Molecular Genotyping Research Articles

Molecular Detection and Genotyping of Cryptosporidium spp. Isolates from Bats in Colombia.

Cryptosporidiosis is a zoonotic infectious disease caused by the protozoan parasite Cryptosporidium spp., frequently found in several animal species, including bats. Several Cryptosporidium genotypes have been described in bats worldwide, suggesting that bats are infected by host-specific Cryptosporidium spp. To date, there are no published reports about Cryptosporidium spp. in bats from Colombia. Therefore, this study aimed to determine the presence and molecular diversity of Cryptosporidium spp. in Colombian bats. A total of 63 gut samples from three bat species served for molecular detection of Cryptosporidium spp. 18S rDNA gene by qPCR. The sequenced amplicons were used in subsequent phylogenetic analyses to identify them as species or genotypes. Cryptosporidium spp. qPCR detection occurred in 9.5% (6/63) of bat intestines, and four sequences represented two new genotypes, called Cryptosporidium bat genotypes XIX and XX, were identified. This study describes the detection of two novel Cryptosporidium bat genotypes, in two species of bats from a region of Colombia, requiring further studies to determine the relationhip between Cryptosporidium and bats in Colombia.

Chlamydia trachomatis Serovar Distribution in Patients with Follicular Conjunctivitis in Iran.

Chlamydia trachomatis infects the urogenital tract and eyes. Anatomical tropism is correlated with serovars which are characterized according to the variation in the major outer membrane proteins encoded by the ompA gene. The aim of the present study was to determine the distribution of C. trachomatis serovars among patients with follicular conjunctivitis in Iran. A total of 68 conjunctival specimens from symptomatic adults were studied for the presence of C. trachomatis using polymerase chain reaction (PCR) analysis. Serovars were determined by Omp1 PCR-RFLP analysis. C. trachomatis was detected in 38 (55.9%) of patients with follicular conjunctivitis, with higher C. trachomatis prevalence in the younger age groups. Twenty-six (38.2%) of these patients had a history of urinary tract infection. Four distinct serovars were identified in the conjunctiva samples using molecular genotyping. The most prevalent was serovar E, followed by G, I, and F. Our serovar distribution indicated that chlamydial follicular conjunctivitis usually has a genital source. Genital serovars may cause eye diseases, especially in sexually active adults. On the other hand, conjunctivitis might be the only sign of sexually transmitted infection. Therefore, genotyping C. trachomatis in ocular and genital specimens could be beneficial for acquiring more detailed epidemiological information about the etiology of the disease and monitoring treatment success.

Obtaining and evaluating breeding material for the creation of F1 hybrids of Chinese cabbage (<i>B. rapa</i> ssp. <i>pekinensis</i>) with resistance to stress factors

Relevance and purpose of the study. Chinese cabbage is a vegetable crop with a high content of vitamins and low calorie content, valued for its ability to receive several crops per year and high productivity. Recently, there has been a reduction in the supply of the vegetable to Russia. There are less than 70 varieties and hybrids of the crop in the State Register of Selection Achievements, many of them are susceptible to the most important diseases. It is necessary to meet consumer demand with new commercial hybrids with resistance to stressors. The aim of the study was to evaluate and obtain genotypes with a set of valuable economic traits, including resistance to clubroot, powdery mildew, and early bolting.Materials and methods. The Chinese cabbage lines of various degrees of inbred and DH, as well as hybrid combinations from crossing these lines, were used as plant material. The lines were obtained in 2019-2020, the breeding material for them was selected on the ground infected with clubroot and with tolerance to tip burn of heads. To achieve the goal, the following methods were applied: plants cultivation; DNA isolation (CTAB method); PCR analysis; electrophoresis and visualization of results; productivity assessment and dispersion analysis; obtaining doubled haploids in culture of isolated microspores in vitro, assessment of resistance/susceptibility to powdery mildew.Results. 2 genotypes with high productivity and alignment in turnover summer-autumn 2022 and a genotype exceeding 3 standards in head weight in turnover spring-summer 2022 were recommended; as a result of molecular genotyping, 41 lines were differentiated according to the clubroot resistance gene CRb; DH plants for 4 genotypes with resistance to early bolting were obtained, their responsiveness to embryogenesis was studied; 18 lines were evaluated for resistance to powdery mildew, 7 genotypes with resistance to the disease were identified.

