A conserved trypanosomatid differentiation regulator controls substrate attachment and morphological development in Trypanosoma congolense.

Trypanosomatid parasites undergo developmental regulation to adapt to the different environments encountered during their life cycle. In Trypanosoma brucei, a genome wide selectional screen previously identified a regulator of the protein family ESAG9, which is highly expressed in stumpy forms, a morphologically distinct bloodstream stage adapted for tsetse transmission. This regulator, TbREG9.1, has an orthologue in Trypanosoma congolense, despite the absence of a stumpy morphotype in that parasite species, which is an important cause of livestock trypanosomosis. RNAi mediated gene silencing of TcREG9.1 in Trypanosoma congolense caused a loss of attachment of the parasites to a surface substrate in vitro, a key feature of the biology of these parasites that is distinct from T. brucei. This detachment was phenocopied by treatment of the parasites with a phosphodiesterase inhibitor, which also promotes detachment in the insect trypanosomatid Crithidia fasciculata. RNAseq analysis revealed that TcREG9.1 silencing caused the upregulation of mRNAs for several classes of surface molecules, including transferrin receptor-like molecules, immunoreactive proteins in experimental bovine infections, and molecules related to those associated with stumpy development in T. brucei. Depletion of TcREG9.1 in vivo also generated an enhanced level of parasites in the blood circulation consistent with reduced parasite attachment to the microvasculature. The morphological progression to insect forms of the parasite was also perturbed. We propose a model whereby TcREG9.1 acts as a regulator of attachment and development, with detached parasites being adapted for transmission.

High level of colonization with third-generation cephalosporin-resistant Enterobacterales in African community settings, Ghana.

A cross-sectional survey was conducted in eight Ghanaian communities to investigate the extent of intestinal colonization with 3rd-generation cephalosporin-resistant Enterobacterales. The study collected faecal samples and corresponding lifestyle data from 736 healthy residents to assess the occurrence of cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae, with a focus on genotypes of plasmid-mediated ESBLs, AmpCs, and carbapenemases. The results showed that 371 participants (50.4%) carried 3rd-generation cephalosporin-resistant E. coli (n=362) and K. pneumoniae (n=9). Most of these were ESBL-producing E. coli (n=352, 94.9%), carrying CTX-M genes (96.0%, n=338/352), mostly for CTX-M-15 (98.9%, n=334/338). Nine participants (1.2%) carried AmpC-producing E. coli that harboured blaDHA-1 or blaCMY-2 genes, and two participants (0.3%) each carried a carbapenem-resistant E. coli that harboured both blaNDM-1 and blaCMY-2. Quinolone-resistant O25b: ST131 E. coli were recovered from six participants (0.8%) and were all CTX-M-15 ESBL-producers. Having a household toilet facility was significantly associated with a reduced risk of intestinal colonization (adjusted odds ratio, 0.71; 95% CI, 0.48-0.99; P-value=0.0095) in multivariate analysis. These findings raise serious public health concerns, and effective control of the spread of antibiotic-resistant bacteria is possible by providing better sanitary conditions for communities.

Divergent metabolism between Trypanosoma congolense and Trypanosoma brucei results in differential sensitivity to metabolic inhibition.

Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of knowledge regarding the biology of T. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism in T. congolense mammalian-infective bloodstream-form parasites, and test whether metabolic differences compared to T. brucei impact upon sensitivity to metabolic inhibition. Like the bloodstream stage of T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly lower in bloodstream stage T. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Transcriptomics analysis showed higher levels of transcripts associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation, and the glycosomal succinate shunt in T. congolense, compared to T. brucei. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and saturated fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with metabolic inhibitors, confirming that electron transport chain activity is not essential in T. congolense. However, the parasite exhibits increased sensitivity to inhibition of mitochondrial pyruvate import, compared to T. brucei. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold higher EC50 for the lipase and fatty acid synthase inhibitor Orlistat, compared to T. brucei. These data highlight that bloodstream form T. congolense diverges from T. brucei in key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stage T. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions.

Reliable, scalable functional genetics in bloodstream-form Trypanosoma congolense in vitro and in vivo.

