Species-specific Identification Research Articles

Bioinformatic Analysis Strategy in Restriction Enzyme Selection for Indonesian Panulirus homarus Identification by PCR-RFLP

The spiny lobster (Panulirus homarus) is a valuable fishery commodity in Indonesia, particularly in its juvenile life form. However, identifying the early life forms of the spiny lobster can be challenging, as it exhibits similar morphological features compared to the juveniles of other Panilurid lobsters. Molecular-based identification, specifically DNA sequencing, is the best method for species identification, but it requires advanced instruments and is costly. An alternative method is proposed here, using the PCR-RFLP technique, which is low-cost, rapid, and has standard instrumentation requirements. The challenge with this method is selecting the appropriate restriction enzyme to determine the targeted species’ identity. This study proposes using the REfind (https://github.com/indriatmoko07/REfind), R package to select the best restriction enzyme for identifying P. homarus, applicable to other species. The bioinformatics workflow used in this study successfully identified BseSI or BmgI as the most suitable restriction enzymes among 739 restriction enzymes to differentiate P. homarus from other Panilurid species. This result was validated by employing a wet lab test using the BseSI enzyme and successfully validated the bioinformatics result. These findings may be useful for biologists in conducting various studies that require rapid, low-cost, and identification of specific species in the future.

Molecular Prevalence of MecA and MecC Genеs in Coagulasе-Positive Staphylococci Isolated From Dogs with Dermatitis and Otitis in Belgrade, Serbia: A One Year Study

Abstract The escalating global concern of antimicrobial resistance in human and veterinary medicine is exacerbated by the inappropriate prescription of antibiotics for bacterial infections in companion animals. This study aimed to determine the distribution of coagulase-positive staphylococci causing clinical skin and ear infections in dogs and to determine methicillin-resistant isolates. A total of 78 staphylococcal strains were isolated from clinical samples taken from patients at the Dermatology Clinic at the Faculty of Veterinary Medicine in Belgrade, Serbia. Multiplex PCR was used for species-specific identification, and mecA and mecC genes were used to determine methicillin resistance, in addition to phenotypic determination, MIC values and detection of PBP2a. Out of the 78 samples analyzed, 65.8% were identified as Staphylococcus pseudintermedius, 22.4% as S. aureus, 7.9% as S. coagulans, and 3.9% as S. intermedius. Four S. aureus isolates exhibited methicillin resistance confirmed by cefoxitin disk diffusion, while five were confirmed with MIC testing and latex agglutination. MecA gene was detected in 29.4% of S. aureus and 30% of S. pseudintermedius isolates. These isolates were classified as methicillin-resistant S. aureus (MRSA) and methicillin-resistant S. pseudintermedius (MRSP), respectively. No isolates carried the mecC gene. This study provides insights into the prevalence of CoPS species and methicillin resistance in isolates from dogs. Continued surveillance is essential to monitor and understand the emergence and dissemination of antimicrobial resistance in veterinary medicine and the results of this study accent the need for establishment of a continuous antimicrobial resistance surveillance program in the Republic of Serbia.

Discovery and identification of interspecies peptide biomarkers in the seahorse species using liquid chromatography tandem mass spectrometry and chemometrics

Seahorses have important edible and medicinal values including strengthening the body, tonifying the liver and kidneys, and reducing swelling. And there are abundant seahorse species on Earth. Many seahorses have large price differences due to the scarcity of resources, and some seahorses with similar appearances appear to be confused for use. While in market trading, Hippocampus is susceptible to loss of specialized morphology characteristics, making it difficult to distinguish between specific species. Here we report an effective method based on peptide biomarkers for the identification of seahorse species. Peptide biomarkers for each species were predicted using nano-liquid chromatography-tandem mass spectrometry (Nano-LC-MS/MS) combined with chemometrics software. One unique biomarker peptide for each species was synthesized and verified, and finally developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) multiple reaction monitoring method. The results indicate that the method has great potential for species-specific identification of seahorses and their preparations, among others.

Phenotypic and Genotypic Methods for the Identification and Quantification of Cyanobacteria in Lake Water.