Genetic Diversity of Toxoplasma gondii by Serological and Molecular Analyzes in Different Sheep and Goat Tissues in Northeastern Iran.

Toxoplasmosis is a parasitic disease caused by compilation protozoan agent Toxoplasma gondii, leading to significant financial and quality-adjusted life-year losses. Overcooked or raw meat consumption has been a considerable transmission route. The present study was conducted to determine the seropositivity rate of T. gondii in sheep and goats by serological and molecular tests and genotyping of obtained isolates in northeast Iran. Blood and tissue samples (diaphragm, heart) of 296 animals (including 168 sheep and 128 goats) were collected from the slaughterhouse in Quchan Country from august 2016 to April 2017. Modified agglutination test (MAT) and the PCR method performed to detect parasite DNA on tissues.PCR-RFLP method of GRA6 gene was used to determine the genotype of T. gondii. In addition, sequencing analysis was performed to evaluate the Toxoplasma type strains. Serum positive for MAT results were found in 27.4% of sheep and 23.4% of goats. Positive PCR of B1 gene results in diaphragm and heart tissues of sheep and goats was 47.8% and 26.1%, 40% and 23.3%, respectively. PCR of GRA6 gene results were positive in 10 samples that RFLP technique results using MseI enzyme revealed genotype I. Sequencing and phylogenetic analysis revealed DNA of all samples was closely related to Toxoplasma type I. Concerning the high seropositivity rate of toxoplasmosis, undertaking an appropriate preventive program for reducing the prevalence of T. gondii infection by raw or undercooked meat consumption of livestock is recommended. Our study supports the notion that these animals' consumption of raw and undercooked meat can be a probable source of human toxoplasmosis.

Virulence and genetic analysis of Puccinia graminis tritici in the Indian sub-continent from 2016 to 2022 and evaluation of wheat varieties for stem rust resistance.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), has re-emerged as one of the major concerns for global wheat production since the evolution of Ug99 and other virulent pathotypes of Pgt from East Africa, Europe, Central Asia, and other regions. Host resistance is the most effective, economic, and eco-friendly approach for managing stem rust. Understanding the virulence nature, genetic diversity, origin, distribution, and evolutionary pattern of Pgt pathotypes over time and space is a prerequisite for effectively managing newly emerging Pgt isolates through host resistance. In the present study, we monitored the occurrence of stem rust of wheat in India and neighboring countries from 2016 to 2022, collected 620 single-pustule isolates of Pgt from six states of India and Nepal, analyzed them on Indian stem rust differentials, and determined their virulence phenotypes and molecular genotypes. The Ug99 type of pathotypes did not occur in India. Pathotypes 11 and 40A were most predominant during these years. Virulence phenotyping of these isolates identified 14 Pgt pathotypes, which were genotyped using 37 Puccinia spp.-specific polymorphic microsatellites, followed by additional phylogenetic analyses using DARwin. These analyses identified three major molecular groups, demonstrating fewer lineages, clonality, and long-distance migration of Pgt isolates in India. Fourteen of the 40 recently released Indian wheat varieties exhibited complete resistance to all 23 Pgt pathotypes at the seedling stage. Twelve Sr genes were postulated in 39 varieties based on their seedling response to Pgt pathotypes. The values of slow rusting parameters i.e. coefficient of infection, area under disease progress curve, and infection rates, assessed at adult plant stage at five geographically different locations during two crop seasons, indicated the slow rusting behavior of several varieties. Six Sr genes (Sr2, Sr57, Sr58, Sr24, Sr31, and Sr38) were identified in 24 wheat varieties using molecular markers closely linked to these genes. These findings will guide future breeding programs toward more effective management of wheat stem rust.

