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1.  Diagnostic performance of the Luminex xTAG gastrointestinal pathogens panel to detect rotavirus in Ghanaian children with and without diarrhoea 
Virology Journal  2016;13:132.
Background
Rotavirus is one of the leading causes of childhood diarrhoea worldwide. The highest disease burden is seen in resource-constrained settings of sub-Saharan Africa. Recently, commercial multiplex PCR panels proved their accuracy to diagnose infectious gastroenteritis in Europe and the USA. However, data on their performance using samples from tropical regions in general and to detect rotavirus in particular remains scant. We aimed to analyse the diagnostic performance of the Luminex xTAG gastrointestinal pathogens panel, a multiplex PCR, to detect rotavirus in stool samples from Ghanaian children.
Methods
A total of 682 stool samples were collected in the Ashanti region of Ghana between 2007 and 2008. Of these, 341 were from cases (children with diarrhoea), and another 341 from controls (children without diarrhoea). All samples were analysed using the Luminex xTAG assay and compared to a rotavirus quantitative reverse-transcription PCR (reference assay). Rotavirus reference assay positive samples were P and G genotyped by sequencing the rotavirus VP4 and VP7 genes.
Results
Overall agreement between the Luminex xTAG and the reference assay was excellent (kappa 0.93). The sensitivity and specificity was 88.2 % (95 % confidence interval [CI] 78.2–94.1) and 100 % (95 % CI 99.2–100), respectively. Of 76 rotavirus reference assay positive samples, 64 were successfully genotyped and the Luminex xTAG assay was able to detect all rotavirus genotypes present in the study.
Conclusion
The Luminex xTAG assay proved a sensitive and highly specific tool to detect rotavirus and may aid clinicians and public health authorities in the diagnosis and surveillance of rotavirus.
doi:10.1186/s12985-016-0588-1
PMCID: PMC4966796  PMID: 27473598
Rotavirus; Luminex xTAG gastrotinestinal pathogens panel; Stool samples; Performance; Sub-saharan Africa
2.  Evidence for an Ancestral Association of Human Coronavirus 229E with Bats 
Journal of Virology  2015;89(23):11858-11870.
ABSTRACT
We previously showed that close relatives of human coronavirus 229E (HCoV-229E) exist in African bats. The small sample and limited genomic characterizations have prevented further analyses so far. Here, we tested 2,087 fecal specimens from 11 bat species sampled in Ghana for HCoV-229E-related viruses by reverse transcription-PCR (RT-PCR). Only hipposiderid bats tested positive. To compare the genetic diversity of bat viruses and HCoV-229E, we tested historical isolates and diagnostic specimens sampled globally over 10 years. Bat viruses were 5- and 6-fold more diversified than HCoV-229E in the RNA-dependent RNA polymerase (RdRp) and spike genes. In phylogenetic analyses, HCoV-229E strains were monophyletic and not intermixed with animal viruses. Bat viruses formed three large clades in close and more distant sister relationships. A recently described 229E-related alpaca virus occupied an intermediate phylogenetic position between bat and human viruses. According to taxonomic criteria, human, alpaca, and bat viruses form a single CoV species showing evidence for multiple recombination events. HCoV-229E and the alpaca virus showed a major deletion in the spike S1 region compared to all bat viruses. Analyses of four full genomes from 229E-related bat CoVs revealed an eighth open reading frame (ORF8) located at the genomic 3′ end. ORF8 also existed in the 229E-related alpaca virus. Reanalysis of HCoV-229E sequences showed a conserved transcription regulatory sequence preceding remnants of this ORF, suggesting its loss after acquisition of a 229E-related CoV by humans. These data suggested an evolutionary origin of 229E-related CoVs in hipposiderid bats, hypothetically with camelids as intermediate hosts preceding the establishment of HCoV-229E.
IMPORTANCE The ancestral origins of major human coronaviruses (HCoVs) likely involve bat hosts. Here, we provide conclusive genetic evidence for an evolutionary origin of the common cold virus HCoV-229E in hipposiderid bats by analyzing a large sample of African bats and characterizing several bat viruses on a full-genome level. Our evolutionary analyses show that animal and human viruses are genetically closely related, can exchange genetic material, and form a single viral species. We show that the putative host switches leading to the formation of HCoV-229E were accompanied by major genomic changes, including deletions in the viral spike glycoprotein gene and loss of an open reading frame. We reanalyze a previously described genetically related alpaca virus and discuss the role of camelids as potential intermediate hosts between bat and human viruses. The evolutionary history of HCoV-229E likely shares important characteristics with that of the recently emerged highly pathogenic Middle East respiratory syndrome (MERS) coronavirus.
doi:10.1128/JVI.01755-15
PMCID: PMC4645311  PMID: 26378164
3.  Application of a multiplex PCR assay for the detection of gastrointestinal pathogens in a rural African setting 
BMC Infectious Diseases  2016;16:150.
Background
Despite high morbidity and mortality, the laboratory diagnosis of gastrointestinal infections is largely neglected in tropical African settings. This study aims to apply the Luminex multiplex PCR assay for the diagnosis of gastrointestinal pathogens in rural Ghana to evaluate its usefulness as a routine method.
Methods
A case–control study was conducted at the Agogo Presbyterian Hospital in Ghana. Stool samples were collected from children below 6 years of age with (cases) and without (controls) diarrhoea. Samples were screened for 15 different diarrhoeal pathogens by the Luminex xTAG GPP assay and associations between diarrhoea and gastrointestinal infections and fractions attributable to diarrhea (AF) were determined.
Results
The Luminex PCR assay identified organisms in 96.6 % (n = 428) of 443 cases and in 92.5 % (n = 221) of 239 selected controls. A mean of 2.5 (standard deviation [SD]: ±1.3) and 2.3 (SD: ±1.3) organisms per sample were detected in cases and controls respectively. An association with diarrhoea was found for rotavirus (adjusted odds ratio [aOR] = 7.2; 95 % confidence interval [CI]: 2.9–18.1), norovirus (aOR = 2.7; 95 % CI: 1.4–5.3) and Shigella spp. (aOR = 1.7; 95 % CI: 1.2–2.4) with respective AFs of 12.5 % (95 % CI: 9.6–15.3), 7.9 % (95 % CI: 3.8–11.7) and 16.9 % (95 % CI: 6.9–25.9).
