Reverse Hybridization Assay Research Articles

HFE and Non-HFE Hereditary Hemochromatosis Based on Screening of 854 Individuals: 12 Years of an Iranian Experience.

Introduction: The genetics of hereditary hemochromatosis (HH) is understudied in Iran. Here, we report the result of genetic screening of 854 individuals, referred as "suspected cases of HH," to a diagnostic laboratory in Iran over a 12-year period. Materials and Methods: From 2011 to 2012, 121 cases were screened for HH using Sanger sequencing of HFE exons. After 2012, this method was replaced by a commercial reverse hybridization assay (RHA) targeting 18 variants in the HFE, TFR2, and FPN1(SLC40A1) genes and 733 cases were screened using this method. Results: From the total studied population, HH was confirmed by genetic diagnosis in only seven cases (0.82%): two homozygotes for HFE:C282Y and five homozygotes for TFR2:AVAQ 594-597 deletion. In 254 cases (29.7%), H63D, C282Y, S65C, and four other HFE variants not targeted by RHA were identified. Although the resulting genotypes in the latter cases did not confirm HH, some of them were known modifying factors of iron overload or could cause HH in combination with a possibly undetected variant. No variant was detected in 593 cases (69.4%). Conclusion: This study showed that the spectrum of genetic variants of HH in the Iranian population includes HFE and TFR2 variants. However, HH was not confirmed in the majority (99.2%) of suspected cases. This could be explained by limitations of our genetic diagnostics and possible inaccuracies in clinical suspicion of HH. A cooperative clinical and genetic investigation is proposed as a solution to this issue.

Genetic variations and clinical features associated with burning sensation due to topical tacrolimus in patients with atopic dermatitis

AbstractBackgroundTopical tacrolimus has been used as one of the treatment options for atopic dermatitis (AD). A burning sensation is the most common side effect of topical tacrolimus.ObjectivesThis study was designed to identify genetic variations and clinical features to predict burning sensation by topical tacrolimus in patients with AD.MethodsPatients with AD who had applied topical tacrolimus were classified according to the presence of a burning sensation. We compared the patients' demographics and blood test results, including serum eosinophil, serum immunoglobulin E and multiple allergen simultaneous test. Furthermore, the frequency of variations in barrier‐ (KLK7, FLG 3321delA, FLG K4022X, SPINK5 1156, SPINK5 1188, SPINK5 2475) and immune response‐ (DEFB1, KDR, CDKAL1, IL‐5RA, IL‐9, IL‐12RB1, IL‐12RB2, IL‐17RA) related genes was compared between two groups by reverse blot hybridization assay of the buccal mucosa.ResultsAmong 206 patients with AD using topical tacrolimus, 34 (16.5%) complained of a burning sensation. The burning sensation was associated with severe AD and a family history of atopic disease. Furthermore, with statistical significance, a heterozygous mutation in the filaggrin gene (FLG K4022X) was more frequent in AD patients with a burning sensation due to topical tacrolimus. However, no other significant genetic differences were observed between them.ConclusionsA burning sensation caused by topical tacrolimus can be expected in AD patients with severe symptoms, a family history of atopy and a genetic variation such as FLG K4022X.

Thalassemia Genotypes and HbA2 levels of Children in Wuzhou, Guangxi

To analyze thalassemia genotypes and distribution of children in Wuzhou Guangxi, and evaluate the diagnostic value of HbA2 in children's thalassemia screening, so as to provide scientific evidence for the prevention and control strategies of thalassemia. Four hundred and fifty-eight children suspected with thalassemia in Wuzhou were enrolled from March 2017 to June 2022. The level of HbA2 was detected using Bio-Rad VARIANT II Hb analysis system. The deletion of α-thalassemia was measured with gap-PCR assay, and the point mutation of α- and β-thalassemia was tested with DNA reverse dot blot hybridization assay. The receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of HbA2 for children's thalassemia. A total of 304 thalassemia carriers were detected in 458 children, accounting for 66.38%. One hundred and seventy-five cases were defined to be α-thalassemia, with the main type of --SEA/αα (54.86%). Thirty-six cases were defined to be intermediate α-thalassemia, with the main type of -α3.7/--SEA (9.72%). In 108 cases with β-thalassemia, βCD41-42/βN was the main type, accounting for 49.07%, followed by βIVS-Ⅱ-654 /βN (14.81%). Seven cases were moderate/severe β-thalassemia (predominantly β-28/β-28 and βCD41-42/βCD17/). Twenty-one genotypes of α- and β-thalassemia were found in the children. There was significant difference of HbA2 level between the children with different types of thalassemia and healthy controls (all P < 0.001). ROC curve analysis showed that the sensitivities of HbA2 for α-thalassemia, β-thalassemia and αβ-thalassemia were 74.3%, 82.4% and 85.7%, with the optimal cut-off values of 2.60%, 3.60% and 3.70%, respectively, the specificities were 64.3%, 96.1% and 96.8%, and the area under the curve were 0.690, 0.887 and 0.916, respectively. The thalassemia genotypes of children in Wuzhou are diverse. It is necessary to further strengthen the prevention and control measure of thalassemia to reduce birth defects and improve birth quality.