Early monitoring of Microcystis, a cyanobacterium that produces microcystin, is paramount in order to confirm the presence of Microcystis spp. Both phenotypic and genotypic methods have been used. The phenotypic methods provide the presence of the microcystis but do not confirm its species type and toxin produced. Additionally, phenotypic methods cannot differentiate toxigenic from non-toxigenic Microcystis. Therefore, the current protocol also describes genetic methods based on PCR to detect toxigenic Microcystis spp. based on microcystin synthetase E (mcy E) gene and 16-23S RNA genes for species-specific identification, which can effectively comprehend distinct lineages and discrimination of potential complexity of microcystin populations. The presence of these microcystin toxins in blood, in most cases, indicates contamination of drinking water by cyanobacteria. The methods presented herein are used to identify microcystin toxins in drinking water and blood.

Detection of volatile organic compounds in headspace of Klebsiella pneumoniae and Klebsiella oxytoca colonies

IntroductionEarly diagnosis of infections and sepsis is essential as adequate therapy improves the outcome. Unfortunately, current diagnostics are invasive and time-consuming, making diagnosis difficult, especially in neonatology. Novel non-invasive analytical methods might be suitable to detect an infection at an early stage and might even allow identification of the pathogen. Our aim is to identify specific profiles of volatile organic compounds (VOCs) of bacterial species.MethodsUsing multicapillary column-coupled ion mobility spectrometry (MCC/IMS), we performed headspace measurements of bacterial cultures from skin and anal swabs of premature infants obtained during weekly screening for bacterial colonization according to KRINKO. We analyzed 25 Klebsiella pneumoniae (KP) cultures on MacConkey (MC) agar plates, 25 Klebsiella oxytoca (KO) cultures on MC agar and 25 bare MC agar plates as a control group.ResultsUsing MCC/IMS, we identified a total of 159 VOC peaks. 85 peaks allowed discriminating KP and bare MC agar plates, and 51 peaks comparing KO and bare MC agar plates and 6 peaks between KP and KO (significance level of p < 0.05 after Bonferroni post hoc analysis), respectively. Peaks P51 (n-Decane) and P158 (Phenylethyl Alcohol), showed the best sensitivity/specificity/ positive predictive value/negative predictive value of 99.9% each (p < 0.001) for KP. P158 showed the best sensitivity/specificity/positive predictive value/negative predictive value of 99.9% each (p < 0.001) for KO. Comparing KP and KO, best differentiation was enabled using peaks P72, P97 and P16 with sensitivity/specificity/positive predictive value/negative predictive value of 76.0%, 84.0%, 82.6%, 77.8%, respectively (p < 0.05).DiscussionWe developed a method for the analysis of VOC profiles of bacteria. Using MCC/IMS, we demonstrated that VOCs derived from bacteria are clearly distinguishable from a bare agar plate. Characteristic peaks obtained by MCC/IMS are particularly suitable for the species-specific identification and differentiation of KP and KO. Thus, MCC/IMS might be a useful tool for in vitro diagnostics. Future studies must clarify whether similar patterns of VOCs can be detected in vivo in patients that are colonized or infected with KP or KO to enable rapid and accurate diagnosis of bacterial colonization.

Phylogenetic Analyses of Lizards from the Chilean Humboldt Archipelago Reveal a New Species for the Chañaral Island (Squamata: Liolaemidae).

The Humboldt Archipelago, situated on Chile's north-central coast, is renowned for its exceptional biodiversity. However, lizards of the Liolaemus genus are a particularly understudied group in this archipelago. Liolaemus genus is divided into two clades: chiliensis and nigromaculatus. Within the nigromaculatus clade the zapallarensis group is restricted to the semi-arid and arid coastal habitats of the Atacama Desert in north-central Chile. While it has been reported that lizards from the zapallarensis group inhabit various islands within the Humboldt Archipelago, there has been limited knowledge regarding their specific species identification. To identify the lizard species inhabiting these islands, we conducted phylogenetic analyses using a mitochondrial gene and examined morphological characteristics. Our findings reveal that lizards from the Damas, Choros, and Gaviota islands belong to Liolaemus silvai. In contrast, the lizards on Chañaral Island form a distinct and previously unrecognised group, clearly distinguishable from Liolaemus silvai. In conclusion, our study not only confirms the presence of L. silvai on the Damas, Choros, and Gaviota islands but also describes a new lizard species on Chañaral Island named Liolaemus carezzae sp. nov. These findings contribute valuable insights into the biodiversity of these islands and introduce a newly discovered endemic taxon to the region, enriching our understanding of Chile's unique island ecosystems.

