) and S. aureus (12.2%). Approximately, 65% of these participants developed ARI-like symptoms. Participants with previous episodes of ARI, did not live alone, HNC and total radiation less than 50 Gy were significantly associated with ARI. All treatment forms were also significantly associated with ARI. CONCLUSION: Data generated from the study suggests that ARI-like symptoms observed among UTC patients receiving radiotherapy in Ghana, could be due to influenza and bacterial single and co-infections in addition to risk factors and not solely the side-effects of radiation as perceived. These findings will be prime importance for diagnosis, prevention, treatment and control for cancer patients who present with such episodes during treatment.

Retrospective Genomic Characterization of a 2017 Dengue Virus Outbreak, Burkina Faso.

Knowledge of contemporary genetic composition of dengue virus (DENV) in Africa is lacking. By using next-generation sequencing of samples from the 2017 DENV outbreak in Burkina Faso, we isolated 29 DENV genomes (5 serotype 1, 16 serotype 2 [DENV-2], and 8 serotype 3). Phylogenetic analysis demonstrated the endemic nature of DENV-2 in Burkina Faso. We noted discordant diagnostic results, probably related to genetic divergence between these genomes and the Trioplex PCR. Forward and reverse1 primers had a single mismatch when mapped to the DENV-2 genomes, probably explaining the insensitivity of the molecular test. Although we observed considerable homogeneity between the Dengvaxia and TetraVax-DV-TV003 vaccine strains as well as B cell epitopes compared with these genomes, we noted unique divergence. Continual surveillance of dengue virus in Africa is needed to clarify the ongoing novel evolutionary dynamics of circulating virus populations and support the development of effective diagnostic, therapeutic, and preventive countermeasures.

Chikungunya viruses containing the A226V mutation detected retrospectively in Cameroon form a new geographical subclade.

BACKGROUND: Chikungunya virus (CHIKV) is a re-emerging arbovirus associated with sporadic outbreaks in Cameroon since 2006. Viral whole genomes were generated to analyze the origins of evolutionary lineages, the potential of emergence/re-emergence, and to infer transmission dynamics of recent Cameroon CHIKV outbreak strains. METHODS: Samples collected between 2016 and 2019 during CHIKV outbreaks in Cameroon were screened for CHIKV using reverse transcription PCR (RT-PCR), followed by whole genome sequencing of positive samples. RESULTS: Three coding-complete CHIKV genomes were obtained from samples, which belong to an emerging sub-lineage of the East/Central/South African genotype and formed a monophyletic taxon with previous Central African strains. This clade, which we have named the new Central African clade, appears to be evolving at 3.0 × 10-4 nucleotide substitutions per site per year (95% highest posterior density (HPD) interval of 1.94 × 10-4 to 4.1 × 10-4). Notably, mutations in the envelope proteins (E1-A226V, E2-L210Q, and E2-I211T), which are known to enhance CHIKV adaptability and infectious potential in Aedes albopictus, were present in all strains and mapped to established high-density Ae. albopictus populations. CONCLUSIONS: These new CHIKV strains constitute a conserved genomic pool of an emerging sub-lineage, reflecting a putative vector host adaptation to Ae. albopictus, which has practically displaced Aedes aegypti from select regions of Cameroon.

Whole Genome Sequencing and Antimicrobial Resistance of Staphylococcus aureus from Surgical Site Infections in Ghana.

Staphylococcus aureus (S. aureus) is a common cause of surgical site infections (SSIs) globally. Data on the occurrence of methicillin-susceptible S. aureus (MSSA) as well as methicillin-resistant S. aureus (MRSA) among patients with surgical site infections (SSIs) in sub-Saharan African are scarce. We characterized S. aureus from SSIs in Ghana using molecular methods and antimicrobial susceptibility testing (AST). Wound swabs or aspirate samples were collected from subjects with SSIs. S. aureus was identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS); AST was performed by Kirby-Bauer disk diffusion, and results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guideline. Detection of spa, mecA, and pvl genes was performed by polymerase chain reaction (PCR). Whole-genome sequencing (WGS) was done using the Illumina MiSeq platform. Samples were collected from 112 subjects, with 13 S. aureus isolates recovered. Of these, 92% were sensitive to co-trimoxazole, 77% to clindamycin, and 54% to erythromycin. Multi-drug resistance was detected in 5 (38%) isolates. The four mecA gene-positive MRSA isolates detected belonged to ST152 (n = 3) and ST5 (n = 1). In total, 62% of the isolates were positive for the Panton-Valentine leukocidin (pvl) toxin gene. This study reports, for the first time, a pvl-positive ST152-t355 MRSA clone from SSIs in Ghana. The occurrence of multi-drug-resistant S. aureus epidemic clones suggests that continuous surveillance is required to monitor the spread and resistance trends of S. aureus in hospital settings in the country.

Whole-genome sequence profiling of antibiotic-resistant Staphylococcus aureus isolates from livestock and farm attendants in Ghana.

OBJECTIVE: Epidemic methicillin-resistant Staphylococcus aureus (MRSA) clones have been described in Ghana, but so far, no typical livestock-associated MRSA isolates (CC398) have been found. In this study we provide baseline information on antimicrobial resistance, population structure, and virulence gene content of S. aureus isolates from livestock and farm attendants. METHODS: Nasal samples were collected from cattle, pigs, goats, sheep, and farm attendants from three farms. Staphylococcus aureus was identified by matrix-assisted laser desorption/ionisation time-of-flight and antimicrobial susceptibility testing was performed using VITEK II (Biomerieux, Marcy l'Etoile, France) and interpreted according to EUCAST guidelines. Whole-genome sequencing was performed using the Illumina, San Diego, CA, USA MiSeq Platform. RESULTS: In total, 401 nasal swab samples were obtained from 57 farm attendants, 208 pigs, 30 goats, 26 sheep, and 80 cattle. The S. aureus isolates (n = 25) recovered (farm attendants: n = 10; pigs: n = 8; and goats: n = 7) were frequently resistant to penicillin (68%), tetracycline (44%), and ciprofloxacin (32%); two human isolates were MRSA. Twelve isolates (48%) were multidrug resistant (MDR) (>3 classes). Genome sequencing of the isolates revealed ST152-t355, ST9-t1430, and ST133-t8662 as dominant clones among farm attendants, pigs, and goats, respectively. The two MRSA isolates detected belonged to ST8-t334 and ST152-t355. The scn and sak genes associated with human-adaption were detected in 10 isolates; 9 from humans and 1 from a goat. Typing results provided evidence of a single potential transmission event (t861, PVL-, scn+). CONCLUSION: No MRSA was detected among livestock, perhaps because of low intensive farming; however, the relatively high prevalence of MDR isolates may be a result of inappropriate antibiotic usage in Ghanaian livestock production.

Antimicrobial resistance (AMR) and molecular characterization of Neisseria gonorrhoeae in Ghana, 2012-2015.

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 ß-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.