Simultaneous detection of seven bacterial pathogens transmitted by flies using the reverse line blot hybridization assay

BackgroundTraditional methods for detecting insect-borne bacterial pathogens are time-consuming and require specialized laboratory facilities, limiting their applicability in areas without access to such resources. Consequently, rapid and efficient detection methods for insect-borne bacterial diseases have become a pressing need in disease prevention and control.MethodsWe aligned the ribosomal 16S rRNA sequences of seven bacterial species (Staphylococcus aureus, Shigella flexneri, Aeromonas caviae, Vibrio vulnificus, Salmonella enterica, Proteus vulgaris, and Yersinia enterocolitica) by DNASTAR Lasergene software. Using DNASTAR Lasergene and Primer Premier software, we designed universal primers RLB-F and RLB-R, two species-specific probes for each pathogen, and a universal probe (catch-all). The PCR products of seven standard strains were hybridized with specific oligonucleotide probes fixed on the membrane for specific experimental procedures. To evaluate the sensitivity of PCR-RLB, genomic DNA was serially diluted from an initial copy number of 1010 to 100 copies/μl in distilled water. These dilutions were utilized as templates for the PCR-RLB sensitivity analysis. Simultaneous detection of seven fly-borne bacterial pathogens from field samples by the established PCR-RLB method was conducted on a total of 1060 houseflies, collected from various environments in Lanzhou, China.ResultsThe established PCR-RLB assay is capable of detecting bacterial strains of about 103 copies/μl for S. aureus, 103 copies/μl for S. flexneri, 105 copies/μl for A. caviae, 105 copies/μl for V. vulnificus, 100 copies/μl for S. enterica, 105 copies/μl for P. vulgaris, and 100 copies/μl for Y. enterocolitica. The results demonstrate that the detection rate of the established PCR-RLB method is higher (approximately 100 times) compared to conventional PCR. This method was applied to assess the bacterial carrier status of flies in various environments in Lanzhou, China. Among the seven bacterial pathogens carried by flies, S. enterica (34.57%), S. flexneri (32.1%), and Y. enterocolitica (20.37%) were found to be the predominant species.ConclusionsOverall, this research shows that the rapid and efficient PCR-RLB detection technology could be a useful for surveillance and therefore effective prevention and control the spread of insect-borne diseases. Meanwhile, the experimental results indicate that urban sanitation and vector transmission sources are important influencing factors for pathogen transmission.Graphical

Exploring the host factors affecting asymptomatic Plasmodium falciparum infection: insights from a rural Burkina Faso study

BackgroundAsymptomatic Plasmodium falciparum parasitaemia forms a reservoir for the transmission of malaria disease in West Africa. Certain haemoglobin variants are known to protect against severe malaria infection. However, data on the potential roles of haemoglobin variants and nongenetic factors in asymptomatic malaria infection is scarce and controversial. Therefore, this study investigated the associations of iron homeostasis, inflammation, nutrition, and haemoglobin mutations with parasitaemia in an asymptomatic cohort from a P. falciparum-endemic region during the high transmission season.MethodsA sub-study population of 688 asymptomatic individuals (predominantly children and adolescents under 15 years, n = 516) from rural Burkina Faso previously recruited by the NOVAC trial (NCT03176719) between June and October 2017 was analysed. Parasitaemia was quantified with conventional haemocytometry. The haemoglobin genotype was determined by reverse hybridization assays targeting a selection of 21 HBA and 22 HBB mutations. Demographics, inflammatory markers (interleukins 6 and 10, hepcidin), nutritional status (mid upper-arm circumference and body mass index), and anaemia (total haemoglobin, ferritin, soluble transferrin receptor) were assessed as potential predictors through logistic regression.ResultsMalaria parasites were detected in 56% of subjects. Parasitaemia was associated most strongly with malnutrition. The effect size increased with malnutrition severity (OR = 6.26, CI95: 2.45–19.4, p < 0.001). Furthermore, statistically significant associations (p < 0.05) with age, cytokines, hepcidin and heterozygous haemoglobin S were observed.ConclusionsAccording to these findings, asymptomatic parasitaemia is attenuated by haemoglobin S, but not by any of the other detected genotypes. Aside from evidence for slight iron imbalance, overall undernutrition was found to predict parasitaemia; thus, further investigations are required to elucidate causality and inform strategies for interventions.