Cord Blood Hematological Parameters of Fetuses Detected Different Thalassemia Genotypes in the Second Trimester of Pregnancy

Hemoglobinopathies are the most common inherited diseases in humans resulting from impaired globin chain synthesis of hemoglobin. The progression of thalassemia rates is prevented with prenatal screening methods. To evaluate the hematological parameters of α- and β-thalassemia and normal fetuses aged 17-25 weeks of gestation. A cross-sectional study. Pregnant women who underwent cordocentesis in the second trimester because of the risk of having a baby with thalassemia were included in the study. Hematological indices and molecular DNA methods were analyzed from the cord blood samples of 129 women who were 17-25 weeks into pregnancy. The HPLC method was used for Hb fraction analysis. Amplification refractory mutation system, restriction enzyme analysis, multiplex polymerase chain reaction, and sequencing methods were used for the molecular analysis. Maternal contamination was eliminated by the short tandem repeat method. In total, 112 of the fetuses carry α- and β-thalassemia heterozygous or homozygous (α: 37, β: 58, mixed: 17) and 17 fetuses had a normal genotype for thalassemia. Significant differences in adult hemoglobin (HbA), fetal hemoglobin (HbF), Hb Barts, MCV, MCH, and RDW were detected in three groups compared with the normal group (p < 0.001, except for RBC, Hb, HCT, and MCHC). Differences in HbF, Hb Barts, MCV, MCH, and RDW were observed in the α-thalassemia groups compared with the normal group (p < 0.001). Among the five β-thalassemia subgroups, only HbA and RDW were different from the normal group (p < 0.001). This study could be a good reference for future studies and prenatal diagnostic applications in emphasizing the importance of changes in the blood parameters of fetuses before molecular genotyping. These hematological data give valuable information to clinicians about the fetus to enlighten families in making appropriate decisions during prenatal diagnosis.

Association Between Availability of Molecular Genotyping Results and Overall Survival in Patients With Advanced Nonsquamous Non-Small-Cell Lung Cancer.

Current guidelines recommend molecular genotyping for patients newly diagnosed with metastatic nonsquamous (mNSq) non-small-cell lung cancer (NSCLC). The association between availability of molecular genotyping before first line (1L) therapy and overall survival (OS) is not known. We conducted a real-world cohort study using electronic health records in patients newly diagnosed with mNSq NSCLC. Cox proportional-hazards multivariable regression models were constructed to examine the association between OS and test result availability before 1L therapy, adjusting for covariates. Additional analyses were conducted to assess the consistency and strength of the relationship. Multivariable logistic regression models were used to examine the association between concurrent tissue and plasma testing (v tissue alone) and result availability. Three hundred twenty-six patients were included, 80% (261/326) with results available before 1L (available testing group), and 20% (65/326) without results available (unavailable testing group). With 14.2-month median follow-up, patients in the available testing group had significantly longer OS relative to the unavailable testing group (adjusted hazard ratio, 0.43; 95% CI, 0.30 to 0.62; P < .0001). The adjusted odds of availability of results before 1L therapy was higher with concurrent tissue and plasma testing (v tissue testing alone; adjusted odds ratio, 2.06; 95% CI, 1.09 to 3.90; P = .026). Among patients with mNSq NSCLC in a real-world cohort, availability of molecular genotyping results before 1L therapy was associated with significantly better OS. Concurrent tissue and plasma testing was associated with a higher odds of availability of results before 1L therapy. These findings warrant renewed attention to the completion of molecular genotyping before 1L therapy.

Prognostic biomarkers for survival in mucosal melanoma.

Mucosal melanoma (MM) is a rare subtype of melanoma with an aggressive clinical course. In cutaneous melanoma (CM), the absence of pigmentation and presence of NRAS/KRAS mutations are biomarkers indicating an aggressive clinical course with shorter overall survival. Similar data for MM are missing. We present the real-world outcome data in a cohort of genotyped MM patients and assessed the prognostic relevance of pigmentation- and NRAS/KRAS mutation status. We correlated pathological reports and clinical data with overall survival of patients with MM. Furthermore, we performed clinically integrated molecular genotyping and analyzed real world treatment regimens for covariates associated with clinical outcome. We identified 39 patients with available clinical and molecular data. Patients with amelanotic MM had a significantly shorter overall survival (p = .003). In addition, the presence of a NRAS or KRAS mutation was significantly associated with poor overall survival (NRAS or KRAS p = .024). Currently, it is unknown if the same prognostic relevance for the lack of pigmentation and RAS mutations in CM, exists in MM. Here we analyzed a cohort of MM for outcome measures and determined that two known prognostic biomarkers for CM are in fact novel prognosticators for MM.