Molecular identification of the brown marmorated stink bug’s egg parasitoids by species-specific PCR collected from Beijing, China

Brown marmorated stink bug (BMSB), Halyomorpha halys (Stål) (Hemiptera: Penta-tomidae), is a highly polyphagous and invasive insect pest with more than 300 plant species as hosts, including a wide range of economic crops. To date, several egg parasitoid species are reported to attack BMSB. In this study, two species of Trissolcus (Hymenoptera: Platygastridae) and one Anastatus (Hymenoptera: Eupelmidae) were recovered from field exposed BMSB sentinel eggs in Beijing, China. The wasps’ small size of only 1.5 mm and 4 mm in length make them difficult to identify morphologically and required taxonomist for identification of specific species. In addition, these parasitoids have morphology sibling species and have been misidentified several times in China. To overcome these problems, a molecular method with species-specific primers designed for the COI gene has been developed to identify Trissolcus, Anastatus and their host samples from field collected samples. After successful morphological confirmation with experts, DNA extractions were carried out from these samples. PCR amplification using published primers for T. japonicus, A. japonicus and BMSB specimens confirmed the species. As for T. cultratus, species specific primers TCYF and TCYR were developed which produce 340-bp PCR products length while no positive amplifications found in other wasps and host. Sensitivity analysis of markers revealed that TCYF and TCYR primers could detect as low a DNA template concentration as 0.00025 ng/μL. This indicates that PCR with these primers specifically and sensitively differentiates T. cultratus specimens from other similar wasp species. All the primers tested in this study could discriminate between parasitized and non-parasitized BMSB eggs. This molecular identification method shows promise for conveniently identifying Trissolcus and Anastatus species in host-parasitoid associations and accurately evaluating parasitism rates in the field.

Metagenomic next-generation sequencing assistance in identifying non-tuberculous mycobacterial infections.

The advent of metagenomics next-generation sequencing (mNGS) has garnered attention as a novel method for detecting pathogenic infections, including Non-Tuberculous Mycobacterial (NTM) and tuberculosis (TB).However, the robustness and specificity of mNGS in NTM diagnostics have not been fully explored. In this retrospective study, we enrolled 27 patients with NTM genomic sequences via mNGS and conducted a comprehensive clinical evaluation. Pulmonary NTM disease was the most commonly observed presentation, with a subset of patients also presenting with extrapulmonary NTM infections.mNGS analysis identified six distinct NTM species, primarily Mycobacteriumavium complex (MAC), followed by Mycobacterium intracellulare andMycobacterium abscessus. Conventional routine culture methods encountered challenges, resulting in negative results for all available 22 samples. Among the 10 patients who underwent quantitative polymerase chain reaction (qPCR) testing, five tested positive for NTM. It is important to note that further species typing is necessary to determine the specific NTM type, as traditional pathogen detection methods serve as an initial step. In contrast, when supplemented with pathogen data, enables the identification of specific species, facilitating precise treatment decisions. In conclusion, mNGS demonstrates significant potential in aidingthe diagnosis of NTMdisease by rapidly detecting NTM pathogens and guiding treatment strategies. Its enhanced performance, faster turnaround time (TAT), and species identification capabilities make mNGS a promising tool for managing NTM infections.

Taxonomic significance of pollen morphology of selected taxa of Bassia, Sedobassia, Spirobassia and Neokochia (Chenopodiaceae/Amaranthaceae sensu APG IV)

ABSTRACT Based on molecular phylogenetic and morphological studies, new genera Sedobassia Freitag & G. Kadereit and Spirobassia Freitag & G. Kadereit were described in the tribe Camphorosmeae (Chenopodiaceae/Amaranthaceae sensu APG IV). Palynomorphological peculiarities are often used in taxonomy as additional diagnostic features. Pollen grains from 26 herbarium specimens belonging to 13 taxa of Bassia, Sedobassia, Spirobassia and Neokochia genera in Eurasia and North America were acetolyzed and studied using both light microscopy and scanning electron microscopy. Dry pollen grains were also studied. The structure of columellae of pollen grains was analyzed for the first time in the current study. The aim of this research was to provide data on their pollen characteristics and to evaluate the taxonomic value of these data for generic and species-specific identification. Pollen grains are pantoporate, spheroidal in shape, circular in outline, slightly undulate, undulate or straight on the edges; medium or small-sized. Pores are circular, with distinct or indistinct margins. Exine sculpture is nanoechinate; tectum psilate or psilate-perforate. Pore membranes with nanoechini. Taxonomic relevance of the most important characters of pollen grains is discussed (pollen diameter, pore number, pore diameter, distance between pores and between pore centers, C/D value, nanoechini density, presenсe/absence of perforations, structure of columellae, number of nanoechini on pore membranes). Pollen morphology proved to be an important additional source of information for species-specific identification. This study provides new data and analyzes the morphological features of pollen grains of Bassia (in the currently accepted circumscription), palynomorphological similarities, and differences between the taxa of Bassia, Sedobassia, Spirobassia, and Neokochia genera.