Type distribution of human papillomaviruses in ThinPrep cytology samples and HPV16/18 E6 gene variations in FFPE cervical cancer specimens in Fars province, Iran

BackgroundThere exists strong evidence that human papillomavirus (HPV) is associated with cervical cancer (CC). HPV E6 is a major oncogene whose sequence variations may be associated with the development of CC. There is not sufficient data on the distribution of HPV types in ThinPrep cytology specimens and HPV 16/18 E6 gene variations among CC patients in the southwest of Iran. This study was conducted to contribute to HPV screening and vaccination in Iran.MethodsA total of 648 women screened for cervicitis, intraepithelial neoplasia or CC were included in the study. All participants underwent ThinPrep cytology testing, single-step HPV DNA detection and allele-specific reverse hybridization assays. Moreover, a total of 96 specimens previously tested positive for single infection with HPV16 or 18 were included for variant analysis. HPV16/18 lineages and sublineages were determined by PCR assays followed by sequencing the E6 gene and the construction of neighbor-joining phylogenetic trees.ResultsOverall, HPV DNA was detected in 62.19% of all the screened subjects. The detection rates of HPV DNA among individuals with normal, ASC-US, ASC-H, LSIL, and HSIL cervical cytology were 48.9%, 93.6%, 100%, 100%, and 100%, respectively. Low-risk HPVs were detected more frequently (46.9%) than high-risk (38.9%) and possible high-risk types (11.1%). Of 403 HPV-positive subjects, 172 (42.7%) had single HPV infections while the remaining 231 (57.3%) were infected with multiple types of HPV. Our results indicated a remarkable growth of high-risk HPV66 and 68 and low-risk HPV81 which have rarely been reported in Iran and HPV90 and 87 that are reported for the first time in the country. In addition, 3 lineages (A, D, and C) and 6 sublineages (A1, A2, A4, C1, D1, and D2) of HPV16, and one lineage and 4 sublineages (A1, A3, A4, and A5) of HPV18 were identified. The studied HPV16 and 18 variants mainly belonged to the D1 and A4 sublineages, respectively.ConclusionThe present study suggests that the prevalence of HPV infection in women of all age groups with or without premalignant lesions in the southwestern Iran is high and the predominant HPV types in the southwest of Iran may differ from those detected in other parts of the country. This study also highlights the necessity of not only initiating HPV vaccination for the general population but also developing new vaccines that confer immunity against the prevalent HPV types in the area and national cervical screening programs using a combination of thinPrep cytology test and HPV detection assays in order to improve the accuracy of the screening.

Head-to-head comparison of genotyping of human papillomavirus by GP5+/6+-PCR-based reverse dot blot hybridization assay and SPF10-PCR-based line probe assay.

