Molecular identification and anti-malarial drug resistance profile of Plasmodium falciparum from patients attending Kisoro Hospital, southwestern Uganda

Background The evolution of malaria infection has necessitated the development of highly sensitive diagnostic assays, as well as the use of dried blood spots (DBS) as a potential source of deoxyribonucleic acid (DNA) yield for polymerase chain reaction (PCR) assays. This study identified the different Plasmodium species in malaria-positive patients, and the anti-malarial drug resistance profile for Plasmodium falciparum using DBS samples collected from patients attending Kisoro Hospital in Kisoro district, Southwestern Uganda. Methods The blood samples were prospectively collected from patients diagnosed with malaria to make DBS, which were then used to extract DNA for real-time PCR and high-resolution melting (HRM) analysis. Plasmodium species were identified by comparing the control and test samples using HRM-PCR derivative curves. Plasmodium falciparum chloroquine (CQ) resistance transporter (pfcrt) and kelch13 to screen the samples for anti-malarial resistance markers. The HRM-PCR derivative curve was used to present a summary distribution of the different Plasmodium species as well as the anti-malarial drug profile. Results Of the 152 participants sampled, 98 (64.5%) were females. The average age of the participants was 34.9 years (range: 2 months–81 years). There were 134 samples that showed PCR amplification, confirming the species as Plasmodium. Plasmodium falciparum (N = 122), Plasmodium malariae (N = 6), Plasmodium ovale (N = 4), and Plasmodium vivax (N = 2) were the various Plasmodium species and their proportions. The results showed that 87 (71.3%) of the samples were sensitive strains/wild type (CVMNK), 4 (3.3%) were resistant haplotypes (SVMNT), and 31 (25.4%) were resistant haplotypes (CVIET). Kelch13 C580Y mutation was not detected. Conclusion The community served by Kisoro hospital has a high Plasmodium species burden, according to this study. Plasmodium falciparum was the dominant species, and it has shown that resistance to chloroquine is decreasing in the region. Based on this, molecular identification of Plasmodium species is critical for better clinical management. Besides, DBS is an appropriate medium for DNA preservation and storage for future epidemiological studies.

The whole-genome sequencing in predicting Mycobacterium tuberculosis drug susceptibility and resistance in Papua, Indonesia

Background Tuberculosis is one of the deadliest disease caused by Mycobacterium tuberculosis. Its treatment still becomes a burden for many countries including Indonesia. Drug resistance is one of the problems in TB treatment. However, a development in the molecular field through Whole-genome sequencing (WGS) can be used as a solution in detecting mutations associated with TB- drugs. This investigation intended to implement this data for supporting the scientific community in deeply understanding any TB epidemiology and evolution in Papua along with detecting any mutations in genes associated with TB-Drugs. Result A whole-genome sequencing was performed on the random samples from TB Referral Laboratory in Papua utilizing MiSeq 600 cycle Reagent Kit (V3). Furthermore, TBProfiler was used for genome analysis, RAST Server was employed for annotation, while Gview server was applied for BLAST genome mapping and a Microscope server was implemented for Regions of Genomic Plasticity (RGP). The largest genome of M. tuberculosis obtained was at the size of 4,396,040 bp with subsystems number at 309 and the number of coding sequences at 4326. One sample (TB751) contained one RGP. The drug resistance analysis revealed that several mutations associated with TB-drug resistance existed. In details, mutations of rpoB gene which were identified as S450L, D435Y, H445Y, L430P, and Q432K had caused the reduced effectiveness of rifampicin; while the mutases in katG (S315T), kasA (312S), inhA (I21V), and Rv1482c-fabG1 (C-15 T) genes had contributed to the resistance in isoniazid. In streptomycin, the resistance was triggered by the mutations in rpsL (K43R) and rrs (A514C, A514T) genes, and, in Amikacin, its resistance was led by mutations in rrs (A514C) gene. Additionally, in Ethambutol and Pyrazinamide, their reduced effectiveness was provoked by embB gene mutases (M306L, M306V, D1024N) and pncA (W119R). Conclusions The results from whole-genome sequencing of TB clinical sample in Papua, Indonesia could contribute to the surveillance of TB-drug resistance. In the drug resistance profile, there were 15 Multi Drugs Resistance (MDR) samples. However, Extensively Drug-resistant (XDR) samples have not been found, but samples were resistant to only Amikacin, a second-line drug.

Drug Resistance Profile and Clinical Features for Hepatitis C Patients Experiencing DAA Failure in Taiwan

About 4% of the population in Taiwan are seropositive for anti-HCV Ab and 70% with HCV RNA. To address this high chronic hepatitis C disease load, Taiwan National Health Insurance started reimbursing genotype-specific DAAs in 2017 and pangenotype DAAs in mid-2018. With a 97% SVR12 rate, there were still 2–3% of patients that failed to clear HCV. To understand the causes of DAA failure in Taiwan, we conducted a multi-center, clinical, and virologic study. A total of 147 DAA-failure patients were recruited, and we searched HCV NS3/4A, NS5A and NS5B for known resistance-associated substitutions (RASs) by population sequencing, and conducted whole genome sequencing (WGS) for those without known RASs. A total of 107 patients received genotype-specific DAAs while 40 had pangenotype DAAs. Clinically, the important cause of failure is poor adherence. Virologically, common RASs in genotype-specific DAAs were NS5A-L31, NS5A-Y93, and NS5B-C316, while common RASs in pangenotype DAAs were NS5A-L31, NS5A-A/Q/R30, and NS5A-Y93. Additionally, new amino acid changes were found by WGS. Finally, we identified 12 cases with inconsistent baseline and post-treatment HCV genotypes, which is suggestive of re-infection rather than treatment failure. Our study described the drug resistance profile for DAA failure in Taiwan, showing differences from other countries.