Conclusion
The high proportion of pathogen-positive stool samples with a high number of co-infections in cases and controls suggests a substantial amount of transient or colonizing microorganisms for which treatment is not necessarily implicated. The use of sequential diagnostic algorithms with pathogen specific or quantitative PCRs might be most appropriate for diagnosing gastrointestinal infections.
doi:10.1186/s12879-016-1481-7
PMCID: PMC4832549  PMID: 27080387
Ghana; West Africa; Multiplex Polymerase Chain Reaction; Gastrointestinal infections
4.  Knowledge and Perceptions of Health Workers’ Training on Ethics, Confidentiality and Medico-Legal Issues 
Introduction
Health care delivery in recent times has become more complicated, as patients expect health personnel to not only provide professional services but be accountable as well. It is thus imperative that health personnel are aware of their responsibility to the patient and also sensitive to medico legal issues if quality health care is to be assured.
Objective
The aim of the study was to assess the knowledge and perception of health care workers on their training in ethics, confidentiality and medico-legal issues. It was expected that the results would inform policy on the training of the health workers.
Method
A cross-sectional survey was conducted among some categories of health workers (Doctors, Nurses and Health care assistants) at the Accident and Emergency directorate of Komfo Anokye Teaching Hospital, Ghana. A self-administered questionnaire was used to elicit information on ethics, confidentiality and medico- legal issues. Data collected was analyzed using SPSS version 16.
Results
A total of 103 health care workers were enrolled on the study representing 96% response rate. The study revealed that 74% had knowledge on ethics, confidentiality and medico- legal concepts; and 35.4% of the respondents indicated that health workers attitudes to ethics, confidentiality and medico- legal concepts was inadequate. About 28.3% indicated that their attitudes were good while 26.3% indicated attitudes were adequate with only 2% indicating that attitudes were very good. Nearly, 49% of the respondents also indicated that training on medico-legal issues should be taught during formal training and also on-the-job.
Conclusion
Knowledge of health workers on ethics confidentiality and medico-legal issues is high and their perceptions are positive. However, regular training to update their knowledge will be necessary in order to ensure continuous improvement of the quality of health care delivery.
doi:10.4172/2155-9627.1000205
PMCID: PMC4795178  PMID: 26998404
Health workers; Ethics; Confidentiality; Medicolegal issues; Knowledge and perception; Training
5.  Spatial heterogeneity of malaria in Ghana: a cross-sectional study on the association between urbanicity and the acquisition of immunity 
Malaria Journal  2016;15:84.
Background
Malaria incidence has declined considerably over the last decade. This is partly due to a scale-up of control measures but is also attributed to increasing urbanization. This study aimed to analyse the association between malaria and urbanization and the effect of urbanicity on the acquisition of semi-immunity.
Methods
In 2012, children with fever presenting to St Michael’s Hospital Pramso/Ghana were recruited. The malaria-positive-fraction (MPF) of fever cases was calculated on community-level to approximate the malaria risk. The mean age of malaria cases was calculated for each community to estimate the acquisition of semi-immunity. The level of urbanicity for the communities was calculated and associations between MPF, urbanicity and immunity were modelled using linear regression.
Results
Twenty-six villages were included into the study with a mean MPF of 35 %. A linear decrease of 5 % (95 % CI: 4–6 %) in MPF with every ten-point increase in urbanicity was identified. The mean age of malaria patients increased by 2.9 months (95 % CI: 1.0–4.8) with every ten-point increase in urbanicity.
Discussion
The results confirm an association between an increase in urbanicity and declining malaria risk and demonstrate that the acquisition of semi-immunity is heterogeneous on a micro-epidemiological scale and is associated with urbanicity.
Electronic supplementary material
The online version of this article (doi:10.1186/s12936-016-1138-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12936-016-1138-4
PMCID: PMC4751679  PMID: 26867774
Malaria; Ghana; Urbanization; Semi-immunity; Children
6.  Highly Divergent Hepaciviruses from African Cattle 
Journal of Virology  2015;89(11):5876-5882.
ABSTRACT
The hepatitis C virus (HCV; genus Hepacivirus) is a highly relevant human pathogen. Unique hepaciviruses (HV) were discovered recently in animal hosts. The direct ancestor of HCV has not been found, but the genetically most closely related animal HVs exist in horses. To investigate whether other peridomestic animals also carry HVs, we analyzed sera from Ghanaian cattle for HVs by reverse transcription-PCR (RT-PCR). Nine of 106 specimens from different sampling sites contained HV RNA (8.5%) at median viral loads of 1.6 × 105 copies/ml. Infection seemed unrelated to cattle age and gender. Near-full-genome sequencing of five representative viruses confirmed taxonomic classifications. Cattle HVs formed two distinct phylogenetic lineages that differed by up to 17.7% on the nucleotide level in the polyprotein-encoding region, suggesting cocirculation of different virus subtypes. A conserved microRNA122-binding site in the 5′ internal ribosomal entry site suggested liver tropism of cattle HVs. Phylogenetic analyses suggested the circulation of HVs in cattle for several centuries. Cattle HVs were genetically highly divergent from all other HVs, including HCV. HVs from genetically related equine and bovine hosts were not monophyletic, corroborating host shifts during the evolution of the genus Hepacivirus. Similar to equine HVs, the genetic diversity of cattle HVs was low compared to that of HCV genotypes. This suggests an influence of the human-modified ecology of peridomestic animals on virus diversity. Further studies should investigate the occurrence of cattle HVs in other geographic areas and breeds, virus pathogenicity in cattle, and the potential exposure of human risk groups, such as farmers, butchers, and abattoir workers.