Species-Specific Analysis of Bacterial Vaginosis-Associated Bacteria.

Vaginal dysbiosis in women reduces the abundance of Lactobacillus species and increases that of anaerobic fastidious bacteria. This dysbiotic condition in the vagina, called bacterial vaginosis (BV), can be symptomatic with odorous vaginal discharges or asymptomatic and affects a third of women of reproductive age. Three unclassified bacterial species designated BV-associated bacteria 1, 2, and 3 (BVAB-1, -2, and -3) in 2005 were found to be highly preponderant in the vagina of females with BV. Here, we used sequence homology and phylogenetics analyses to identify the actual species of BVAB-1, -2, and -3 and found BVAB-1 to be Clostridiales genomosp. BVAB-1, BVAB-2 to be Oscillospiraceae bacterium strain CHIC02, and BVAB-3 to be Mageeibacillus indolicus, respectively. These are anaerobic and uncultured species that can be identified only through metagenomics. Long-read sequencing of BV specimens can also enable a genomic reassembly of these species' genomes from metagenomes. Species-specific identification of these pathogens and the availability of their genomes from assembled metagenomes will advance our understanding of their biology, facilitate the design of sensitive diagnostics and drugs, and enhance the treatment of BV. IMPORTANCE For many years since 2005, BVAB, an important pathogen of the female vaginal tract that is associated with BV, has been identified using PCR without knowing its actual species. Without a full genome of these pathogens, a better understanding of their pathogenicity, treatment, resistance, and diagnostics cannot be reached. In this analysis, we use the DNA of BVAB-1, -2, and -3 to determine their actual species to enhance further research into their pathogenicity, resistance, diagnosis, and treatment.

Preliminary results of the recombinase polymerase amplification technique for the detection of Haemonchus contortus from Hungarian field samples

Haemonchus contortus is a parasitic nematode of small ruminants responsible for significant economic losses and animal health concerns globally. Detection of gastrointestinal nematode (GIN) infection in veterinary practice typically relies on microscopy-based methods such as the faecal egg count and morphological identification of larval culture. However, mixed co-infections are common and species-specific identification is typically time-consuming and expertise-intensive. Compounded by increasing anthelmintic resistance, there is an urgent need to implement the molecular diagnosis of GIN in the livestock industry, preferably in field settings. Advances in isothermal amplification techniques including recombinase polymerase amplification (RPA) assays could improve this. Yet, constraints in RPA kit availability and amplicon detection systems limit the use of this technology in point of care settings. In this study, we present an early-stage, proof-of-concept demonstration of RPA targeting the internal transcribed spacer (ITS2) region of H. contortus. Having tested against eight closely related nematodes and also against five farm isolates in Eastern Hungary, preliminary results derived from a comparative analysis of 3 primer sets showed the assay detects H. contortus DNA and has a limit of detection of 10−5 ng/μl. We also tested an end-result naked eye detection system using various DNA binding dyes, of which EvaGreen® dye was successful for a qualitative RPA detection that could be adaptable at farm sites.

Polymerase chain reaction in the diagnosis of invasive aspergillosis: approaches for appropriate use

Detection of aspergillus by PCR is a helpful tool for early diagnosis. The test has excellent sensitivity and specificity with a high negative predictive value. Well-accepted, standardized method for DNA extraction for PCR testing is to be adopted for all commercial assays and conclusive validation data are awaited in varied clinical settings. This perspective offers guidance for utilizing PCR testing while awaiting such data. Quantification by PCR, species-specific identification assays and detection of resistance genetic markers are of future promise. Herein, we summarize the available data on aspergillus PCR and describe its potential utility through a clinical case scenario-based approach.