Animal African trypanosomiasis (AAT) is a severe, wasting disease of domestic livestock and diverse wildlife species. The disease in cattle kills millions of animals each year and inflicts a major economic cost on agriculture in sub-Saharan Africa. Cattle AAT is caused predominantly by the protozoan parasites Trypanosoma congolense and T. vivax, but laboratory research on the pathogenic stages of these organisms is severely inhibited by difficulties in making even minor genetic modifications. As a result, many of the important basic questions about the biology of these parasites cannot be addressed. Here we demonstrate that an in vitro culture of the T. congolense genomic reference strain can be modified directly in the bloodstream form reliably and at high efficiency. We describe a parental single marker line that expresses T. congolense-optimized T7 RNA polymerase and Tet repressor and show that minichromosome loci can be used as sites for stable, regulatable transgene expression with low background in non-induced cells. Using these tools, we describe organism-specific constructs for inducible RNA-interference (RNAi) and demonstrate knockdown of multiple essential and non-essential genes. We also show that a minichromosomal site can be exploited to create a stable bloodstream-form line that robustly provides >40,000 independent stable clones per transfection-enabling the production of high-complexity libraries of genome-scale. Finally, we show that modified forms of T. congolense are still infectious, create stable high-bioluminescence lines that can be used in models of AAT, and follow the course of infections in mice by in vivo imaging. These experiments establish a base set of tools to change T. congolense from a technically challenging organism to a routine model for functional genetics and allow us to begin to address some of the fundamental questions about the biology of this important parasite.

Household cockroaches carry CTX-M-15-, OXA-48- and NDM-1-producing enterobacteria, and share beta-lactam resistance determinants with humans.

BACKGROUND: This study was designed to investigate whether household cockroaches harbor cephalosporin-resistant enterobacteria that share resistance determinants with human inhabitants. From February through July 2016, whole cockroach homogenates and human fecal samples from 100 households were cultured for cephalosporin-resistant enterobacteria (CRe). The CRe were examined for plasmid-mediated AmpC, ESBL, and carbapenemase genes; antibiotic susceptibility patterns; and conjugative transfer of antibiotic resistance mechanisms. Clonal associations between CRe were determined by multi-locus sequence typing (MLST). RESULTS: Twenty CRe were recovered from whole cockroach homogenates from 15 households. The prevalence of households with cockroaches that harbored CRe, AmpC- (based on phenotype, with no identifiable blaAmpC genes), ESBL-, and carbapenemase-producers were 15, 4, 5%(2 blaCTX-M-15/TEM-1; 1 blaCTX-M-15/TEM-4; 1 blaTEM-24; 1 blaSHV-4) and 3%(2 blaNDM-1 genes and 1 blaOXA-48 gene), respectively. Overall, 20 CRe were recovered from 61 fecal samples of inhabitants from all 15 households that had cockroach samples positive for CRe. Of these, 5CRe (1 per household) were positive for ESBLs (blaTEM-24, blaTEM-14, blaCTX-M-15/TEM-4, blaSHV-3, blaCTX-M-15/TEM-1) and none carried AmpCs or carbapenemases. From 4% of households, the pair of cockroach and human CRe shared the same sequence type (ST), clonal complex (CC), antibiogram, and conjugable bla gene sequence (house 34, E. coli ST9/CC20-blaTEM-4; house 37, E. coli ST44/CC10-blaCTX-15/TEM-4; house 41, E. coli ST443/CC205-blaCTX-15/TEM-1; house 49, K. pneumoniae ST231/CC131-blaSHV-13). CONCLUSION: The findings provide evidence that household cockroaches may carry CTX-M-15-, OXA-48- and NDM-1-producers, and share clonal relationship and beta-lactam resistance determinants with humans.

Detection of herpes viruses in Ghanaian patients with periodontitis.

AIM: The complexity of periodontitis in both etiology and progression has raised many questions, necessitating enormous research in recent years. The aim of the present study was to detect the presence of herpes viruses in Ghanaian patients diagnosed with periodontitis. METHODS: Thirty-one patients were included in the study; 21 with periodontitis classified into localized chronic, generalized, and aggressive periodontitis, and 10 without the disease were used as controls. Subgingival samples were collected, followed by DNA extraction. Multiplex polymerase chain reaction was used to amplify viral DNA for the detection of herpes viruses. Data was analyzed using Stata 14. RESULTS: The mean age for patients with aggressive periodontitis was 32.2 years (standard deviation [SD]: 8.50), while those for localized chronic periodontitis and generalized chronic periodontitis were 40.6 years (SD: 7.83) and 46.3 years (SD: 12.12), respectively. Viruses were detected only among patients clinically diagnosed with aggressive periodontitis. Of the total number of aggressive periodontitis patients, herpes simplex virus 1 (HSV-1) and Epstein-Barr virus (HBV) were found in four (44%) and one (11%), respectively. The mean age for patients found to have HSV-1 or EBV was 29 years (SD: 6.93). CONCLUSION: We found HSV-1 and EBV in the subgingival plaque samples of Ghanaian patients clinically diagnosed with aggressive periodontitis. While our finding requires further investigation, the role of HSV in periodontitis, if elucidated, could transform and inform the clinical management of the condition.