IMPORTANCE HCV (genus Hepacivirus) is a major human pathogen, causing liver failure and cancer. Unique hepaciviruses (HVs) were discovered over the last few years in animals, but the direct ancestor of HCV has not been found. The animal HV most closely related to HCV so far originated from horses, suggesting that other livestock animals also harbor HVs. Therefore, we investigated African cattle and discovered previously unknown HVs at high prevalence and viral loads. Because of the agricultural importance of cattle, it may be relevant to investigate HV pathogenicity. The frequent exposure of humans to cattle also may warrant investigations of the zoonotic potential of these viruses. Evolutionary analyses suggested that cattle HVs have existed for centuries. Despite the genetic relatedness of their animal hosts, HVs from cattle and horses were not phylogenetically related, corroborating frequent host shifts during the evolution of the genus Hepacivirus.
doi:10.1128/JVI.00393-15
PMCID: PMC4442428  PMID: 25787289
7.  A Novel Rhabdovirus Isolated from the Straw-Colored Fruit Bat Eidolon helvum, with Signs of Antibodies in Swine and Humans 
Journal of Virology  2015;89(8):4588-4597.
ABSTRACT
Bats have been implicated as reservoirs of emerging viruses. Bat species forming large social groups and roosting in proximity to human communities are of particular interest. In this study, we sampled a colony of ca. 350,000 individuals of the straw-colored fruit bat Eidolon helvum in Kumasi, the second largest city of Ghana. A novel rhabdovirus (Kumasi rhabdovirus [KRV]) was isolated in E. helvum cell cultures and passaged to Vero cells as well as interferon-competent human and primate cells (A549 and MA104). Genome composition was typical for a rhabdovirus. KRV was detected in 5.1% of 487 animals, showing association with the spleen but not the brain. Antibody prevalence was 11.5% by immunofluorescence and 6.4% by plaque reduction virus neutralization test (PRNT). Detection throughout 3 sampling years was pronounced in both annual wet seasons, of which only one overlaps the postparturition season. Juvenile bats showed increased viral prevalence. No evidence of infection was obtained in 1,240 female mosquitos (6 different genera) trapped in proximity to the colony to investigate potential vector association. Antibodies were found in 28.9% (5.4% by PRNT) of 107 swine sera but not in similarly large collections of sheep, goat, or cattle sera. The antibody detection rate in human subjects with occupational exposure to the bat colony was 11% (5/45 persons), which was significantly higher than in unexposed adults (0.8% [1/118]; chi square, P < 0.001). KRV is a novel bat-associated rhabdovirus potentially transmitted to humans and swine. Disease associations should be investigated.
IMPORTANCE Bats are thought to carry a huge number of as-yet-undiscovered viruses that may pose epidemic threats to humans and livestock. Here we describe a novel dimarhabdovirus which we isolated from a large colony of the straw-colored fruit bat Eidolon helvum in Ghana. As these animals are exposed to humans and several livestock species, we looked for antibodies indicating infection in humans, cattle, swine, sheep, and goats. Signs of infection were found in swine and humans, with increased antibody findings in humans who are occupationally exposed to the bat colony. Our data suggest that it is worthwhile to look for diseases caused by the novel virus in humans and livestock.
doi:10.1128/JVI.02932-14
PMCID: PMC4442353  PMID: 25673701
8.  16S rRNA Gene Sequence-Based Identification of Bacteria in Automatically Incubated Blood Culture Materials from Tropical Sub-Saharan Africa 
PLoS ONE  2015;10(8):e0135923.
Background
The quality of microbiological diagnostic procedures depends on pre-analytic conditions. We compared the results of 16S rRNA gene PCR and sequencing from automatically incubated blood culture materials from tropical Ghana with the results of cultural growth after automated incubation.
Methods
Real-time 16S rRNA gene PCR and subsequent sequencing were applied to 1500 retained blood culture samples of Ghanaian patients admitted to a hospital with an unknown febrile illness after enrichment by automated culture.
Results
Out of all 1500 samples, 191 were culture-positive and 98 isolates were considered etiologically relevant. Out of the 191 culture-positive samples, 16S rRNA gene PCR and sequencing led to concordant results in 65 cases at species level and an additional 62 cases at genus level. PCR was positive in further 360 out of 1309 culture-negative samples, sequencing results of which suggested etiologically relevant pathogen detections in 62 instances, detections of uncertain relevance in 50 instances, and DNA contamination due to sample preparation in 248 instances. In two instances, PCR failed to detect contaminants from the skin flora that were culturally detectable. Pre-analytical errors caused many Enterobacteriaceae to be missed by culture.
Conclusions
Potentially correctable pre-analytical conditions and not the fastidious nature of the bacteria caused most of the discrepancies. Although 16S rRNA gene PCR and sequencing in addition to culture led to an increase in detections of presumably etiologically relevant blood culture pathogens, the application of this procedure to samples from the tropics was hampered by a high contamination rate. Careful interpretation of diagnostic results is required.
doi:10.1371/journal.pone.0135923
PMCID: PMC4535881  PMID: 26270631
9.  Human–Bat Interactions in Rural West Africa 
Emerging Infectious Diseases  2015;21(8):1418-1421.
Because some bats host viruses with zoonotic potential, we investigated human–bat interactions in rural Ghana during 2011–2012. Nearly half (46.6%) of respondents regularly visited bat caves; 37.4% had been bitten, scratched, or exposed to bat urine; and 45.6% ate bat meat. Human–bat interactions in rural Ghana are frequent and diverse.
doi:10.3201/eid2108.142015
PMCID: PMC4517717  PMID: 26177344
bats; virus reservoir; Ebola; SARS; severe acute respiratory syndrome; MERS; Middle East respiratory syndrome; Nipah; viruses; West Africa; zoonoses
10.  Serological Evidence of Influenza A Viruses in Frugivorous Bats from Africa 
PLoS ONE  2015;10(5):e0127035.