Prevalence of Salmonella in Free-Range Pigs: Risk Factors and Intestinal Microbiota Composition

Extensive pig systems are gaining importance as quality production systems and as the standard for sustainable rural development and animal welfare. However, the effects of natural foods on Salmonella epidemiology remain unknown. Herein, we assessed the presence of Salmonella and the composition of the gut microbiota in pigs from both Salmonella-free and high Salmonella prevalence farms. In addition, risk factors associated with the presence of Salmonella were investigated. The pathogen was found in 32.2% of animals and 83.3% of farms, showing large differences in prevalence between farms. Most isolates were serovars Typhimurium monophasic (79.3%) and Bovismorbificans (10.3%), and exhibited a multi-drug resistance profile (58.6%). Risk factor analysis identified feed composition, type/variety of vegetation available, and silos’ cleaning/disinfection as the main factors associated with Salmonella prevalence. Clear differences in the intestinal microbiota were found between Salmonella-positive and Salmonella-negative populations, showing the former with increasing Proteobacteria and decreasing Bacteroides populations. Butyrate and propionate producers including Clostridium, Turicibacter, Bacteroidaceae_uc, and Lactobacillus were more abundant in the Salmonella-negative group, whereas acetate producers like Sporobacter, Escherichia or Enterobacter were more abundant in the Salmonella-positive group. Overall, our results suggest that the presence of Salmonella in free-range pigs is directly related to the natural vegetation accessible, determining the composition of the intestinal microbiota.

Urinary Tract Infection, Drug Resistance Profile And Fetal Outcomes Among Pregnant Women In Selected Health Facilites Of Addis Ababa, Ethiopia: A CrossSectional Study Design

Background: Urinary tract infection (UTI) is a commonly encountered health problem among pregnant women. Untreated UTI may result in fetal complications like preterm birth, low birth weight, intra uterine growth retardation and intrauterine fetal death. One of the major problems with bacterial pathogens is their ability to develop drug resistance. Determining drug susceptibility pattern of bacteria from urinary tract helps to identify effective drugs and minimize further adverse perinatal outcomes. The purpose of this study was to describe the magnitude and drug resistance profile of pregnant women and related fetal outcomes. Methods:A cross-sectional study was conducted from March to June 2019. Socio-demographic data of the study participants was collected by administering structured questionnaire after obtaining full consent of the participants. Clean catch mid-stream urine was collected from the study participants and the samples were transported to the laboratory with screw-capped container. Blood and MacConkey agar were used to cultivate bacteria from urine sample. Bacterial colonies were isolated and identified using standard biochemical properties. Antibacterial susceptibility test was done on Muller-Hinton agar using antibiotic discs. At the time of delivery, infant’s birth weight and gestational age was recorded on the log book as part of the daily work. Fetal outcome data were recorded from the log book and entered to EPI data management software then transferred to SPSS for analysis. Results:Out of 424 urine samples processed, 63 (14.9%) yielded significant bacteriuria. Fifty-nine out of 63 (93.7%) cases of UTIs were recorded among age group of 15-34 years. Escherichia coli, Klebsiellapneumoniae, and Staphylococcus aureus were the three dominant bacterial isolates. Most of the gram negative bacterial isolates were resistant to ampicillin and ceftazidime (43.2% each) while gram positive was resistant for tetracycline (36.8%). The result of this study showed that UTI had statistical association with the occurrence of Intrauterine Fetal Death (IUFD) and Prerupture of Membrane (PROM) (P<.05). Conclusion: Adverse fetal outcomes are significantly linked with the presence of UTI during pregnancy. The condition can be managed if early diagnosis and treatments are made for the mother

The Magnitude and Drug Resistance Profile of Extended Spectrum Β-Lactamase (Esbl) Producing Gram-Negative Bacteria from Different Inanimate Objects at Tikur Anbessa Specialized Hospital, Addis Ababa, Ethiopia

Background: Infections caused by gram-negative bacteria are causing morbidity and mortality worldwide. The production of extended-spectrum β-lactamases (ESBLs) is an important mechanism that is responsible for resistance to the third-generation cephalosporin. The purpose of this study was to determine the magnitude and drug resistance profile of ESBL producing gram-negative bacteria isolated from various inanimate objects at Tikur Anbessa Specialized Hospital. Methods: Laboratory based cross-sectional study was conducted involving a total of 216 isolates from January to March 2019. The samples were taken from different inanimate objects at Tikur Anbessa Specialized Hospital using pre-moistened sterile swabs. Screening of ESBLs was done using ESBL CHROME agar and confirmed with combined disk diffusion test. Antimicrobial susceptibility testing was done by disc diffusion method. The data were analyzed using SPSS software version 20 and descriptive statistical tests were performed. Results: 33/216 (15.3%) isolates were found to be ESBL producers based on the confirmatory test (combined disk method). Different ESBL producing gram-negative bacteria were isolated from the various inanimate objects of TASH including, Klebsiella ozaenae, Klebsiella oxytoca, Klebsiella pneumoniae, Klebsiella rhinoscleromatis, Citrobacter spp, Escherichia coli, Serriatia spp and Acinetobacter spp. The isolates were found to be 100% resistant to ceftazidime and ceftriaxone. Conclusion: It is worrisome to detect ESBL producing gram-negative bacteria from the inanimate objects of TASH, calling for systematic screening of inanimate objects for ESBL and other multidrug-resistant bacteria in the hospital. Furthermore, strengthening the infection prevention practice is vital to halt the transmission of these microorganisms.