A pandemic within a pandemic? Admission to COVID-19 wards in hospitals is associated with increased prevalence of antimicrobial resistance in two African settings.

BACKGROUND: Patients who develop severe illness due to COVID-19 are more likely to be admitted to hospital and acquire bacterial co-infections, therefore the WHO recommends empiric treatment with antibiotics. Few reports have addressed the impact of COVID-19 management on emergence of nosocomial antimicrobial resistance (AMR) in resource constrained settings. This study aimed to ascertain whether being admitted to a COVID-19 ward (with COVID-19 infection) compared to a non-COVID-19 ward (as a COVID-19 negative patient) was associated with a change in the prevalence of bacterial hospital acquired infection (HAI) species or resistance patterns, and whether there were differences in antimicrobial stewardship (AMS) and infection prevention and control (IPC) guidelines between COVID-19 and non-COVID-19 wards. The study was conducted in Sudan and Zambia, two resource constrained settings with differing country-wide responses to COVID-19. METHODS: Patients suspected of having hospital acquired infections were recruited from COVID-19 wards and non-COVID-19 wards. Bacteria were isolated from clinical samples using culture and molecular methods and species identified. Phenotypic and genotypic resistance patterns were determined by antibiotic disc diffusion and whole genome sequencing. Infection prevention and control guidelines were analysed for COVID-19 and non-COVID-19 wards to identify potential differences. RESULTS: 109 and 66 isolates were collected from Sudan and Zambia respectively. Phenotypic testing revealed significantly more multi-drug resistant isolates on COVID-19 wards in both countries (Sudan p = 0.0087, Zambia p = 0.0154). The total number of patients with hospital acquired infections (both susceptible and resistant) increased significantly on COVID-19 wards in Sudan, but the opposite was observed in Zambia (both p = ≤ 0.0001). Genotypic analysis showed significantly more ß-lactam genes per isolate on COVID-19 wards (Sudan p = 0.0192, Zambia p = ≤ 0.0001). CONCLUSIONS: Changes in hospital acquired infections and AMR patterns were seen in COVID-19 patients on COVID-19 wards compared to COVID-19 negative patients on non-COVID-19 wards in Sudan and Zambia. These are likely due to a potentially complex combination of causes, including patient factors, but differing emphases on infection prevention and control, and antimicrobial stewardship policies on COVID-19 wards were highlighted.

Molecular and morphological identification of suspected Plasmodium vivax vectors in Central and Eastern Sudan.

BACKGROUND: In spite of the global effort to eliminate malaria, it remains the most significant vector-borne disease of humans. Plasmodium falciparum is the dominant malaria parasite in sub-Saharan Africa. However, Plasmodium vivax is becoming widely spread throughout Africa. The overuse of vector control methods has resulted in a remarkable change in the behaviour of mosquito that feeds on human as well as on vector composition. The aim of this study was to identify Anopheles mosquito species in vivax malaria endemic regions and to investigate their role in P. vivax circumsporozoite protein (Pvcsp) allele diversity. METHODS: Mosquito samples were collected from Central Sudan (Rural Khartoum and Sennar) and Eastern Sudan (New Halfa, Kassala state) using pyrethrum spray catch (PSC) and CDC light traps. Mosquitoes were identified using appropriate morphological identification keys and Anopheles gambiae complex were confirmed to species level using molecular analysis. A subset of blood-fed anopheline mosquitoes were dissected to determine the presence of natural infection of malaria parasites. In addition, the rest of the samples were investigated for the presence of Pvcsp gene using nested-PCR. RESULTS: A total of 1037 adult anopheline mosquitoes were collected from New Halfa (N = 467), Rural Khartoum (N = 132), and Sennar (N = 438). Morphological and molecular identification of the collected mosquitoes revealed the presence of Anopheles arabiensis (94.2%), Anopheles funestus (0.5%), and Anopheles pharoensis (5.4%). None of the dissected mosquitoes (N = 108) showed to be infected with malaria parasite. Overall P. vivax infectivity rate was 6.1% (63/1037) by Pvcsp nested PCR. Co-dominance of An. arabiensis and An. pharoensis is reported in Sennar state both being infected with P. vivax. CONCLUSION: This study reported P. vivax infection among wild-caught anopheline mosquitoes in Central and Eastern Sudan. While An. arabiensis is the most abundant vector observed in all study areas, An. funestus was recorded for the first time in New Halfa, Eastern Sudan. The documented Anopheles species are implicated in Pvcsp allele diversity. Large-scale surveys are needed to identify the incriminated vectors of P. vivax malaria and determine their contribution in disease transmission dynamics.