Bats are likely natural hosts for a range of zoonotic viruses such as Marburg, Ebola, Rabies, as well as for various Corona- and Paramyxoviruses. In 2009/10, researchers discovered RNA of two novel influenza virus subtypes – H17N10 and H18N11 – in Central and South American fruit bats. The identification of bats as possible additional reservoir for influenza A viruses raises questions about the role of this mammalian taxon in influenza A virus ecology and possible public health relevance. As molecular testing can be limited by a short time window in which the virus is present, serological testing provides information about past infections and virus spread in populations after the virus has been cleared. This study aimed at screening available sera from 100 free-ranging, frugivorous bats (Eidolon helvum) sampled in 2009/10 in Ghana, for the presence of antibodies against the complete panel of influenza A haemagglutinin (HA) types ranging from H1 to H18 by means of a protein microarray platform. This technique enables simultaneous serological testing against multiple recombinant HA-types in 5μl of serum. Preliminary results indicate serological evidence against avian influenza subtype H9 in about 30% of the animals screened, with low-level cross-reactivity to phylogenetically closely related subtypes H8 and H12. To our knowledge, this is the first report of serological evidence of influenza A viruses other than H17 and H18 in bats. As avian influenza subtype H9 is associated with human infections, the implications of our findings from a public health context remain to be investigated.
doi:10.1371/journal.pone.0127035
PMCID: PMC4429104  PMID: 25965069
12.  Clinical Indicators for Bacterial Co-Infection in Ghanaian Children with P. falciparum Infection 
PLoS ONE  2015;10(4):e0122139.
Differentiation of infectious causes in severely ill children is essential but challenging in sub- Saharan Africa. The aim of the study was to determine clinical indicators that are able to identify bacterial co-infections in P. falciparum infected children in rural Ghana. In total, 1,915 severely ill children below the age of 15 years were recruited at Agogo Presbyterian Hospital in Ghana between May 2007 and February 2011. In 771 (40%) of the children malaria parasites were detected. This group was analyzed for indicators of bacterial co-infections using bivariate and multivariate regression analyses with 24 socio-economic variables, 16 terms describing medical history and anthropometrical information and 68 variables describing clinical symptoms. The variables were tested for sensitivity, specificity, positive predictive value and negative predictive value. In 46 (6.0%) of the children with malaria infection, bacterial co-infection was detected. The most frequent pathogens were non-typhoid salmonellae (45.7%), followed by Streptococcus spp. (13.0%). Coughing, dehydration, splenomegaly, severe anemia and leukocytosis were positively associated with bacteremia. Domestic hygiene and exclusive breastfeeding is negatively associated with bacteremia. In cases of high parasitemia (>10,000/μl), a significant association with bacteremia was found for splenomegaly (OR 8.8; CI 1.6–48.9), dehydration (OR 18.2; CI 2.0–166.0) and coughing (OR 9.0; CI 0.7–118.6). In children with low parasitemia, associations with bacteremia were found for vomiting (OR 4.7; CI 1.4–15.8), severe anemia (OR 3.3; CI 1.0–11.1) and leukocytosis (OR 6.8 CI 1.9–24.2). Clinical signs of impaired microcirculation were negatively associated with bacteremia. Ceftriaxone achieved best coverage of isolated pathogens. The results demonstrate the limitation of clinical symptoms to determine bacterial co-infections in P. falciparum infected children. Best clinical indicators are dependent on the parasitemia level. Even with a moderate sensitivity of >60%, only low positive predictive values can be obtained due to low prevalence of bacteremia. Rapid testing for distinguishing parasitemia and bacteremia is essential.
doi:10.1371/journal.pone.0122139
PMCID: PMC4391931  PMID: 25856341
13.  Drinking Water from Dug Wells in Rural Ghana — Salmonella Contamination, Environmental Factors, and Genotypes 
Salmonellosis is an important but neglected disease in sub-Saharan Africa. Food or fecal-oral associated transmissions are the primary cause of infections, while the role of waterborne transmission is unclear. Samples were collected from different dug wells in a rural area of Ghana and analyzed for contamination with bacteria, and with Salmonella in particular. In addition, temporal dynamics and riks factors for contamination were investigated in 16 wells. For all Salmonella isolates antibiotic susceptibility testing was performed, serovars were determined and strains from the same well with the same serovar were genotyped. The frequency of well water contamination with Gram-negative rod-shaped bacteria was 99.2% (n = 395). Out of 398 samples, 26 (6.5%) tested positive for Salmonella spp. The serovar distribution was diverse including strains not commonly isolated from clinical samples. Resistance to locally applied antibiotics or resistance to fluoroquinolones was not seen in the Salmonella isolates. The risk of Salmonella contamination was lower in wells surrounded by a frame and higher during the rainy season. The study confirms the overall poor microbiological quality of well water in a resource-poor area of Ghana. Well contamination with Salmonella poses a potential threat of infection, thus highlighting the important role of drinking water safety in infectious disease control.
doi:10.3390/ijerph120403535
PMCID: PMC4410201  PMID: 25826395
Salmonella; disease transmission; drinking water; dug wells; risk factor
14.  Factors associated with failure to screen for syphilis during antenatal care in Ghana: a case control study 
BMC Infectious Diseases  2015;15:125.
Background
There is little data regarding the effect of ramping up new screening interventions on their uptake by target populations into routine care services in developing countries. This study aimed to determine patient-level factors associated with failure of pregnant women to get screened for syphilis during antenatal care, in the context of a national rollout of rapid syphilis point of care tests (POCTs) in Ghana.
Methods
An unmatched 1:2 case control study conducted among women admitted for delivery in two district hospitals in the Ashanti Region of Ghana from August to October 2010, 7 to 9 months after the introduction of POCTs in the region. Cases were women who had not been screened for syphilis during antenatal care and controls were women who had been screened. Patient-reported factors for being unscreened were examined using logistic regression to obtain odds ratios (ORs) and 95% confidence intervals (CIs).