Genome-Wide Resequencing Reveals Genetic Relationships between Main Gardenia jasminoides Ellis Cultivars

Nineteen samples of Gardenia jasminoides Ellis from China were used for genome resequencing. The clean Q30 base rates ranged from 91.19% to 92.94%. When mapping with the reference genome, a total of 7,568,199 inDel sites and 61,535,595 SNP sites were detected, with variations of heterozygosity ratios ranging from 44.86% to 92.69% and from 41.53% to 90.38%, respectively. Genome location and function annotation of SNP and inDel in different samples were carried out. SNP and inDel were mainly located in the intergenic region, with the ratios of 67.2360% and 62.6415%, respectively. Based on the phylogenetic tree constructed by the maximum likelihood method, the 19 samples could be divided into four categories. Among them, Y_10 and Y_38 were put into one category, while Y_2 was put into a separate category. These two were first differentiated from other cultivars and species. Similar conclusions can be drawn from the results of genetic structure and principal component analysis. This study provided sequence foundation for the identifying of the molecular marker and genotype of G. jasminoides. It also provided theoretical foundation for subsequent resource conservation and utilization, the genetic localization of important agronomical traits, and high-quality variety formulation and breeding.

Implementation of a molecular genotyping protocol for patients with warm autoantibodies.

Warm autoantibodies (WAAs) cause delays and additional expenses while determining suitable products when using a traditional protocol (TP). In 2013, Carter BloodCare Immunohematology Reference Laboratory (IRL) introduced a molecular protocol (MP) for patients with WAAs. Retrospective review of records for samples referred to the IRL from November 2004 to September 2020, was performed. Referrals, alloantibody(ies), gender, and age were recorded. Additionally, the count of common clinically significant antigens needed for phenotypically matched red blood cells (RBCs) were recorded for patients in MP. To further analyze charges and time spent testing patients with WAAs, 300 patients were selected. Analysis of average charges to the referring hospital and time spent testing in the IRL determined savings at two or more referrals. Overall, 219 of 300 (73%) of patients in the study met or exceeded the number of referrals. Further analysis shows that while the population of patients with WAA (n = 300) shared similar demographics, there was a statistically significant difference between the average time testing patients in TP (M = 264.18, SD = 15.06) and MP (M = 156.00, SD = 90.37), t(157) = 14.46, p < .001, 95% confidence interval [CI] (93.41-122.97). Additionally, the assumption that each patient received two RBCs per referral provided no statistically significant difference between average charges to the hospitals of patients in TP (M = 1222.58, SD = 165.69) and MP (M = 1269.78, SD = 433.52), t(192) = -1.25, p = .214, 95% CI (-121.95-27.54). The MP has been effective in saving time spent testing patients with WAAs, which benefits referring hospitals, patients, and IRLs. Charges for prophylactic phenotypically matched blood were negligible and a MP would alleviate some of the current laboratory difficulties while providing safe products to patients.

Significant of Molecular Genotyping Over Serological Phenotyping Techniques in Determination of Blood Group Systems among Multiple Transfused Patients and Blood Donors to Prevent Alloimmunization: A Review Article

Significant of Molecular Genotyping Over Serological Phenotyping Techniques in Determination of Blood Group Systems among Multiple Transfused Patients and Blood Donors to Prevent Alloimmunization: A Review Article

Clonal diversity of Staphylococcus aureus isolates in clinical specimens from selected health facilities in Ethiopia