Pf7: an open dataset of Plasmodium falciparum genome variation in 20,000 worldwide samples.

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network.  It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented.  For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations.  We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent.  We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines.  Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website.

Geographical distribution and genetic diversity of Plasmodium vivax reticulocyte binding protein 1a correlates with patient antigenicity.

Plasmodium vivax is the most widespread cause of human malaria. Recent reports of drug resistant vivax malaria and the challenge of eradicating the dormant liver forms increase the importance of vaccine development against this relapsing disease. P. vivax reticulocyte binding protein 1a (PvRBP1a) is a potential vaccine candidate, which is involved in red cell tropism, a crucial step in the merozoite invasion of host reticulocytes. As part of the initial evaluation of the PvRBP1a vaccine candidate, we investigated its genetic diversity and antigenicity using geographically diverse clinical isolates. We analysed pvrbp1a genetic polymorphisms using 202 vivax clinical isolates from six countries. Pvrbp1a was separated into six regions based on specific domain features, sequence conserved/polymorphic regions, and the reticulocyte binding like (RBL) domains. In the fragmented gene sequence analysis, PvRBP1a region II (RII) and RIII (head and tail structure homolog, 152-625 aa.) showed extensive polymorphism caused by random point mutations. The haplotype network of these polymorphic regions was classified into three clusters that converged to independent populations. Antigenicity screening was performed using recombinant proteins PvRBP1a-N (157-560 aa.) and PvRBP1a-C (606-962 aa.), which contained head and tail structure region and sequence conserved region, respectively. Sensitivity against PvRBP1a-N (46.7%) was higher than PvRBP1a-C (17.8%). PvRBP1a-N was reported as a reticulocyte binding domain and this study identified a linear epitope with moderate antigenicity, thus an attractive domain for merozoite invasion-blocking vaccine development. However, our study highlights that a global PvRBP1a-based vaccine design needs to overcome several difficulties due to three distinct genotypes and low antigenicity levels.

Quantitative Analysis of Candidate Endodontic Pathogens and Their Association with Cause and Symptoms of Apical Periodontitis in a Sudanese Population.

OBJECTIVE: To investigate the prevalence of key endodontic pathogens and their association with the clinical features and the cause of apical periodontitis. METHODS: The study population included patients referred to Khartoum Dental teaching Hospital, Sudan for endodontic treatment. Samples were collected from single-rooted teeth carious or traumatised teeth with clinical and radiographic evidence of apical periodontitis. The endodontic pathogens Porphyromonas endodontalis, Fusobacterium nucleatum and Treponema denticola were quantified by real time polymerase chain reaction (qPCR). The prevalence of each species was identified at both a low detection threshold (>50 bacteria) and a high detection threshold (>1000 bacteria). RESULTS: 75 patients (mean age 30.1 yrs SD 10.1) were included in the analysis. The most prevalent bacterium at both the low and high threshold was F. nucleatum followed by T. denticola at the low threshold and P. endodontalis at the high threshold. There was no association with symptoms at the low detection threshold, but at high threshold P. endodontalis was associated with swelling, adjusted odds ratio (OR), 9.32 95%CI 1.11- 78.66, P=0.04. All species were more prevalent in apical periodontitis due to caries only at the low detection threshold, OR=5.01 (P=0.006) for T. denticola; 4.84 (P=0.01) for F. nucleatum; and 3.62 (P=0.03) for P. endodontalis. CONCLUSION: There was a high prevalence of the F. nucleatum, T. denticola and P. endodontalis in apical periodontitis associated with caries. None of these bacterial were associated with pain but the presence of P. endodontalis at high levels was associated with swelling.