Grass flowering times determined using herbarium specimens for modeling grass pollen under a warming climate

The grass family is responsible for most of peoples pollen allergies, and the severity of pollen-based asthma and allergies is expected to increase with global climate change. Identifying grass species through standard pollen monitoring techniques have limitations due to challenges in species-specific pollen identification. As a result, these monitoring methods end up grouping all Poaceae species together, even though there are hundreds of grass species in Europe with flowering times that may vary drastically among species. Given this lack of specificity, it is hard to know which grass species are responsible for causing allergies over the pollen season, and how different species are affected by climate change. To address these issues, we obtained phenological data from thousands of herbarium specimens collected across Denmark spanning 190 years and used pollen monitoring data collected over the last four decades to determine the response of flowering time to climate change for 12 allergenic grass species, and identify which species are likely the biggest contributors to grass pollen loads throughout the pollen season. We find that pollen season duration is lasting longer and starting earlier, and the maximum pollen loads are occurring earlier in response to climate warming. Herbarium specimens provide taxonomic resolution and reveal that many grass species are flowering earlier in response to warmer spring temperatures. Seven out of the 12 species studied in Denmark are identified as major contributors to airborne pollen based on their flowering times, relative abundance and overlap with the time of the year when maximum pollen loads are detected. Four species (Poa pratensis, Dactylis glomerata, Festuca rubra, Holcus lanatus) significantly shifted their flowering time in response to warming temperatures and are flagged as of particular concern to allergy sufferers. Using data derived from natural history collections can contribute to the advancement of pollen forecasting for asthma and allergy patients under both current conditions and amidst future global changes.

Misidentification of Plasmodium Species by Cross-Reacting Primers and Cerebral Malaria Caused by Plasmodium vivax

Abstract Introduction The clinical presentation of a case as cerebral malaria with molecular identification confirming it as Plasmodium vivax underlines the importance of using molecular tools to identify the species and type of malaria. The possibility of the relationship between the complication observed during clinical diagnosis and the multifactorial molecular changes could likely be the reason for terming it cerebral malaria. Methods We report four cases analyzed using the quantitative buffy coat technique followed by classical Giemsa stained thick-film microscopy, and nested polymerase chain reaction for the genus-specific region of Plasmodium targeting 18S rDNA followed by species-specific identification with a different set of primers and products confirmation with sequencing. Results Primers targeting P. knowlesi generated the expected product size of 153 base pairs that, upon sequencing, matched with the P. vivax sequence reflecting the relatedness of the species. Likewise, primers targeting P. ovale generated a 456 product whose sequence matched the P. vivax sequence. Conclusion Infection with P. vivax can potentially cause cerebral malaria, and P. vivax can cause severe malaria complications alone or mixed with other species and can show cerebral malaria signs, which are typically associated with P. falciparum infections. The sequence relatedness reflects the genome similarity between P. knowlesi and P. ovale with P. vivax. The need to reconfirm with an additional set of newly reported primers is mandatory.

Diagnosis, Clinical and Molecular Delineation of Human Plasmodium Species from Mangaluru, Southwest India

Malaria is a global threat and a never-ending battle without appropriate identification and differentiation of the parasite species. This work compared the diagnostic methods including the thick film microscopy technique, quantitative buffy coat, and polymerase chain reaction. The inaccuracy of species determination by microscopy and the consequent treatment regime underlines the necessity to upgrade routine diagnostic methods with molecular techniques. In the study, 436 samples were collected; venous blood was processed for the quantitative buffy coat technique followed by classical Giemsa staining of thin and thick smears and nested Polymerase Chain Reaction (nPCR) for the genus-specific region of Plasmodium targeting 18S rDNA followed by species-specific identification. Of 436 samples screened for malaria, results in PCR showed 78.7% (100/127) to be P. vivax, 4.8% (6/127) as P. falciparum and 16.5% (21/127) to be mixed infection (P. vivax + P. falciparum). The prevalence of malaria was 0.29, and there was good concordance between the methods for detecting Plasmodium (Kappa:0.77). In our investigation, nested PCR and TFM exhibited a sensitivity of 97.7% and a specificity of 100% for malaria detection compared to QBC. Clinical parameters- thrombocytopenia and anemia, were compared in this study. A positive association was observed between thrombocytopenia and malaria (p<0.05), but the association between anemia and malaria infection remains unclear. Primer cross-reactions were also observed in the primer sequence of P. ovale and P. knowlesi, but sequencing confirmed it as P. vivax and the study of phylogeny paved a new way in analyzing the relatedness of the sequences.