Results
160 consecutive unscreened and 327 screened women were recruited. Most women had good knowledge of syphilis (58.7% among unscreened women vs. 64.2% among screened; P = 0.24). Factors associated with failure to get screened were: attending antenatal care in a private health facility (adjusted OR, 11.09; 95% CI 5.48-22.48), previous adverse pregnancy outcome (adjusted OR, 1.98; 95% CI 1.22-3.23) and not being screened for HIV during the current pregnancy (adjusted OR, 2.78; 95% CI 1.50-5.13). The odds of being unscreened also increased with decreasing doses of intermittent preventive treatment for malaria in pregnancy received (P trend < 0.001) and decreasing education level (P trend = 0.02).
Conclusion
Significant risk factors for not being screened, following the national rollout of syphilis POCTs, related to the type of health facility where antenatal care was received and some of the women’s personal characteristics. Targeting of private medical facilities to include syphilis POCTs and support other neglected public health interventions should be a priority.
doi:10.1186/s12879-015-0868-1
PMCID: PMC4364573  PMID: 25888254
Syphilis screening; Antenatal care; Point of care tests; Ghana
15.  Molecular Characterization of Cryptosporidium spp. among Children in Rural Ghana 
PLoS Neglected Tropical Diseases  2015;9(3):e0003551.
Background
The relevance of Cryptosporidium infections for the burden of childhood diarrhoea in endemic settings has been shown in recent years. This study describes Cryptosporidium subtypes among symptomatic and asymptomatic children in rural Ghana to analyse subtype-specific demographic, geographical, seasonal and clinical differences in order to inform appropriate control measures in endemic areas.
Methodology/Principal Findings
Stool samples were collected from 2232 children below 14 years of age presenting with and without gastrointestinal symptoms at the Agogo Presbyterian Hospital in the rural Ashanti region of Ghana between May 2007 and September 2008. Samples were screened for Cryptosporidium spp. by PCR and isolates were classified into subtypes based on sequence differences in the gp60 gene. Subtype specific frequencies for age, sex, location and season have been determined and associations with disease symptoms have been analysed within a case-control study.
Cryptosporidium infections were diagnosed in 116 of 2232 (5.2%) stool samples. Subtyping of 88 isolates revealed IIcA5G3 (n = 26, 29.6%), IbA13G3 (n = 17, 19.3%) and IaA21R3 (n = 12, 13.6%) as the three most frequent subtypes of the two species C. hominis and C. parvum, known to be transmitted anthroponotically. Infections peak at early rainy season with 67.9% and 50.0% of infections during the months April, May and June for 2007 and 2008 respectively. C. hominis infection was mainly associated with diarrhoea (odds ratio [OR] = 2.4; 95% confidence interval [CI]: 1.2–4.9) whereas C. parvum infection was associated with both diarrhoea (OR = 2.6; CI: 1.2–5.8) and vomiting (OR = 3.1; 95% CI: 1.5–6.1).
Conclusions/Significance
Cryptosporidiosis is characterized by seasonal anthroponotic transmission of strains typically found in Sub-Saharan Africa. The infection mainly affects young infants, with vomiting and diarrhoea being one of the leading symptoms in C. parvum infection. Combining molecular typing and clinical data provides valuable information for physicians and is able to track sources of infections.
Author Summary
Cryptosporidium spp. are a frequent cause of diarrhoea worldwide. While both animal-to-human and human-to-human transmission has been reported from industrialized countries, human-to-human transmission clearly prevails in Sub-Saharan Africa. However, data on the distribution of zoonotic and human subtypes is limited for rural African regions, where children are in very close contact to animals. We conducted a case-control study with 2232 stool samples from symptomatic and asymptomatic children living in the rural Ashanti region of Ghana. The combination of molecular typing results and clinical data helped to untangle transmission routes and to analyze the association of clinical symptoms with specific Cryptosporidium subtypes. Our study results demonstrate seasonal transmission with no clusters of specific subtypes. All subtypes detected have so far been only encountered from human specimens, strongly suggesting a predominantly human-human transmission among children living in the rural Ghana, despite close contact to livestock. Therefore, public health control programmes need to primarily focus on hygienic conditions among young infants below the age of two years. Of interest for practicing physicians, vomiting is a frequent symptom, especially in C. parvum infections.
doi:10.1371/journal.pntd.0003551
PMCID: PMC4352007  PMID: 25749411
16.  Gastrointestinal Infections and Diarrheal Disease in Ghanaian Infants and Children: An Outpatient Case-Control Study 
PLoS Neglected Tropical Diseases  2015;9(3):e0003568.
Introduction
Diarrheal diseases are among the most frequent causes of morbidity and mortality in children worldwide, especially in resource-poor areas. This case-control study assessed the associations between gastrointestinal infections and diarrhea in children from rural Ghana.
Methods
Stool samples were collected from 548 children with diarrhea and from 686 without gastrointestinal symptoms visiting a hospital from 2007–2008. Samples were analyzed by microscopy and molecular methods.
Results
The organisms most frequently detected in symptomatic cases were Giardia lamblia, Shigella spp./ enteroinvasive Escherichia coli (EIEC), and Campylobacter jejuni. Infections with rotavirus (adjusted odds ratio [aOR] = 8.4; 95% confidence interval [CI]: 4.3–16.6), C. parvum/hominis (aOR = 2.7; 95% CI: 1.4–5.2) and norovirus (aOR = 2.0; 95%CI: 1.3–3.0) showed the strongest association with diarrhea. The highest attributable fractions (AF) for diarrhea were estimated for rotavirus (AF = 14.3%; 95% CI: 10.9–17.5%), Shigella spp./EIEC (AF = 10.5%; 95% CI: 3.5–17.1%), and norovirus (AF = 8.2%; 95% CI 3.2–12.9%). Co-infections occurred frequently and most infections presented themselves independently of other infections. However, infections with E. dispar, C. jejuni, and norovirus were observed more often in the presence of G. lamblia.
Conclusions
Diarrheal diseases in children from a rural area in sub-Saharan Africa are mainly due to infections with rotavirus, Shigella spp./EIEC, and norovirus. These associations are strongly age-dependent, which should be considered when diagnosing causes of diarrhea. The presented results are informative for both clinicians treating gastrointestinal infections as well as public health experts designing control programs against diarrheal diseases.