Prevalence of glucose-6-phosphate dehydrogenase deficiency (G6PDd), CareStart qualitative rapid diagnostic test performance, and genetic variants in two malaria-endemic areas in Sudan.

Glucose-6-phosphate dehydrogenase deficiency (G6PDd) is the most common enzymopathy globally, and deficient individuals may experience severe hemolysis following treatment with 8-aminoquinolines. With increasing evidence of Plasmodium vivax infections throughout sub-Saharan Africa, there is a pressing need for population-level data at on the prevalence of G6PDd. Such evidence-based data will guide the expansion of primaquine and potentially tafenoquine for radical cure of P. vivax infections. This study aimed to quantify G6PDd prevalence in two geographically distinct areas in Sudan, and evaluating the performance of a qualitative CareStart rapid diagnostic test as a point-of-care test. Blood samples were analyzed from 491 unrelated healthy persons in two malaria-endemic sites in eastern and central Sudan. A pre-structured questionnaire was used which included demographic data, risk factors and treatment history. G6PD levels were measured using spectrophotometry (SPINREACT) and first-generation qualitative CareStart rapid tests. G6PD variants (202 G>A; 376 A>G) were determined by PCR/RFLP, with a subset confirmed by Sanger sequencing. The prevalence of G6PDd by spectrophotometry was 5.5% (27/491; at 30% of adjusted male median, AMM); 27.3% (134/491; at 70% of AMM); and 13.1% (64/490) by qualitative CareStart rapid diagnostic test. The first-generation CareStart rapid diagnostic test had an overall sensitivity of 81.5% (95%CI: 61.9 to 93.7) and negative predictive value of 98.8% (97.3 to 99.6). All persons genotyped across both study sites were wild type for the G6PD G202 variant. For G6PD A376G all participants in New Halfa had wild type AA (100%), while in Khartoum the AA polymorphism was found in 90.7%; AG in 2.5%; and GG in 6.8%. Phenotypic G6PD B was detected in 100% of tested participants in New Halfa while in Khartoum, the phenotypes observed were B (96.2%), A (2.8%), and AB (1%). The African A- phenotype was not detected in this study population. Overall, G6PDd prevalence in Sudan is low-to-moderate but highly heterogeneous. Point-of-care testing with the qualitative CareStart rapid diagnostic test demonstrated moderate performance with moderate sensitivity and specificity but high negative predicative value. The two sites harbored primarily the African B phenotype. A country-wide survey is recommended to understand GP6PD deficiencies more comprehensively in Sudan.

Identification of Risk Factors Associated with Resistant Escherichia coli Isolates from Poultry Farms in the East Coast of Peninsular Malaysia: A Cross Sectional Study.

Antimicrobial resistance is of concern to global health security worldwide. We aimed to identify the prevalence, resistance patterns, and risk factors associated with Escherichia coli (E. coli) resistance from poultry farms in Kelantan, Terengganu, and Pahang states of east coast peninsular Malaysia. Between 8 February 2019 and 23 February 2020, a total of 371 samples (cloacal swabs = 259; faecal = 84; Sewage = 14, Tap water = 14) were collected. Characteristics of the sampled farms including management type, biosecurity, and history of disease were obtained using semi-structured questionnaire. Presumptive E. coli isolates were identified based on colony morphology with subsequent biochemical and PCR confirmation. Susceptibility of isolates was tested against a panel of 12 antimicrobials and interpreted alongside risk factor data obtained from the surveys. We isolated 717 E. coli samples from poultry and environmental samples. Our findings revealed that cloacal (17.8%, 46/259), faecal (22.6%, 19/84), sewage (14.3%, 2/14) and tap water (7.1%, 1/14) were significantly (p < 0.003) resistant to at least three classes of antimicrobials. Resistance to tetracycline class were predominantly observed in faecal samples (69%, 58/84), followed by cloacal (64.1%, 166/259), sewage (35.7%, 5/14), and tap water (7.1%, 1/84), respectively. Sewage water (OR = 7.22, 95% CI = 0.95-151.21) had significant association with antimicrobial resistance (AMR) acquisition. Multivariate regression analysis identified that the risk factors including sewage samples (OR = 7.43, 95% CI = 0.96-156.87) and farm size are leading drivers of E. coli antimicrobial resistance in the participating states of east coast peninsular Malaysia. We observed that the resistance patterns of E. coli isolates against 12 panel antimicrobials are generally similar in all selected states of east coast peninsular Malaysia. The highest prevalence of resistance was recorded in tetracycline (91.2%), oxytetracycline (89.1%), sulfamethoxazole/trimethoprim (73.1%), doxycycline (63%), and sulfamethoxazole (63%). A close association between different risk factors and the high prevalence of antimicrobial-resistant E. coli strains reflects increased exposure to resistant bacteria and suggests a concern over rising misuse of veterinary antimicrobials that may contribute to the future threat of emergence of multidrug-resistant pathogen isolates. Public health interventions to limit antimicrobial resistance need to be tailored to local poultry farm practices that affect bacterial transmission.