Trichomonas vaginalis infection and the diagnostic significance of detection tests among Ghanaian outpatients.

BACKGROUND: There is little data on Trichomonas vaginalis infection in Ghana. This study evaluated the prevalence of trichomoniasis using different diagnostic methods and determined the risk factors for infection in patients. METHODS: A structured questionnaire was administered. Vaginal swabs, urethral swabs and urine specimens were obtained from consenting patients; and the samples processed following standard protocols. The presence of T. vaginalis was determined using wet mount microscopy and polymerase chain reaction (PCR) as gold standard. We also assessed the diagnostic performance the JD's Trichomonas V® rapid antigen test to inform clinical practice. RESULTS: The PCR assay detected T. vaginalis positivity in 64 of 150 patients (42.6, 95%CI:35.0, 50.6) including all positive samples of wet mount microscopy and JD's Trichomonas V® test. Wet mount microscopy showed low sensitivity (31.6%), high specificity (100%), moderate positive predictive value (75.0%), moderate positive likelihood ratio (3.0), and weak agreement (Cohen's kappa, 0.283) with PCR assay. The JD's Trichomonas V® test displayed lower sensitivity (25.0%), specificity (83.3%), and weaker measure of agreement (Cohen's kappa, 0.233) with PCR. In multivariate analysis, the strongest independent predictor for T. vaginalis was female gender [adjusted odds ratio (AOR), 24.89; 95% confidence interval (CI): 10.58, 51.21; P-value< 0.001]. Knowledge of STI showed a protective effect against infection with the parasite (AOR, 0.13; 95%CI: 0.07, 0.29; P-value< 0.017). CONCLUSION: The sensitivity of wet mount microscopy was low for T. vaginalis screening in our region. The JD's Trichomonas V® test should not be considered as an alternative test. We recommend mandatory PCR assay for confirmation of negative wet mount results.

Trichomoniasis and associated co-infections of the genital tract among pregnant women presenting at two hospitals in Ghana.

BACKGROUND: Trichomonas vaginalis (TV) infection is the most prevalent non-viral sexually transmitted pathogen worldwide. Among pregnant women, the infection may cause adverse birth outcomes such as premature rupture of membranes and premature labour. In view of the paucity of information relating to TV among Ghanaian pregnant women, this study investigated its prevalence and associated co-infections among pregnant women. METHODS: High vaginal swabs were obtained from 99 pregnant women using sterile cotton swab sticks. Wet preparation, Grams staining, culturing, coagulase and sensitivity testing were carried out to determine the presence of TV and associated microorganisms. RESULTS: The prevalence of TV among the pregnant women was found to be 20.2% (n = 20). Concurring with Trichomoniasis, 75% (n = 15) of participants had other infections such as Candida with prevalence of 53% (n = 8), Proteus infection - 20% (n = 3), Streptococcus infection - 13% (n = 2) and other GNRs and Gonococci having 7% each (n = 1). Moreover, there was 86.9% (n = 86) prevalence of Staphylococcus spp. among study participants. There was statistically significant correlation between TV and Gonococci infection at a correlation co-efficient of 0.107 (P < 0.05) as well as significant correlation between TV and Proteus spp. at a correlation co-efficient of 0.189 (P < 0.05). TV infection was high (60%) among the most sexually active age group (19 to 29 yrs). CONCLUSION: There was 20.2% prevalence of TV among the pregnant women presenting at the hospitals, with Gonococci and Proteus infections being statistically significant associated infections.

Trends in paediatric and adult bloodstream infections at a Ghanaian referral hospital: a retrospective study.