Author Summary
Gastrointestinal infections are frequent in many low-income countries. However, their role in diarrheal diseases is still under discussion. Many epidemiological studies focus on individuals with diarrheal symptoms only, ignoring the fact that infections may progress asymptomatically as well. In order to identify infectious agents associated with diarrhea it is imperative to consider cases without symptoms as a control group. We conducted a case-control study, including 548 children with diarrhea and 651 children without gastrointestinal symptoms in order to untangle the role of gastrointestinal infections in diarrheal disease. As shown in other studies infections with rotavirus, Shigella spp./EIEC and norovirus are responsible for the main diarrhea burden. Co-infections are frequently observed in our study group and some organisms occur more frequently in the presence of a second one. Especially Giardia lamblia, which is not associated with diarrhea, is more often observed along with Campylobacter jejuni and norovirus, which are responsible for a high number of diarrheal episodes. This may be of particular interest since G. lamblia is, with a frequency of 40% within the study group, the most prevalent organism observed. Furthermore, the high number of co-infections challenged the identification of causative pathogens since diagnosing a particular isolate may not rule out the effect of another potentially infectious agent in diarrheal disease. We observed a strong effect of age on the course of an infection, which may guide clinicians when diagnosing causes of diarrhea.
doi:10.1371/journal.pntd.0003568
PMCID: PMC4349824  PMID: 25738935
17.  Predictors of Condom Use among Peer Social Networks of Men Who Have Sex with Men in Ghana, West Africa 
PLoS ONE  2015;10(1):e0115504.
Ghanaian men who have sex with men (MSM) have high rates of HIV infection. A first step in designing culturally relevant prevention interventions for MSM in Ghana is to understand the influence that peer social networks have on their attitudes and behaviors. We aimed to examine whether, in a sample of Ghanaian MSM, mean scores on psychosocial variables theorized to influence HIV/STI risk differed between peer social networks and to examine whether these variables were associated with condom use. We conducted a formative, cross-sectional survey with 22 peer social networks of MSM (n = 137) in Ghana. We assessed basic psychological-needs satisfaction, HIV/STI knowledge, sense of community, HIV and gender non-conformity stigmas, gender equitable norms, sexual behavior and condom use. Data were analyzed using analysis of variance, generalized estimating equations, and Wilcoxon two sample tests. All models were adjusted for age and income, ethnicity, education, housing and community of residence. Mean scores for all psychosocial variables differed significantly by social network. Men who reported experiencing more autonomy support by their healthcare providers had higher odds of condom use for anal (AOR = 3.29, p<0.01), oral (AOR = 5.06, p<0.01) and vaginal (AOR = 1.8, p<0.05) sex. Those with a stronger sense of community also had higher odds of condom use for anal sex (AOR = 1.26, p<0.001). Compared to networks with low prevalence of consistent condom users, networks with higher prevalence of consistent condom users had higher STD and HIV knowledge, had norms that were more supportive of gender equity, and experienced more autonomy support in their healthcare encounters. Healthcare providers and peer social networks can have an important influence on safer-sex behaviors in Ghanaian MSM. More research with Ghanaian MSM is needed that considers knowledge, attitudes, and norms of their social networks in the development and implementation of culturally relevant HIV/STI prevention intervention strategies.
doi:10.1371/journal.pone.0115504
PMCID: PMC4312093  PMID: 25635774
18.  A Cross-Sectional Study of ‘Yaws’ in Districts of Ghana Which Have Previously Undertaken Azithromycin Mass Drug Administration for Trachoma Control 
PLoS Neglected Tropical Diseases  2015;9(1):e0003496.
Yaws, caused by Treponema pallidum ssp. pertenue, is reportedly endemic in Ghana. Mass distribution of azithromycin is now the cornerstone of the WHO yaws eradication campaign. Mass distribution of azithromycin at a lower target dose was previously undertaken in two regions of Ghana for the control of trachoma. Ongoing reporting of yaws raises the possibility that resistance may have emerged in T. pallidum pertenue, or that alternative infections may be responsible for some of the reported cases. We conducted a cross-sectional survey in thirty communities in two districts of Ghana where MDA for trachoma had previously been conducted. Children aged 5–17 years with ulcerative lesions compatible with yaws were enrolled. Samples for treponemal serology and lesion PCR were collected from all children. 90 children with 98 lesions were enrolled. Syphilis serology was negative in all of them. PCR for T. pallidum ssp pertenue was negative in all children, but Haemophilus ducreyi DNA was detected in 9 lesions. In these communities, previously treated for trachoma, we found no evidence of ongoing transmission of yaws. H. ducreyi was associated with a proportion of skin lesions, but the majority of lesions remain unexplained. Integration of diagnostic testing into both pre and post-MDA surveillance systems is required to better inform yaws control programmes.
Author Summary
Yaws is a chronic bacterial infection of the skin and bones that is endemic in Ghana. Since 2012, mass distribution of the antibiotic azithromycin has become the cornerstone of the WHO yaws eradication campaign. Accurate surveillance data are necessary to guide decisions about where MDA is required. Many national surveillance systems report only clinical cases without laboratory confirmation, which may lead to misdiagnosis. Mass distribution of azithromycin, at a lower dose than is used for yaws, is also used in the control of the eye disease trachoma. Between 2001 and 2008, two regions of Ghana undertook distribution of azithromycin to eliminate trachoma. These regions still report cases of yaws, and it is unclear if these are due to drug resistant yaws or other causes. We conducted a survey of patients with skin ulcers that looked like yaws in one of these regions of Ghana. Blood tests for yaws were negative in all the individuals examined and molecular testing of the ulcers did not show evidence of yaws caused by drug resistant Treponema pallidum ssp. pertenue. Our data suggest that previous treatment for trachoma may have also treated yaws in these districts. These findings highlight the need for yaws control programmes to integrate diagnostic testing into surveillance.
doi:10.1371/journal.pntd.0003496
PMCID: PMC4310597  PMID: 25632942
19.  Etiology of Bacteremia in Young Infants in Six Countries 
Background:
Neonatal illness is a leading cause of death worldwide; sepsis is one of the main contributors. The etiologies of community-acquired neonatal bacteremia in developing countries have not been well characterized.