Studies of host preferences of wild-caught Phlebotomus orientalis and Ph. papatasi vectors of leishmaniasis in Sudan.

INTRODUCTION: Understanding the feeding behavior and host choice of sand flies provides valuable information on vector-host relationships and elucidates the epidemiological patterns of leishmaniasis transmission. Blood meal analysis studies are essential for estimating the efficiency of pathogen transmission, assessing the relative human disease risk, and assist in identifying the other potential hosts of leishmaniasis. In Sudan and most of East Africa, there are large remaining gaps in knowledge regarding the feeding habits of phlebotomine vectors. The study aimed to identify the blood meal sources and, therefore, the host preferences of the principal vectors Phlebotomus orientalis and Ph. papatasi in leishmaniasis endemic areas of eastern and central Sudan. MATERIALS AND METHODS: Sand flies were collected from two endemic villages in eastern and central Sudan using CDC light traps and sticky traps. The phlebotomine sand flies were morphologically and then molecularly identified. The source of blood meal of the engorged females was determined using a multiplex PCR methodology and specific primers of cytochrome b gene of mitochondrial DNA for human, goat, cow, and dog. The detection of the Leishmania parasite was done using PCR. RESULTS: The total number of collected female phlebotomine sand flies was 180. Morphological identification revealed the abundance of Ph. orientalis 103 (57.2%), Ph. papatasi 42 (23.3%), Ph. bergeroti 31 (17.2%), Ph. rodhaini 2 (1.1%) and Ph. duboscqi 2 (1.1%) in the study sites. Out of the 180 collected, 31 (17%) were blood-fed flies. Three species were blood-fed and molecularly identified: Ph. papatasi (N = 7, 22.6%), Ph. bergeroti (N = 9, 26%), and Ph. orientalis (N = 15, 48.4%). Blood meal analysis revealed human DNA in two Ph. orientalis (6.4%), hence, the anthropophilic index was 13.3%. CONCLUSIONS: Multiplex PCR protocol described here allowed the identification of blood meal sources of many vertebrate species simultaneously. The results indicate that wild-caught Ph. orientalis are anthropophilic in the study areas. Further studies on larger blood-fed sample size are required to validate the potential applications of this technique in designing, monitoring and evaluating control programs, particularly in investigating the potential non-human hosts of leishmaniasis.

Antimalarial drug resistance molecular makers of Plasmodium falciparum isolates from Sudan during 2015-2017.