The SPF10-PCR-based line probe assay (LiPA-25) for human papillomavirus (HPV) genotyping with high analytical sensitivity and specificity was widely used in HPV vaccine clinical trials and epidemiologic studies. In the study, we aimed to compare a novel GP5+/6+-PCR-based reverse dot blot hybridization assay (Yaneng-23) with LiPA-25. The performance of two assays was evaluated in 1735 cervical swab and 117 tissue samples, with a focus on 19 common HPV types (14 high-risk: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68/73; 5 low-risk: 6, 11, 42, 43, and 53). A total of 1197 (69.0%) swab samples were identified as HPV-positive by two assays. Of these, 878 (73.4%) samples displayed absolute agreement (concordant), 255 (21.3%) showed additional or fewer types (compatible), and the remaining 64 (5.3%) samples were discordant. Additionally, the two assays showed an excellent strength of agreement for 19 HPV-combined detection (κ = 0.886) and 17 individual HPV types (all κ > 0.800), and displayed a good agreement for HPV39 (κ = 0.780) and 42 (κ = 0.699). Yaneng-23 was more sensitive than LiPA-25 for HPV58, 59, 68/73, 42, 43 and 53 (McNemar's test: all p < 0.05), while LiPA-25 was more sensitive for HPV31, 39, 52, and 66 than Yaneng-23 (all p < 0.05). In 113 HPV-positive tissue specimens, the identification of genotypes was 82.3% identical and 17.7% compatible. The agreement between the tests for HPV45 (κ = 0.796) and 51 (κ = 0.742) was good, and for other types (all κ > 0.843) and 19 HPV-combined detection (κ = 0.929) was perfect (all p > 0.05). In conclusion, Yaneng-23 and LiPA-25 are comparable. Yaneng-23 could be used for the detection and genotyping of HPV in cervical samples for epidemiological and vaccine studies worldwide.

Development and validation of a TaqMan® probe- based real-time PCR assay for detection of Ehrlichia canis

Ehrlichiosis is a potentially fatal zoonotic tick-borne disease, caused by a pleomorphic Gram-negative bacterium. It occurs worldwide and affects humans, domestic and wild animals. Dogs infected with Ehrlichia canis develop canine monocytic ehrlichiosis (CME), a significant infectious disease of canines. TaqMan® based real-time PCR assays to detect Ehrlichia spp. affecting dogs were developed and a real-time PCR assay specific for E. canis validated. The efficiency of the assay was 93% and the 95% limit of detection was 33 E. canis plasmid copies/µl of blood (95% confidence interval: 23 - 58). The assay was specific for E. canis when tested against other haemoparasites. Consistent repeatability was observed, with an inter-run standard deviation (SD) range between 0.33 and 1.29 and an intra-run SD range between 0.04 and 1.14. Field samples were tested in parallel by both the E. canis real-time PCR assay and a reverse line blot hybridization assay. The results were in agreement for the two assays, with an exception of two out of 121 samples. Bayesian latent class analysis was used to calculate a diagnostic sensitivity of the E. canis real-time PCR assay of 90% and a specificity of 92%. This assay is a sensitive and reliable molecular detection method for E. canis and will be a useful tool for early diagnosis and timely treatment for this haemoparasite.

Substantial Improvement in Nontuberculous Mycobacterial Identification Using ASTA MicroIDSys Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry with an Upgraded Database.

Identifying Mycobacterium using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is challenging. We evaluated the performance of MALDI-TOF MS in identifying nontuberculous mycobacteria (NTM) using the ASTA MicroIDSys system (ASTA Inc., Suwon, Korea) with the MycoDB v1.95s and upgraded MycoDB v2.0-beta databases. We tested 124 NTM isolates collected from Ogawa medium at Severance Hospital, Seoul, Korea, between January and April 2019. MicroIDSys scores were categorized into three groups: ≥140, reliable identification; 130–139, ambiguous identification; and <130, invalid identification. To validate the results, we used the reverse blot hybridization assay (Molecutech REBA MycoID, YD Diagnostics Corp., Korea). Initial analysis using MycoDB v1.95s resulted in 26.6% (33/124) reliable, 43.5% (54/124) ambiguous, and 29.8% (37/124) invalid identifications. Re-analysis using the upgraded MycoDB v2.0-beta database resulted in 94.4% (117/124) reliable, 4.0% (5/124) ambiguous, and 1.6% invalid (2/124) identifications. The percentage of reliable identifications that matched with the reference increased from 26.6% (33/124) with MycoDB v1.95s to 93.5% (116/124) with MycoDB v2.0-beta. The upgraded databases enable substantially improved NTM identification through deep learning in the inference algorithm and by considering more axes in the correlation analysis. MALDI-TOF MS using the upgraded database unambiguously identified most NTM species. Our study lays a foundation for applying MALDI-TOF MS for the simple and rapid identification of NTM isolated from solid media.

Trends in cocirculation of oncogenic HPV genotypes in single and multiple infections among theunvaccinated community.

