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.

Gel express: a novel frugal method quantifies gene relative expression in conventional RT-PCR

Background Real-time PCR system is a valuable scientific mainstream needed for quantifying specific gene expression. Nevertheless, compared with conventional PCR, the real-time PCR system is extremely expensive and not affordable for limited or mid-budget research laboratories. Here, a novel, doable and low-cost recipe (referred to as gel express) is developed to quantify gene expression using conventional RT-PCR assay. The novelty of the gel express method is based on replacing crossing point (CP) values with integrated density (IntDen) values of PCR amplicon bands in real-time PCR regular mathematical formulas. Results In this work, gene expression profiles of two different rice stress-marker genes (OsCYP94C2a and OsLOX8) were quantified in response to mechanical wounding at different time points (0, 30, 60, and 150 min). In the gel express method, the free software ImageJ was employed to measure integrated density (IntDen) values of PCR amplicon bands in agarose gel images. IntDen values were then used instead of crossing point (CP) values according to the following modified formula: [EIntDen(ref)/EIntDen(target)]sample ÷ [EIntDen(ref)/EIntDen(target)]control. Gene relative expression profiles (dynamic expression pattern) quantified by gel express method in both genes were highly comparable with real-time RT-PCR. R2 values were 0.9976 and 0.9975 in OsCYP94C2a and OsLOX, respectively. PCR amplification efficiency (E) for all studied genes could be calculated depending on IntDen values through experimentally designed calibration curves. PCR amplification efficiencies with all studied genes obtained by gel express were all in the accepted range. For better-visualized PCR amplicons thus detectable biological effects between treatments, the number of PCR cycles applied in gel express method (IntCyc) was experimentally estimated to be 29 cycles. Conclusions Gel express is a novel, cost-effective and feasible recipe for quantifying gene relative expression in conventional RT-PCR. The expression pattern quantified by gel express is highly comparable and fits the expression data revealed by the used real-time PCR system.

Metabarcoding reveals low prevalence of microsporidian infections in castor bean tick (Ixodes ricinus)

Background Microsporidia is a large group of eukaryotic obligate intracellular spore-forming parasites, of which 17 species can cause microsporidiosis in humans. Most human-infecting microsporidians belong to the genera Enterocytozoon and Encephalitozoon. To date, only five microsporidian species, including Encephalitozoon-like, have been found in hard ticks (Ixodidae) using microscopic methods, but no sequence data are available for them. Furthermore, no widespread screening for microsporidian-infected ticks based on DNA analysis has been carried out to date. Thus, in this study, we applied a recently developed DNA metabarcoding method for efficient microsporidian DNA identification to assess the role of ticks as potential vectors of microsporidian species causing diseases in humans. Methods In total, 1070 (493 juvenile and 577 adult) unfed host-seeking Ixodes ricinus ticks collected at urban parks in the city of Poznan, Poland, and 94 engorged tick females fed on dogs and cats were screened for microsporidian DNA. Microsporidians were detected by PCR amplification and sequencing of the hypervariable V5 region of 18S rRNA gene (18S profiling) using the microsporidian-specific primer set. Tick species were identified morphologically and confirmed by amplification and sequencing of the shortened fragment of cytochrome c oxidase subunit I gene (mini-COI). Results All collected ticks were unambiguously assigned to I. ricinus. Potentially zoonotic Encephalitozoon intestinalis was identified in three fed ticks (3.2%) collected from three different dogs. In eight unfed host-seeking ticks (0.8%), including three males (1.1%), two females (0.7%) and three nymphs (0.7%), the new microsporidian sequence representing a species belonging to the genus Endoreticulatus was identified. Conclusions The lack of zoonotic microsporidians in host-seeking ticks suggests that I. ricinus is not involved in transmission of human-infecting microsporidians. Moreover, a very low occurrence of the other microsporidian species in both fed and host-seeking ticks implies that mechanisms exist to defend ticks against infection with these parasites. Graphical abstract