BACKGROUND: Current malaria control and elimination strategies rely mainly on efficacious antimalarial drugs. However, drug resistance is a major threat facing malaria control programs. Determination of drug resistance molecular markers is useful in the monitoring and surveillance of malaria drug efficacy. This study aimed to determine the mutations and haplotypes frequencies of different genes linked with antimalarial drug resistance in certain areas in Sudan. METHODS: A total of 226 dried blood spots (DBS) of microscopically diagnosed P. falciparum isolates were collected from Khartoum and three other areas in Sudan during 2015-2017. Plasmodium falciparum confirmation and multiplicity of infection was assessed using the Sanger's 101 SNPs-barcode and speciation was confirmed using regions of the parasite mitochondria. Molecular genotyping of drug resistance genes (Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, exonuclease, Pfk13, parasite genetic background (PGB) (Pfarps10, ferredoxin, Pfcrt, Pfmdr2)) was also performed. All genotypes were generated by selective regions amplicon sequencing of the parasite genome using the Illumina MiSeq platform at the Wellcome Sanger Institute, UK then genotypes were translated into drug resistance haplotypes and species determination. FINDINGS: In total 225 samples were confirmed to be P. falciparum. A higher proportion of multiplicity of infection was observed in Gezira (P<0.001) based on the Sanger 101 SNPs -barcode. The overall frequency of mutant haplotype Pfcrt 72-76 CVIET was 71.8%. For Pfmdr1, N86Y was detected in 53.6%, Y184F was observed in 88.1% and D1246Y was detected in 1.5% of the samples. The most frequently observed haplotype was YFD 47.4%. For Pfdhfr (codons 51, 59,108,164), the ICNI haplotype was the most frequent (80.7%) while for Pfdhps (codons 436, 437, 540, 581, 613) the (SGEAA) was most frequent haplotype (41%). The Quadruple mutation (dhfr N51I, S108N + dhps A437G, K540E) was the highest frequent combined mutation (33.9%). In Pfkelch13 gene, 18 non-synonymous mutations were detected, 7 of them were detected in other African countries. The most frequent Pfk13 mutation was E433D detected in four samples. All of the Pfk13 mutant alleles have not been reported to belong to mutations associated with delayed parasite clearance in Southeast Asia. PGB mutations were detected only in Pfcrt N326S\I (46.3%) and Pfcrt I356T (8.2%). The exonuclease mutation was not detected. There was no significant variation in mutant haplotypes between study areas. CONCLUSIONS: There was high frequency of mutations in Pfcrt, Pfdhfr and Pfdhps in this study. These mutations are associated with chloroquine and sulfadoxine-pyrimethamine (SP) resistance. Many SNPs in Pfk13 not linked with delayed parasite clearance were observed. The exonuclease E415G mutation which is linked with piperaquine resistance was not reported.

Seroprevalence and associated risk factors of Dengue fever in Kassala state, eastern Sudan.

Dengue is a rapidly growing public health threat in Kassala state, eastern Sudan. The objective of this study was to determine the seroprevalence, entomological transmission indices, and socioeconomic risk factors associated with dengue in this region. A cross-sectional community-based study was conducted in four dengue-endemic sites; Khatmia, West Gash, Thoriba, and Shokriya between March 2016 to March 2017. Enzyme-linked immunosorbent assay (ELISA) of immunoglobulin G (IgG) was used to determine the prevalence of dengue virus among the study participants. An entomological survey was conducted using pyrethrum spray catch and dipping for the collection of adults and aquatic stages of Aedes aegypti, respectively. Ribonucleic acid was extracted from the buffy coat of participants as well as from adult female Ae. aegypti to assess the possible circulation of dengue virus using Reverse Transcription Polymerase Chain Reaction (RT-PCR). Multiple logistic regression model was used to estimate the association between potential risk factors and dengue seropositivity. A total of 409 persons were recruited to the study: 45.5% were in the 20-39 years' age category; 57.9% were living in houses with 6-10 persons; and 29.1% had at most secondary school education. In the majority (65.8%) of the households, the socioeconomic status was low (P<0.001). Long-lasting insecticide-treated bed nets were used in 56.5% of the households. Over three-quarters (77.8%) claimed not to have experienced febrile illness in the last three months. Routine entomological survey across Kassala state identified a total of 3,304 larvae and 390 pupae Ae. aegypti, respectively. The overall house index was 32.8% and Breteau Index was 35.96% (146/406). The overall pupal demographic index was 13.31%, and the pupal children index was 97.26%. Antibodies against IgG were detected from 66 (42.04%) out of a total of 157 sera. Twenty-two positive sera (75.9%) were collected from Khatmia. A total of 329 adults Ae. aegypti were identified but only one (0.3%) was positive for DENV in Khatmia. Finally, four independent risk factors were identified to derive dengue circulation in Kassala: elder age (> 60 years) (OR 6.31, CI 1.09-36.36); type of bathroom (OR 3.52, CI 1.35-9.20); using water-based air conditioner (OR 6.90, CI 1.78-26.85) and previous infection of any household member with dengue (OR 28.73, CI 3.31-249.63). Our findings suggest that Kassala state is facing an increasing occurrence of dengue and emphasizes the need for developing appropriate interventions to address the identified risk factors, and place control programs into actions. Establishment of routine dengue epidemiological and entomological surveillance, and climate warning systems will contribute to early warning and timely detection and response to emerging outbreaks.