Staphylococcus aureus is among the top three causative agents of nosocomial infection in Ethiopia. The majority of studies in Ethiopia have focused on the epidemiology of S. aureus in hospital settings, with limited molecular genotyping results. Molecular characterization of S. aureus is essential for identification of strains, and contributes to the control and prevention of S. aureus infection. The aim of the current study was to determine the molecular epidemiology of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates recovered from clinical specimens in Ethiopia. A total of 161 MSSA and 9 MRSA isolates were characterized using pulsed-field gel electrophoresis (PFGE) and staphylococcal protein A (spa) typing. Based on the PFGE analysis, MSSA isolates were grouped into eight pulso-types groups (from A to I), while MRSA isolates clustered into three (A, B and C) pulso-types with more than 80% similarity. The spa typing analysis showed diversity of S. aureus with 56 distinct spa types. Spa type t355 was most prevalent (56/170, 32.9%), while eleven new spa types were detected including t20038, t20039, and t20042. The identified spa types were clustered into 15 spa-clonal complexes (spa-CCs) using BURP analysis; novel/unknown spa types were further subjected to MLST analysis. The majority of isolates belonged to spa-CC 152 (62/170, 36.4%), followed by spa-CC 121 (19/170, 11.2%), and spa-CC 005 (18 /170, 10.6%). Of the nine MRSA isolates, 2 (22.2%) were spa-CC 239 with staphylococcal cassette chromosome (SCC)mec III. These findings highlight the diversity of S. aureus strains in Ethiopia, as well as the presence of potentially epidemic strains circulating in the country necessitating further characterization of S. aureus for antimicrobial resistance detection and infection prevention purposes.

Evaluating St. Augustinegrass [Stenotaphrum Secundatum (Waltz.) Kuntze] Cultivar Morphological Differences

Current trends toward energy conservation in home landscaping present problems in warm-season turfgrass selection since all species grow best in full sunlight. Previous research has identified several St. Augustinegrass [Stenotaphrum secundatum (Waltz.) Kuntze] selections with excellent shade and cold tolerance. To determine if germplasm samples are truly different than established cultivars, morphological traits were evaluated. The objective of this study was to determine if experimental St. Augustinegrass germplasm samples possessed different morphological differences compared to industry standards, ‘Raleigh’ and ‘Palmetto’. Turf height measurements, visual seedhead density counts and morphological measurements were taken to quantify leaf width, leaf length, and internode distance. Measurements were taken at the third internode of each stolon. Internode distance was measured between the third and fourth internode. Significant differences between leaf width, leaf length, and internode distance occurred as did differences between turf height and seedhead density when compared to the industry standards, ‘Raleigh’ and ‘Palmetto’ St. Augustinegrasses. Findings suggest these germplasm samples have sufficient different morphological characteristics to indicate they are probably different cultivars. Differences in morphological characteristics is one tool used to determine differences between cultivars. Additional verification is justifiable if technology advances allow genetic sequencing to be definitive enough to determine if these grasses are truly different. As homeowners trend towards greater energy efficiency, demands for better performing, more shade tolerant turfgrasses also increase. Current trends toward energy conservation in home landscaping present problems in warm-season (C4) turfgrass selection since all species grow best in full sunlight. As the use of shade for cooling homes and buildings has increased, the need for a shade and cold tolerant turfgrass by homeowners and landscapers has arrived [1-3]. To determine if germplasm samples are truly different cultivars than established ones, morphological traits are typically evaluated to justify further research such as genetic sequencing. In addition, turf height and seedhead production differences would be valuable information for those turf managers interested in cultivars not requiring as frequent mowing as others. Genotypic sequencing, however, isn’t always able to delineate between cultivars. In hybrid bermudagrasses (Cynodon dactylon (L.) Pers. x C. transvaalensis Burtt-Davy), genotyping-by-sequencing was unable to consistently distinguish triploid standard cultivars from one another [4]. Researchers also were not able to distinguish these selections from their parents due to similar origin and clonal propagation but could distinguish triploids from diploid and tetraploid samples. To the author’s knowledge, molecular genotyping of St. Augustinegrasses has yet been published. Busey [5] classified 94 gentotypes based on 26 characters into five groups and developed a key based on their geography, chromosome number, and adaptation characteristics. The objective of this study was to determine if experimental St. Augustinegrass germplasm samples possessed different morphological differences compared to industry standards ‘Raleigh’ and ‘Palmetto’

First report of race TTRTF of the Wheat Stem Rust pathogen Puccinia graminis f. sp. tritici in Sweden.