Cyanogenesis in the Sorghum Genus: From Genotype to Phenotype

Domestication has resulted in a loss of genetic diversity in our major food crops, leading to susceptibility to biotic and abiotic stresses linked with climate change. Crop wild relatives (CWR) may provide a source of novel genes potentially important for re-gaining climate resilience. Sorghum bicolor is an important cereal crop with wild relatives that are endemic to Australia. Sorghum bicolor is cyanogenic, but the cyanogenic status of wild Sorghum species is not well known. In this study, leaves of wild species endemic in Australia are screened for the presence of the cyanogenic glucoside dhurrin. The direct measurement of dhurrin content and the potential for dhurrin-derived HCN release (HCNp) showed that all the tested Australian wild species were essentially phenotypically acyanogenic. The unexpected low dhurrin content may reflect the variable and generally nutrient-poor environments in which they are growing in nature. Genome sequencing of six CWR and PCR amplification of the CYP79A1 gene from additional species showed that a high conservation of key amino acids is required for correct protein function and dhurrin synthesis, pointing to the transcriptional regulation of the cyanogenic phenotype in wild sorghum as previously shown in elite sorghum.

Antimicrobial Susceptibility Profiles among Pseudomonas aeruginosa Isolated from Professional SCUBA Divers with Otitis Externa, Swimming Pools and the Ocean at a Diving Operation in South Africa

SCUBA divers are predisposed to otitis externa caused by Pseudomonas aeruginosa, which is becoming increasingly multi-drug resistant (MDR). The present work assessed the antibiotic resistance profiles of P. aeruginosa obtained from SCUBA divers and their environment in Sodwana Bay, South Africa. Bacterial isolates from a total of 137 random water and ear swab samples were identified using biochemical and molecular methods. P. aeruginosa strains were further evaluated for antibiotic susceptibility using the Kirby–Bauer assay. Double disk synergy test (DDST) to confirm metallo-β-lactamase (MBL) production and PCR amplification of specific antibiotic resistance genes was performed. All (100%) 22 P. aeruginosa isolates recovered were resistant to 6 of the β-lactams tested including imipenem but exhibited susceptibility to trimethoprim–sulfamethoxazole. MBL production was observed in 77% of isolates while the most prevalent extended-spectrum β-lactamase (ESBL) genes present included blaAmpC (86.9%) followed by blaTEM (82.6%). Sulfonamide resistance was largely encoded by sul1 (63.6%) and sul2 (77.3%) genes with a high abundance of class 1 integrons (77.3%) of which 18.2% carried both Intl1 and Intl2. P. aeruginosa found in Sodwana Bay exhibits multi-drug resistance (MDRce) to several pharmaceutically important drugs with the potential to transfer antibiotic resistance to other bacteria if the judicious use of antibiotics for their treatment is not practiced.

The Nasal Microbiome of Predicting Bronchopulmonary Dysplasia in Preterm Infants

Bronchopulmonary dysplasia (BPD) is chronic lung disease of prematurity and associated with substantial long-term disabilities. To characterize and compare the nasal swabs microbiome of early stage in premature infants and determine whether microbial diversity or composition in the airway associated with BPD disease. We performed a prospective observational cohort design. Preterm neonates less than 32 weeks of gestation were recruited from NICU, Children's Hospital, Zhejiang University School of Medicine from 2019 to 2020. Sterile foam swabs were collected from anterior nares at 1 and 3 weeks of postnatal age. We used PCR amplification and 16S rDNA sequencing. Neonatal demographic data including gestational age, birth weight, medication administration history were recorded. A total of 98 nasal swabs samples were collected from 54 preterm infants, 13 developed BPD infants and 41 control infants were finally involved in the study. Birth weights ranged from 700 to 2,050 g. Gestational age ranged from 25 2/7to 31 6/7. We found increased in the expression of Prevotella, Marinomonas, Enterobacteriaceae, Weissella, Selenomonas, Oribacterium, Nubsella and Antricoccus in BPD group at two time points. Prevotella was correlated with the severity of BPD (Spearman r=0.361, P=0.000). Given possible roles for noninvasive upper airway microbiota in BPD pathobiology, the nasal microbiome in BPD is a compelling area of research to continue to expand.