New data on pollen morphology of the genus Camphorosma (Chenopodiaceae)

Recent molecular phylogenetic studies of Camphorosmoideae (Chenopodiaceae) demonstrated that Camphorosma is monophyletic. Palynomorphological peculiarities are used in taxonomy as additional diagnostic features and pollen analysis. Pollen morphology was studied using light and scanning electron microscopy, based on 13 specimens belonging to four species (C. annua, C. lessingii, C. monspeliaca and C. songorica). The aim of this research was to provide new data on pollen characteristics of the above mentioned species and to evaluate their taxonomic value. The results obtained show that pollen grains are pantoporate, spheroidal, circular in outline, undulate or slightly undulate on the edges; small or medium-sized. Pores are circular, with distinct margins. Exine sculpture is nanoechinate, tectum psilate or psilate-perforate. Pore membranes are nanoechinate. Taxonomic relevance of the most important characters of pollen grains (pollen diameter, pore number, pore diameter, distance between pores and between pore centres, C/D value, nanoechini density, presence/absence of perforations, number of nanoechini on pore membranes, structure of columellae) is discussed. The exine structure of pollen grains of all species was analysed for the first time in the current study. Pollen morphology proved to be an important additional source of information for taxonomy for species-specific identification and pollen analysis within Camphorosma. Palynomorphological data are interpreted in the existing phylogenetic framework.

Pollen morphology of selected taxa of Valeriana (Caprifoliaceae): taxonomic and evolutionary implications

Pollen morphology of 34 herbarium specimens belonging to 17 taxa of the genus Valeriana was studied using light and scanning electron microscopy. The aim of this research was to evaluate the taxonomical value of palynomorphological data for species-specific identification. Pollen grains are monads, radially symmetrical, isopolar, tricolpate, oblate to prolate (P/E = 0.69–1.59); medium- or large-sized (P = 26.60–66.50 µm; E = 23.94–57.19 µm). Colpi are long, medium-length or short, with acute, obtuse or rounded ends. Four types and eight subtypes of exine sculpture were recognised: type I—echinate-verrucate (5 spp.), type II—echinate-nanoechinate-verrucate (10), type III—echinate-microechinate-nanoechinate-verrucate (1), type IV—echinate-microechinate-nanoechinate (1). Additional characters of taxa of Valeriana diagnostic at species level for the purposes of taxonomy are: size of pollen and colpi, exine sculpture: presence/absence of verrucae, their form, size and form of echini/microechini/nanoechini, their number, pattern of tectum in areas between echini, exine thickness in mesocolpia and apocolpia. Our palynomorphological data support the results of molecular phylogenetic studies. The close phylogenetic relationships of the species V. montana, V. tripteris, V. supina and V. pyrenaica are also supported by their palynomorphological characters, such as exine sculpture (type I). Cluster analysis mainly supports the definition of pollen types and subtypes (based on qualitative data), since species are grouped as five separate branches. The pollen grains of five taxa were analysed for the first time in the current study.

Targeting a highly repetitive genomic sequence for sensitive and specific molecular detection of the filarial parasite Mansonella perstans from human blood and mosquitoes.

Mansonella perstans is among the most neglected of the neglected tropical diseases and is believed to cause more human infections than any other filarial pathogen in Africa. Based largely upon assumptions of limited infection-associated morbidity, this pathogen remains understudied, and many basic questions pertaining to its pathogenicity, distribution, prevalence, and vector-host relationships remain unanswered. However, in recent years, mounting evidence of the potential for increased Mansonella infection-associated disease has sparked a renewal in research interest. This, in turn, has produced a need for improved diagnostics, capable of providing more accurate pictures of infection prevalence, pathogen distribution, and vector-host interactions. Utilizing a previously described pipeline for the discovery of optimal molecular diagnostic targets, we identified a repetitive DNA sequence, and developed a corresponding assay, which allows for the sensitive and species-specific identification of M. perstans in human blood samples. Testing also demonstrated the ability to utilize this assay for the detection of M. perstans in field-collected mosquito samples. When testing both sample types, our repeat-targeting index assay outperformed a ribosomal sequence-targeting reference assay, facilitating the identification of additional M. perstans-positive samples falsely characterized as "negative" using the less sensitive detection method. Through the development of an assay based upon the systematic identification of an optimal DNA target sequence, our novel diagnostic assay will provide programmatic efforts with a sensitive and specific testing platform that is capable of accurately mapping M. perstans infection and determining prevalence. Furthermore, with the added ability to identify the presence of M. perstans in mosquito samples, this assay will help to define our knowledge of the relationships that exist between this pathogen and the various geographically relevant mosquito species, which have been surmised to represent potential secondary vectors under certain conditions. Detection of M. perstans in mosquitoes will also demonstrate proof-of-concept for the mosquito-based monitoring of filarial pathogens not vectored primarily by mosquitoes, an approach expanding opportunities for integrated surveillance.