Wheat (Triticum aestivum L.) stem rust caused by Puccinia graminis f. sp. tritici (Pgt) was generally insignificant in Europe from the 1960s until 2016, where a new race (TTRTF) caused damage on huge areas of durum wheat and bread wheat in Sicily (Bhattacharya, 2017). During the following five years, TTRTF was detected in eight additional countries in south- and central Europe (Patpour et al. 2022). In July 2021, seven wheat stem rust samples collected from spring wheat and one from barley in Svalov (55° 54' 10.8″ N, 13° 6' 54″ E) and Alnarp (55° 39' 39.6″ N, 13° 4' 40.8″ E), Sweden. Both cereal fields had a total disease incidence of 50% or higher. The samples were sent to the Global Rust Reference Center (GRRC, Denmark). Urediniospores of each sample were recovered on two susceptible cultivars, Line E and Morocco, which were used as susceptible controls in all experiments. Single pustular isolates were extracted and race typing was generally repeated two to three times based on Patpour et al. (2022) using 20 North American stem rust differential lines. Seedling infection types (IT) were scored on the first and second leaf 17 days post-inoculation using a 0-4 scale (Stakman et al. 1962; McIntosh et al. 1995). Isolates conferring 'low' ITs (i.e., 0, 0; 1, 1+, 2, and 2+), or combinations thereof, were considered 'avirulent' (incompatible), whereas ITs of 3-, 3, 3+, and 4 were considered 'high' (i.e., compatible, 'virulent'). Race nomenclature was based on a modified letter code proposed by Jin et al. (2008). We conducted DNA extraction and molecular genotyping using seventeen Simple Sequence Repeat (SSR) primer pairs derived from Stoxen (2012) and applied at large scale by Patpour et al. (2022). Based on the results from pathotyping and genotyping, two samples from wheat showed Pgt race TKTTF (Clade IV-B), three from wheat TKKTF (Clade IV- F), and three samples from wheat and barley were identified as TTRTF (Clade III-B). This is the first report of race TTRTF in northern Europe, particularly, Sweden, which significantly extends the known distribution of this race. The TTRTF race is a serious threat to wheat productivity and evaluation of resistance of commercial European wheat varieties to the TTRTF race confirmed that 70% of the cultivars were susceptible (Patpour et al. 2022). Therefore, if the conditions are suitable for the establishment and development of stem rust, the disease can cause significant damage to the wheat crop in these countries. Susceptibility of European wheat varieties stress an urgent need to initiate new breeding efforts to identify effective sources of resistance to wheat stem rust in breeding programs. Funding: The work is funded by the European Union's Horizon 2020 research and innovation programme under grant agreement No 773311 (RustWatch).

Molecular identification and genotyping of Blastocystis sp. in sheep and goats from some areas in Inner Mongolia, Northern China.

Blastocystis sp. is a kind of unicellular intestinal commensal which is widely distributed in humans and animals, and frequently found in the people who are in close contact with animals. To investigate the prevalence and evaluate the zoonotic potential of Blastocystis sp. in sheep and goats from Inner Mongolia, China, a total of 1037 samples were collected from them, and subjected to nested PCR amplification based on the small subunit ribosomal RNA (SSU rRNA) gene of Blastocystis sp. The sanger sequencing was used for Blastocystis sp. subtype identification. The results indicated that the average infection rate of Blastocystis sp. was 10.70% [95CI: 8.82%-12.58%] (111/1037), including 11.30% [95CI: 7.96%-14.64%] for sheep (39/345) and 10.40% [95CI: 8.13%-12.67%] for goats (72/692). Five Blastocystis subtypes (ST5, ST10, ST14, ST21 and ST26) were identified in the present study. Among them, ST10 was the most dominant subtype in sheep and goats, accounting for 70.27% (78/111) of the total identified positive samples. This is the first report regarding Blastocystis sp. subtypes ST21 and ST26 in goats in China. This study has provided a detail epidemiological data on the prevalence and subtypes distribution of Blastocystis sp. in sheep and goats in Inner Mongolia, China. Our results indicated that sheep and goats could be reservoir host for multiple Bastocystis subtypes, including the zoonotic subtypes. Further studies among humans, livestock and wild animals are needed to better understand their role in the spread of Blastocystis sp.