A Real-Time PCR Assay for Detection of Low Pneumocystis jirovecii Levels

Here we report a new real-time PCR assay using SYBR Green which provides higher sensitivity for the specific detection of low levels of Pneumocystis jirovecii. To do so, two primer sets were designed, targeting the family of genes that code for the most abundant surface protein of Pneumocystis spp., namely the major surface glycoproteins (Msg), and the mitochondrial large subunit rRNA (mtLSUrRNA) multicopy gene, simultaneously detecting two regions. PCR methods are instrumental in detecting these low levels; however, current nested-PCR methods are time-consuming and complex. To validate our new real-time Msg-A/mtLSUrRNA PCR protocol, we compared it with nested-PCR based on the detection of Pneumocystis mitochondrial large subunit rRNA (mtLSUrRNA), one of the main targets used to detect this pathogen. All samples identified as positive by the nested-PCR method were found positive using our new real-time PCR protocol, which also detected P. jirovecii in three nasal aspirate samples that were negative for both rounds of nested-PCR. Furthermore, we read both rounds of the nested-PCR results for comparison and found that some samples with no PCR amplification, or with a feeble band in the first round, correlated with higher Ct values in our real-time Msg-A/mtLSUrRNA PCR. This finding demonstrates the ability of this new single-round protocol to detect low Pneumocystis levels. This new assay provides a valuable alternative for P. jirovecii detection, as it is both rapid and sensitive.

Disproportionate Distribution of HBV Genotypes A and D and the Recombinant Genotype D/E in the High and Low HBV Endemic Regions of Uganda: A Wake-Up Call for Regional Specific HBV Management

Background. Hepatitis B virus (HBV) is the leading cause of liver-related diseases. In Uganda, there is a regional disparity in the HBV burden. Our study was aimed at establishing the circulating genotypes in a low and a high endemic region to give plausible explanations for the differences in regional burden and guide the future management of the disease. Methods. A total of 200 HBsAg-seropositive subjects were recruited into the study by convenience sampling. The HBsAg Rapid Test Strip (Healgen Scientific Limited Liability Company, Houston, TX77047- USA) was used to screen for HBsAg while the Roche machine (Roche, Basel Switzerland/Abbot Technologies (USA)) was used to determine the viral load. The Chemistry Analyzer B120 (Mindray, China) was used for chemistry analysis. For HBV genotyping, total DNA was extracted from whole blood using the QIAamp® DNA extraction kit. Nested PCR amplification was performed using Platinum Taq DNA Polymerase (Invitrogen Corporation, USA) to amplify the 400 bp HBV polymerase gene. Purification of nested PCR products was performed using Purelink PCR product purification kit (Life Technologies, USA). Automated DNA sequencing was performed using BigDye Terminator v3.1 Cycle Sequencing Kit on 3130 Genetic Analyzer (Applied Biosystems, USA). The NCBI HBV genotyping tool (https://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.cgi) was used for determination of genotype for each HBV sequence. Pearson’s chi-square, multinomial logistic regression, and Mann–Whitney U tests were used for the analysis. All the analyses were done using SPSS version 26.0 and MedCalc software version 19.1.3 at 95% CI. A p < 0.05 was considered statistically significant. Results. Majority of our study subjects were female (64.5%), youth (51.0%), and married (62.0%). Overall, genotype A was the most prevalent (46%). Genotype D and the recombinant genotype D/E were proportionately more distributed in the high endemic (38.2%) and low endemic (36.5%) regions, respectively. Genotype D was significantly more prevalent in the high endemic region and among the elderly ( p < 0.05 ). Genotype D was significantly associated with elevated viral load and direct bilirubin ( p < 0.05 ). The recombinant genotype D/E was significantly associated with elevated viral load ( p < 0.05 ). Similarly, genotype A was significantly associated with elevated AST and GGT, lowered viral load, and normal direct bilirubin levels ( p < 0.05 ). Conclusion. There is disproportionate distribution of genotypes A and D and the recombinant genotype D/E in the low and high endemic regions of Uganda. This probably could explain the differences in endemicity of HBV in our country signifying the need for regional specific HBV management and control strategies.