Cross sectional study to determine chloroquine resistance among Plasmodium falciparum clinical isolates from Khartoum, Sudan.

Background:  Malaria continues to present a global health threat; the World Health Organization (WHO) reported 214 million cases of malaria by the year 2015 with a death rate of 438000. Sudan is endemic to malaria with over 95% of malaria cases due to Plasmodium falciparum. Chloroquine is a well-established drug in the treatment of P. falciparum malaria although its use has declined since its introduction as the drug of choice in treatment of malaria in Sudan. The mechanism of resistance has been attributed to mutations in P. falciparum Chloroquine resistance transporter gene coding for a key food vacuole proteins. In current study we aimed at verifying the genetic cause of resistance to Chloroquine in field isolates of P. falciparum. Methods: Twenty P. falciparum cases were diagnosed from East Nile hospital in Khartoum and recruited in the investigation. Nested PCR was conducted to isolate mutation region in the PfCRT gene and the amplicons were sequenced using Sanger sequencing technique (Macrogen, Soule Korea). Results: 16/20 (80%) of the field isolates contained base pair mutation of codon 76 in the pfcrt gene thus being resistant to chloroquine treatment and only 4/20 (20%) did not contain such mutation. Conclusions: High treatment failures associated with Chloroquine treatment is evident of the high prevalence of mutant strains of P. falciparum field isolates thus suggesting the reduced relevance of Chloroquine as a treatment choice in the management of P. falciparum malaria.

The prevalence of Hymenolepis nana among preschool children of displacement communities in Khartoum state, Sudan: a cross-sectional study.

BACKGROUND: Hymenolepis nana is among the most common intestinal parasitic infections causing a public health threat in poor communities in Sub-Saharan Africa. The present study was conducted to determine the prevalence of H. nana infections and associated risk factors among preschool children of displacement communities in Khartoum state, Sudan. METHODS: A cross-sectional survey was conducted in May 2013 in displacement camps, Khartoum state, Sudan. A simple random sample of preschool children from the displacement camps, aged between 1 and 5 years, were included. Information was collected by presenting a questionnaire and taking 500 fresh stool specimens which were examined microscopically for the presence of eggs, using direct saline and formal-ether concentration techniques. RESULTS: The prevalence of H. nana was determined to be 32.6% (163/500), 95% CI (28.5%-36.9%). Infections of H. nana were more prevalent among males than females, and this association was statistically significant (P < 0.001, OR = 2.125, 95% CI = 1.452-3.108). H. nana infections were significantly prevalent among the older age group (2.6-5.0 years) (P < 0.001, OR = 2.909, 95% CI = 1.914-4.420). Approximately 76.7% of infected preschool children had diarrhea and it was significantly associated with H. nana infection (P < 0.001, OR = 9.45, 95% CI = 6.10-14.64). None of the preschool children had access to a clean water supply. No significant association was found between use of latrines and infections of H. nana (P = 0.56, OR = 0.880, 95% CI = 0.73-1.763). CONCLUSIONS: There was a high prevalence rate of H. nana infection among preschool children of displacement camps in Khartoum state, Sudan. Being male, aged between 2.6 and 5.0 years, and having diarrhea were identified as important risk factors for H. nana infection. Measures including health education, environmental hygiene, water supply and treatment should be taken into account to reduce the high prevalence of H. nana.