Prognostic biomarkers for survival in mucosal melanoma.

e21576 Background: Mucosal Melanoma is a rare subtype of melanoma with a more aggressive clinical course when compared to cutaneous melanoma. In cutaneous melanoma, the absence of pigmentation and presence of NRAS/KRAS mutations are known as biomarkers indicating an aggressive clinical course with significantly shorter overall survival times. Similar data for mucosal melanoma are currently missing. Here, we present real world outcome data in a large cohort of genotyped mucosal melanoma patients and assessed the prognostic relevance of pigmentation- and NRAS/KRAS mutation status. Methods: We correlated pathological reports and clinical data with overall survival of patients with mucosal melanoma. Furthermore, we performed clinically integrated molecular genotyping and analyzed real world treatment regimens for covariates associated with clinical outcome. For institutional data analysis, we ran a source query and only included patients with the diagnosis of mucosal melanoma, available genotyping data and comprehensive clinical information. As a population-based reference and to directly compare overall survival differences we first queried the National Cancer Institute’s Surveillance, Epidemiology and End Results (SEER) database. Pigmentation status was assessed by pathological review. Treatment regimens were identified by chart review. Results: We identified a cohort of 39 patients with mucosal melanoma where clinical and molecular data was available. Patients with amelanotic mucosal melanoma had a significantly shorter overall survival ( p = 0.003). In addition, the presence of a NRAS or KRAS mutation was significantly associated with poor overall survival (NRAS or KRAS p = 0.024). Therapeutic regimens were heterogeneous with a mean of 5 treatment lines per patient. Patients with low stage tumors who received immunotherapy had a significantly shorter overall survival (p &lt; 0.001), although numbers were small. Conclusions: The absence of pigmentation and the presence of NRAS or KRAS mutations are poor prognostic indicators for survival in CM. Both biomarkers are have been described in MM. Currently, it is unknown if the same prognostic relevance for the lack of pigmentation and RAS mutations exists in MM. Here we analyzed a cohort of MM for outcome measures and determined that two known prognostic biomarkers for CM are in fact novel prognosticators for MM.

Molecular Characterization and Prevalence of Antimicrobial-Resistant Escherichia coli Isolates Derived from Clinical Specimens and Environmental Habitats.

Antibiotic-resistant bacteria (ARB) are present in wastewaters as their elimination during treatment in wastewater treatment plants (WWTPs) is often impossible. Water plays an important role in the spread of these microorganisms among humans, animals and the environment. This study aimed to assess the antimicrobial resistance patterns, resistance genes and molecular genotypes by means of phylogenetic groups of E. coli isolates in aquatic habitats, including sewage and receiving water bodies, as well as clinical settings in the Boeotia regional district of Greece. The highest resistance rates among both environmental and clinical isolates were observed to be for penicillins, ampicillin and piperacillin. Resistance patterns related to extended spectrum β-lactamases (ESBL) production and ESBL genes were also detected in both environmental and clinical isolates. Phylogenetic group B2 was predominant in clinical settings and the second most frequent among wastewaters, whereas group A was dominant in all environmental isolates. In conclusion, the studied river water and wastewaters may serve as reservoirs of resistant E. coli isolates that pose potential threats to both human and animal health.

Reproductive potential of the black bean aphid (Aphis fabae Scop.) on a range of faba bean (Vicia faba L.) accessions

AbstractThe black bean aphid is one of the main insect pests of faba bean, leading to yield losses in many countries. The aphid inflicts damage to faba bean through direct phloem feeding and indirectly by transmission of several viruses. Sources of genetic resistance or tolerance to the aphid have been hard to identify and to use in breeding because of the high environmental variability of the attack, the weak and partial nature of the resistance when found, and the low repeatability of experiments. A range of molecular genotyping tools is now available for identifying the genes underlying key traits in faba bean. Hence, we screened the responses of eight inbred lines (Columbo, Albus, Closed‐flower, Diana, Hedin/2, Icarus, ILB938/2, and Mélodie/2) of faba bean, derived from commercial cultivars and experimental germplasm, to aphid infestation in controlled (no‐choice‐test) and field (preference test) conditions. In the controlled environment, aphid performance was assessed by measuring fecundity and determining the intrinsic rate of natural increase (rm). In the field experiment, population growth was examined on Columbo, Closed‐flower, Hedin/2, and ILB938/2 during June and July. Each week, 10 plants in each plot, randomly chosen at the start, were screened for aphid colonization and number of plants attacked. For each plant, an index number was given reflecting the severity of aphid presence. Our results demonstrate a clear conformity between the results obtained from the indoor pot experiments and the field experiments. In both experiments, ILB938/2 showed partial resistance with significantly lower fecundity, rm, and field infestation when compared with the known susceptible line Columbo. The consistently strong performance of ILB938/2 against the black bean aphid suggests that it carries a factor or factors rendering it unconducive to aphid landing, feeding, and/or reproduction. Further investigation into the resistance mechanisms of ILB938/2 is planned.