BACKGROUND: Bloodstream infections (BSI) are life-threatening emergencies. Identification of the common pathogens and their susceptibility patterns is necessary for timely empirical intervention. METHODS: We conducted a 4-year retrospective analysis of blood cultures from all patients excluding neonates at the Korle-Bu Teaching hospital, Ghana, from January 2010 through December 2013. Laboratory report data were used to determine BSI, blood culture contamination, pathogen profile, and antimicrobial resistance patterns. RESULTS: Overall, 3633 (23.16 %) out of 15,683 blood cultures were positive for various organisms. Pathogen-positive cultures accounted for 1451 (9.3 %, 95 % CI 8.5-9.8 %). Infants recorded the highest true blood culture positivity (20.9 %, n = 226/1083), followed by the elderly (13.3 %, n = 80/601), children (8.9 %, n = 708/8000) and adults (7.2 %, n = 437/6000) (p = 0.001 for Marascuilo's post hoc). Overall occurrence of BSI declined with increasing age-group (p = 0.001) but the type of isolates did not vary with age except for Citrobacter, Escherichia coli, Klebsiella, Salmonella, and Enterococcus species. Gram negative bacteria predominated in our study (59.8 %, n = 867/1451), but the commonest bacterial isolate was Staphylococcus aureus (21.9 %, n = 318/1451)-and this trend run through the various age-groups. From 2010 to 2013, we observed a significant trend of yearly increase in the frequency of BSI caused by cephalosporin-resistant enterobacteria (Chi square for trend, p = 0.001). Meropenem maintained high susceptibility among all Gram-negative organisms ranging from 96 to 100 %. Among Staphylococcus aureus, susceptibility to cloxacillin was 76.6 %. CONCLUSION: Our study shows a significantly high blood culture positivity in infants as compared to children, adults and the elderly. There was a preponderance of S. aureus and Gram-negative bacteria across all age-groups. Meropenem was the most active antibiotic for Gram-negative bacteria. Cloxacillin remains a very useful anti-staphylococcal agent.

Malaria, helminths and malnutrition: a cross-sectional survey of school children in the South-Tongu district of Ghana.

BACKGROUND: As part of malaria characterization study in the South-Tongu district of Ghana, the current study was conducted to explore relationships between malaria, schistosomiasis, soil transmitted helminths and malnutrition in riparian community settings that had hitherto encountered episodes of mass deworming exercises. METHODS: School-age children were enrolled in a cross-sectional study from April through July 2012. Stool and urine samples were examined respectively for helminths and Schistosoma haematobium. Blood samples were analyzed for malaria parasites and haemoglobin (Hb) concentrations, respectively. Anthropometric indices were measured. Relationships were determined using generalized linear models. RESULTS: The results show low numbers of asymptomatic Plasmodium falciparum (9.2%, n = 37/404) and S. haematobium (2.5%, n = 10/404) infections. The associations between significance terms in the multivariate analysis for P. falciparum infections were further assessed to test the significance of the product terms directly i.e., age in years [adjusted odds ratio (AOR), 3.1; 95% confidence interval (CI) 1.1-5.6], Hb concentration (AOR = 0.71; 95% CI 0.42-2.3), and stunted malnutrition (AOR, 8.72; 95% CI 4.8-25.1). The P. falciparum-associated decrease in mean Hb concentration was 2.82 g/dl (95% CI 1.63-4.1 g/dl; P = 0.001) in stunted children, and 0.75 g/dl (95% CI 1.59-0.085 g/dl; P = 0.076) in the non-stunted cohort. The anaemia-associated decrease in mean parasitaemia in stunted children was 3500 parasites/µl of blood (95% CI 262.46-6737.54 parasites/µl of blood; P = 0.036), and in non-stunted children 2127 parasites/µl of blood (95% CI -0.27 to 4.53; P = 0.085). Stunted malnutrition was the strongest predictor of S. haematobium infection (AOR = 11; 95% CI 3.1-33.6) but significant associations as described for P. falciparum infections were absent. The population attributable risk of anaemia due to P. falciparum was 6.3% (95% CI 2.5-9.3), 0.9% (95% CI 0.4-2.3) for S. haematobium, and 12.5% (95% CI 9.11-19.52) for stunted malnutrition. CONCLUSION: Plasmodium falciparum, S. haematobium, intestinal helminths and their co-infections were uncommon in our school-age children. Stunting exacerbated the extent to which malaria was associated with loss in Hb concentration.