Methods:
Infants <2 months of age brought with illness to selected health facilities in Bangladesh, Bolivia, Ghana, India, Pakistan and South Africa were evaluated, and blood cultures taken if they were considered ill enough to be admitted to hospital. Organisms were isolated using standard culture techniques.
Results:
Eight thousand eight hundred and eighty-nine infants were recruited, including 3177 0–6 days of age and 5712 7–59 days of age; 10.7% (947/8889) had a blood culture performed. Of those requiring hospital management, 782 (54%) had blood cultures performed. Probable or definite pathogens were identified in 10.6% including 10.4% of newborns 0–6 days of age (44/424) and 10.9% of infants 7–59 days of age (39/358). Staphylococcus aureus was the most commonly isolated species (36/83, 43.4%) followed by various species of Gram-negative bacilli (39/83, 46.9%; Acinetobacter spp., Escherichia coli and Klebsiella spp. were the most common organisms). Resistance to second and third generation cephalosporins was present in more than half of isolates and 44% of the Gram-negative isolates were gentamicin-resistant. Mortality rates were similar in hospitalized infants with positive (5/71, 7.0%) and negative blood cultures (42/557, 7.5%).
Conclusions:
This large study of young infants aged 0–59 days demonstrated a broad array of Gram-positive and Gram-negative pathogens responsible for community-acquired bacteremia and substantial levels of antimicrobial resistance. The role of S. aureus as a pathogen is unclear and merits further investigation.
doi:10.1097/INF.0000000000000549
PMCID: PMC4272225  PMID: 25389919
Neonatal sepsis; infant; neonate; bacteremia; Staphylococcus aureus
20.  Human Coronaviruses Associated with Upper Respiratory Tract Infections in Three Rural Areas of Ghana 
PLoS ONE  2014;9(7):e99782.
Background
Acute respiratory tract infections (ARI) are the leading cause of morbidity and mortality in developing countries, especially in Africa. This study sought to determine whether human coronaviruses (HCoVs) are associated with upper respiratory tract infections among older children and adults in Ghana.
Methods
We conducted a case control study among older children and adults in three rural areas of Ghana using asymptomatic subjects as controls. Nasal/Nasopharyngeal swabs were tested for Middle East respiratory syndrome coronavirus (MERS-CoV), HCoV-22E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 using Reverse Transcriptase Real-Time Polymerase Chain Reaction.
Results
Out of 1,213 subjects recruited, 150 (12.4%) were positive for one or more viruses. Of these, single virus detections occurred in 146 subjects (12.0%) and multiple detections occurred in 4 (0.3%). Compared with control subjects, infections with HCoV-229E (OR = 5.15, 95%CI = 2.24–11.78), HCoV-OC43 (OR = 6.16, 95%CI = 1.77–21.65) and combine HCoVs (OR = 2.36, 95%CI = 1.5 = 3.72) were associated with upper respiratory tract infections. HCoVs were found to be seasonally dependent with significant detections in the harmattan season (mainly HCoV-229E) and wet season (mainly HCoV-NL63). A comparison of the obtained sequences resulted in no differences to sequences already published in GenBank.
Conclusion
HCoVs could play significant role in causing upper respiratory tract infections among adults and older children in rural areas of Ghana.
doi:10.1371/journal.pone.0099782
PMCID: PMC4117488  PMID: 25080241
21.  Multisite Laboratory Evaluation of a Dual Human Immunodeficiency Virus (HIV)/Syphilis Point-of-Care Rapid Test for Simultaneous Detection of HIV and Syphilis Infection 
Open Forum Infectious Diseases  2014;1(1):ofu015.
Background.
 Recently, test developers have created rapid point-of-care tests that can simultaneously detect multiple infections within the same specimen using a single device. The SD BIOLINE Duo HIV/Syphilis rapid point-of-care test uses a solid-phase immunochromatographic assay to detect immunoglobulin (Ig)G, IgM, and IgA antibodies to human immunodeficiency virus (HIV)-specific antigens (HIV-1 gp41, sub O, HIV-2 gp36) and recombinant Treponema pallidum antigen (17 kDa) in human serum. This study was a multisite laboratory-based evaluation of the performance of SD BIOLINE HIV/Syphilis Duo test using previously characterized sera in 6 countries.
Methods.
 Laboratories in Ghana, Mexico, Laos, Togo, Kenya, and Myanmar participated in the evaluation during 2012–2013. Each site characterized sera using T pallidum particle agglutination assay or T pallidum hemagglutination assay and HIV enzyme immunoassay, Western blot, and/or HIV antibody rapid tests. Those gold standard test results were compared with SD BIOLINE Duo test results. We calculated the sensitivity and specificity of test performance and used the exact binomial method to calculate 95% confidence intervals (CIs).
Results.
 The sensitivity and specificity for the HIV antibody test component (n = 2336) were estimated at 99.91% (95% CI, 99.51% and 100%) and 99.67% (95% CI, 99.16% and 99.91%), respectively. For the T pallidum test component (n = 2059), the sensitivity and specificity were estimated at 99.67% (95% CI, 98.82% and 99.96%) and 99.72% (95% CI, 99.29% and 99.92%), respectively.
Conclusions.
 The sensitivity and specificity of the SD BIOLINE HIV/Syphilis Duo test were consistently high across sera specimens from 6 countries around the world. Dual rapid tests should be considered for improved HIV and syphilis screening coverage.
doi:10.1093/ofid/ofu015
PMCID: PMC4324189  PMID: 25734088
HIV; Syphilis; Dual testing; test evaluation; rapid test
22.  Kinetics of mycolactone in human subcutaneous tissue during antibiotic therapy for Mycobacterium ulcerans disease 
BMC Infectious Diseases  2014;14:202.