The evolution of insect visual opsin genes with specific consideration of the influence of ocelli and life history traits

Background Visual opsins are expressed in the compound eyes and ocelli of insects and enable light detection. Three distinct phylogenetic groups of visual opsins are found in insects, named long (LW), short (SW) and ultraviolet (UV) wavelength sensitive opsins. Recently, the LW group was found to be duplicated into the LW2b and the LW2a opsins. The expression of LW2b opsins is ocelli specific in some insects (e.g., bees, cricket, scorpion flies), but the gene was not found in other orders possessing three or less ocelli (e.g., dragonflies, beetles, moths, bugs). In flies, two LW2b homologs have been characterised, with one expressed in the ocelli and the other in the compound eyes. To date, it remains unclear which evolutionary forces have driven gains and losses of LW opsins in insects. Here we take advantage of the recent rapid increase in available sequence data (i.e., from insect genomes, targeted PCR amplification, RNAseq) to characterize the phylogenetic relationships of 1000 opsin sequences in 18 orders of Insects. The resulting phylogeny discriminates between four main groups of opsins, and onto this phylogeny we mapped relevant morphological and life history traits. Results Our results demonstrate a conserved LW2b opsin only present in insects with three ocelli. Only two groups (Brachycera and Odonata) possess more than one LW2b opsin, likely linked to their life history. In flies, we hypothesize that the duplication of the LW2b opsin occurred after the transition from aquatic to terrestrial larvae. During this transition, higher flies (Brachycera) lost a copy of the LW2a opsin, still expressed and duplicated in the compound eyes of lower flies (Nematocera). In higher flies, the LW2b opsin has been duplicated and expressed in the compound eyes while the ocelli and the LW2b opsin were lost in lower flies. In dragonflies, specialisation of flight capabilities likely drove the diversification of the LW2b visual opsins. Conclusion The presence of the LW2b opsin in insects possessing three ocelli suggests a role in specific flight capabilities (e.g., stationary flight). This study provides the most complete view of the evolution of visual opsin genes in insects yet, and provides new insight into the influence of ocelli and life history traits on opsin evolution in insects.

Genetic variability and population structure of Leucinodes orbonalis (Guenée), a severe insect pest of brinjal in India

Aim: The present study was undertaken to provide valuable insights regarding population genetic structure of Leucinodes orbonalis from diverse agro-ecologies of India. Methodology: Molecular characterization of L. orbonalis populations collected from five major agro-climatic zones of India was carried out using mitochondrial cytochrome oxidase I (COI) gene. Collected specimens were subjected to DNA extractions, partial PCR amplification and sequencing of the target gene, and multiple sequence alignments. Results: The results showed very less diversity in the nucleotide positions of the COI sequences of 79 studied specimens, with a low number of segregating sites (30), nucleotide diversity (0.00438) and overall mean genetic distance (0.004 ± 0.001). The significant negative values of neutrality tests and unimodal mismatch distribution supported the demographic expansion theory in Indian L. orbonalis. Analysis of the molecular variance revealed that 93.13% of the genetic variation was within populations, and the variation among populations was only 6.87%. The pairwise genetic differentiation was also found to be low to moderate between most of the populations. Multiple haplotypes were recorded from all the populations, and both neighbour-joining tree as well as the haplotype network showed that clustering of the haplotypes was independent of the geographical location. Interpretation: Thus, it can be inferred that Indian populations of L. orbonalis have very low genetic variation levels concerning the COI gene. There is a possible occurrence of stable inherited gene flow among populations, thereby reducing genetic variation in India.