Insecticide resistance intensity and efficacy of synergists with pyrethroids in Anopheles gambiae (Diptera: Culicidae) from Southern Togo

BackgroundThis study was designed to provide insecticide resistance data for decision-making in terms of resistance management plans in Togo.MethodsThe susceptibility status of Anopheles gambiae sensu lato (s.l.) to insecticides used in public health was assessed using the WHO tube test protocol. Pyrethroid resistance intensity bioassays were performed following the CDC bottle test protocol. The activity of detoxification enzymes was tested using the synergists piperonyl butoxide, S.S.S-tributlyphosphorotrithioate and ethacrinic acid. Species-specific identification of An. gambiae s.l. and kdr mutation genotyping were performed using PCR techniques.ResultsLocal populations of An. gambiae s.l. showed full susceptibility to pirimiphos methyl at Lomé, Kovié, Anié, and Kpèlè Toutou. At Baguida, mortality was 90%, indicating possible resistance to pirimiphos methyl. Resistance was recorded to DDT, bendiocarb, and propoxur at all sites. A high intensity of pyrethroid resistance was recorded and the detoxification enzymes contributing to resistance were oxidases, esterases, and glutathione-s-transferases based on the synergist tests. Anopheles gambiae sensu stricto (s.s.) and Anopheles coluzzii were the main species identified. High kdr L1014F and low kdr L1014S allele frequencies were detected at all localities.ConclusionThis study suggests the need to reinforce current insecticide-based malaria control interventions (IRS and LLINs) with complementary tools.

16S-ITS-23S rRNA operon segment sequencing provides necessary and sufficient conditions for bacterial species-specific identification

Introduction. Sequencing of the 16S rRNA gene is the predominant method for assessing microbial communities and strain molecular identification. The short reads (2nd generation sequencing)-based technology does not allow analysis beyond the 16S rRNA gene. The taxonomic verification level of samples usually remains at the genus or even family level. Currently, there have been proposed the latest versions of long-read technologies (Oxford Nanopore MinION, PacBio) for amplicon sequencing of near-complete ribosomal operon, including genes 16S, 23S, 5S, and internal transcribed spacer (ITS). At the moment, this approach has not been sufficiently studied, in addition, it involves PCR amplification of a very extended DNA region (more than 4000 bp-long). Materials and methods. The collection of non-tuberculous mycobacteria strains and their primary identification was carried out in the years 20192021. The strains were obtained by inoculation of positive cultures from the Bactec MGIT 960 bacteriological analyzer lacking MPT64 antigen in the MGIT TB Identification Test (Becton Dickinson, USA) on Lowenstein-Jensen medium. Preliminary species strain identification was carried out with the Speed-oligo Mycobacteria kit (Vircell, Spain) according to the manufacturers protocol. In this work, both known and newly developed universal bacterial primers flanking the near-complete 16S rRNA gene, ITS, and the beginning of the 23S rRNA gene are used. In the present study, both known and newly developed universal bacterial primers are used to flank the near-complete 16S rRNA gene, ITS, and start of the 23S rRNA gene. Results and discussion. Sanger sequencing of the amplicons (about 2000 bp) obtained shows the taxonomic level sufficient to determine species up to 8 strains of non-tuberculous mycobacteria isolated from humans that caused clinically and bacteriologically confirmed diseases. The method proposed for PCR amplification of a bacterial operon a fragment containing most of the 16S rRNA gene, ITS, and the beginning of the 23S rRNA gene is considered by us as an approbation of a methodological approach to study microbial communities in material with a high degree of degradation (necrotic foci, etc.). The results obtained indicate a significantly higher resolution of the approach used than the classical 16S rRNA gene sequencing.