Background
Mycobacterium ulcerans (M. ulcerans) causes a devastating necrotising infection of skin tissue leading to progressive ulceration. M. ulcerans is the only human pathogen that secretes mycolactone, a polyketide molecule with potent cytotoxic and immunomodulatory properties. These unique features make mycolactone an attractive biomarker for M. ulcerans disease. We sought to measure the concentration of mycolactone within lesions of patients with Buruli ulcer before, during and after antibiotic treatment to evaluate its association with the clinical and bacteriological response to therapy.
Methods
Biopsies of M. ulcerans infected skin lesions were obtained from patients before, during and after antibiotic therapy. Lipids were extracted from the biopsies and concentration of mycolactone was assayed by mass spectrometry and a cytotoxicity assay and correlated with clinical and bacteriological response to therapy.
Results
Baseline concentration of mycolactone measured by mass spectrometry predicted time to complete healing of small nodules and ulcers. Even though intra-lesional concentrations of mycolactone declined with antibiotic treatment, the toxin was still present after antibiotic treatment for 6 weeks and also 4 weeks after the end of treatment for 8 weeks in a subgroup of patients with slowly healing lesions. Additionally viable bacilli were detected in a proportion of these slowly healing lesions during and after treatment.
Conclusions
Our findings indicate that baseline intra-lesional mycolactone concentration and its kinetics with antibiotic therapy are important prognostic determinants of clinical and bacteriological response to antibiotic treatment for Mycobacterium ulcerans disease. Mycolactone may be a useful biomarker with potential utility in optimising antibiotic therapy.
doi:10.1186/1471-2334-14-202
PMCID: PMC4021496  PMID: 24731247
Mycobacterium ulcerans; Mycolactone; Biomarker; Antibiotic therapy; Prognosis; Treatment response
23.  Health Care Utilization and Symptom Severity in Ghanaian Children – a Cross-Sectional Study 
PLoS ONE  2013;8(11):e80598.
The aim of this study was to identify factors influencing health care utilization behavior for children with mild or severe disease symptoms in rural Ghana. Between March and September 2008 a cross-sectional health care utilization survey was conducted and 8,715 caregivers were interviewed regarding their intended behavior in case their children had mild or severe fever or diarrhea. To show associations between hospital attendance and further independent factors (e.g. travel distance or socio-economic status) prevalence ratios were calculated for the four disease symptoms. A Poisson regression model was used to control for potential confounding. Frequency of hospital attendance decreased constantly with increasing distance to the health facility. Being enrolled in the national health insurance scheme increased the intention to attend a hospital. The effect of the other factors diminished in the Poisson regression if modeled together with travel distance. The observed associations weakened with increasing severity of symptoms, which indicates that barriers to visit a hospital are less important if children experience a more serious illness. As shown in other studies, travel distance to a health care provider had the strongest effect on health care utilization. Studies to identify local barriers to access health care services are important to inform health policy making as they identify deprived populations with low access to health services and to early treatment.
doi:10.1371/journal.pone.0080598
PMCID: PMC3828249  PMID: 24244698
25.  Evidence for Novel Hepaciviruses in Rodents 
PLoS Pathogens  2013;9(6):e1003438.
Hepatitis C virus (HCV) is among the most relevant causes of liver cirrhosis and hepatocellular carcinoma. Research is complicated by a lack of accessible small animal models. The systematic investigation of viruses of small mammals could guide efforts to establish such models, while providing insight into viral evolutionary biology. We have assembled the so-far largest collection of small-mammal samples from around the world, qualified to be screened for bloodborne viruses, including sera and organs from 4,770 rodents (41 species); and sera from 2,939 bats (51 species). Three highly divergent rodent hepacivirus clades were detected in 27 (1.8%) of 1,465 European bank voles (Myodes glareolus) and 10 (1.9%) of 518 South African four-striped mice (Rhabdomys pumilio). Bats showed anti-HCV immunoblot reactivities but no virus detection, although the genetic relatedness suggested by the serologic results should have enabled RNA detection using the broadly reactive PCR assays developed for this study. 210 horses and 858 cats and dogs were tested, yielding further horse-associated hepaciviruses but none in dogs or cats. The rodent viruses were equidistant to HCV, exceeding by far the diversity of HCV and the canine/equine hepaciviruses taken together. Five full genomes were sequenced, representing all viral lineages. Salient genome features and distance criteria supported classification of all viruses as hepaciviruses. Quantitative RT-PCR, RNA in-situ hybridisation, and histopathology suggested hepatic tropism with liver inflammation resembling hepatitis C. Recombinant serology for two distinct hepacivirus lineages in 97 bank voles identified seroprevalence rates of 8.3 and 12.4%, respectively. Antibodies in bank vole sera neither cross-reacted with HCV, nor the heterologous bank vole hepacivirus. Co-occurrence of RNA and antibodies was found in 3 of 57 PCR-positive bank vole sera (5.3%). Our data enable new hypotheses regarding HCV evolution and encourage efforts to develop rodent surrogate models for HCV.
Author Summary
The hepatitis C virus (HCV) is one of the most relevant causes of liver disease and cancer in humans. The lack of a small animal models represents an important hurdle on our way to understanding, treating, and preventing hepatitis C. The investigation of small mammals could identify virus infections similar to hepatitis C in animals that can be kept in laboratories, such as rodents, and can also yield insights into the evolution of those ancestral virus lineages out of which HCV developed. Here, we investigated a worldwide sample of 4,770 rodents, 2,939 bats, 210 horses and 858 cats and dogs for HCV-related viruses. New viruses were discovered in European bank voles (Myodes glareolus) and South African four-striped mice (Rhabdomys pumilio). The disease in bank voles was studied in more detail, suggesting that infection of the liver occurs with similar symptoms to those caused by HCV in humans. These rodents might thus enable the development of new laboratory models of hepatitis C. Moreover, the phylogenetic history of those viruses provides fascinating new ideas regarding the evolution of HCV ancestors.
doi:10.1371/journal.ppat.1003438
PMCID: PMC3688547  PMID: 23818848

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