Phytochemical and genetic marker (SCoT and CBDP) based study of genetic diversity and population structure in natural populations of Cannabis sativa L.: A high-value sustainable biodiversity of North-Indian Himalaya

Cannabis sativa is a versatile, sustainable cash crop grown in vast agro-climatic zones of India. The present study used comprehensive datasets involving phytochemical and molecular genotyping to characterize forty-two wild cannabis accessions collected from fourteen geographical locations of the North Indian Himalayas. Two gene-targeted dominant molecular markers, i.e., start codon targeted (SCoT) polymorphism and CAAT-box derived polymorphism (CBDP), were used for molecular genotyping to evaluate genetic assortment and interrelatedness among and within wild inhabitants of C.sativa. Multiple t-tests indicated significant differences among the studied cannabis populations for the phytochemical traits. Five major cannabinoids were identified in Indian Cannabis sativa germplasm, and most of the populations were of drug/marijuana types. The considerable diversity in drug types was observed at the chemical and genetic levels. Twenty-six SCoT and twenty-one CBDP markers generated a sum of 134, and 80 alleles averaged with 3.08 and 6.65 alleles per primer, respectively. The average polymorphic information content (PIC) and resolving power per primer indicated both SCoT and CBDP as ideal markers system for genetic diversity study in Cannabis sativa. The population diversity analysis based on SCoT, CBDP, and cumulative marker dataset revealed a high genetic differentiation (Gst>0.15), and a low estimated gene flow (Nm<1.0) between the studied populations. The highest gene diversity (H) and Shannon diversity Index (I) were observed for the population of Matiyal location (Pop3) using SCoT and cumulative marker dataset. However, Population 7 (Pop7) of location Kanatal was found to be highly diverse using the CBDP marker dataset. The multivariate analyses, i.e., Neighbour-joining tree based phenetic analysis, Principal Coordinate Analysis (PCoA), and Structure, revealed that there was no individual geographical location based grouping among the cannabis populations using both markers data set individually and their cumulative datasets. However, the allocation of accessions in common groups corresponding to their geographical locations was maximum in the case of Analysis with a cumulative marker data set. Analysis of molecular variance (AMOVA) also demonstrated a maximum percentage of molecular variation at the intra-population level (77–86%) rather than the inter-population level (14–23%). Altogether, our data suggest that both SCoT and CBDP markers are the ideal markers to study the genetic relationship among geographically different populations of C.sativa, and populations identified as highly diverse can be utilized as parents in Indian cannabis breeding programs.

Molecular diagnosis and genotyping of Orientia tsutsugamushi in Maesot and Chiangrai, Thailand.

Scrub typhus is a neglected tropical disease with an estimated 1 million cases annually. The Asia-Pacific region is an endemic area for scrub typhus, especially in Thailand. Between June 2018 and December 2019, 31 patients with acute undifferentiated febrile illness (AUFI) were recruited for clinical trials and tested positive by a scrub typhus IgM RDT. Of the 17 buffy coat patient samples tested by 47kDa real-time PCR and 56kDa type-specific antigen (TSA) nested PCR, 94% (16/17) were positive, and of the 11 patients that presented with eschar lesions, 100% (11/11) of the eschar samples were confirmed positive. Genetic analysis of the 560 bp partial 56-kDa TSA gene demonstrated that most Orientia tsutsugamushi (Ot) infections were with Karp, Gilliam, Taiwan, P23, and CM606-like strains. This is the second occasion that the CM606-like and P23-like strains were reported in northern Thailand (first reported in 2011 and 2013, respectively). This study demonstrates that 1) the eschar remains the most reliable biological sample for PCR diagnosis of scrub typhus and 2) Northwestern Thailand has significant diversity of Ot strains, which underlines the requirement for ongoing surveillance to increase our understanding of Ot diversity to ensure accurate diagnostics and treatment.