Cocirculation of multiple human papillomavirus (HPV) infections with low, probably high, and high‐risk genotypes are to be associated with various grades of infections and cancer progression. The oncogenic high‐risk HPVs are distributed and cocirculated throughout the world. This study was investigated to identify HPV genotypes related to genital disorders in unvaccinated women. The subjects were referred from clinics to a molecular lab for HPV testing in Iran as a low‐coverage vaccinated country. HPVs DNAs of cervical scrapping and genital tissue specimens of 1,133 un‐vaccinated women were genotyped using an in vitro diagnostic line probe (reverse hybridization) assay. In addition, phylogenetic trees were constructed on 100 MY09/MY11 polymerase chain reaction (PCR) amplicons of common genotypes of HPV L1 gene by Sanger sequencing. The mean age of the population study was 32.7 ± 8.0 and the mean age of HPV‐positive cases was 31.6 ± 7.8. HPV DNA was detected in 57.8% (655/1133) of women subjects and 42.2% (478/1133) of cases were undetected. Among 655 HPV‐positive cases, 639 subjects (56.4%) were related to defined genotypes and 16 subjects (1.4%) were untypeable. The highest prevalence rate of HPV genotypes was identified in the 25–34 years. The top 6 dominant HPVs in single and multiple genotypes were HPV6 (284/655 [43.4%]), HPV16 (111/655 [16.9%]), HPV31 (72/655 [11%]), HPV53 (67/655 [10.2%]), HPV11 (62/655 [9.5%]), and HPV52 (62/655 [9.5%]). Moreover, single, multiple and untypeable HPV genotypes were diagnosed as follows: 1 type (318/655 [48.5%]), 2 types (162/655 [24.8%]), 3 types (83/655 [12.7%]), 4 types (42/655 [6.5%]), more than 5 types (34/655 [5.3%]), and 1.4% un‐typeable subjects. The sequenced partial L1 gene of HPV genotypes (GenBank databases under the accession numbers: MH253467‐MH253566) confirmed and determined the cocirculated HPV genotypes' origins and addressed helpful insights into the future viral epidemiology investigations. Multiple HPV infections and cocirculation of various oncogenic HPV genotypes among the normal population (women and men) with asymptomatic forms are still challenging in unvaccinated communities. The preventive and organized surveillance programs for HPV screening are needed to be considered and compiled by health policy makers of low or unvaccinated countries.

Reverse dot blot hybridization assay - An expeditious diagnostic tool for drug resistant TB.

Tuberculosis (TB) is one of the leading infectious diseases that is highly transmissible. It is reported to cause approximately 10 million infections and 1.4 million deaths globally in the year of 2019.¹ Prompt detection and treatment of TB are essential to intercept the proliferation of the disease. In the case of drug- resistant TB, failure to detect the resistance of anti TB drugs may give rise to extensively drug- resistant tuberculosis (XDR-TB). Thus, the rapid detection of drug resistance is vital;, however, the current standard drug susceptibility tests (DST) may take up to 12 weeks raising an alarming concern.² A study was done in China by Li Wan et al, on the accuracy of the reverse dot blot hybridization assay (RDBH) for rapidly detecting the resistance of four anti TB drugs (rifampicin (RIF), isoniazid (INH), streptomycin (SM) and ethambutol (EMB)) in mycobacterium tuberculosis (MTB) isolates.³ Continuous...

Clinical and genetic characteristics of hemoglobin H disease in Iran

Hemoglobin H (Hb H) disease is a subtype of α-thalassemia caused by deletional and/or non-deletional mutations in three alpha-globin genes in which the various genotypes determine the disease severity. This study was aimed to investigate the frequency of alpha gene mutations and genotypes and their correlation with hematological and clinical characteristics in Iran. Among 202 patients diagnosed with Hb H disease through a national study in Iran according to standard methods, we had access to the hematologic and clinical findings and genetic data of 101 patients in whom genetic study was performed. Genomic DNA from peripheral blood was extracted and analyzed for identification of α-globin gene mutations using Multiplex Gap Polymerase Chain Reaction, Reverse Hybridization Assay, and finally Direct DNA Sequencing method. Twenty-one different mutations and thirty genotypes were detected in 101 patients with Hb H disease. In total, 39 patients (38.6%) were deletional and 62 patients (61.4%) were non-deletional type of the disease. The -- MED mutation was highly prevalent in almost half of the patients (56.4%). Among various genotypes, –MED/-a3.7 (29.7%) and -α20 .5/-α5NT (6.9%) were the most prevalent genotypes found in the studied group. Patients with non-deletional type presented with more severe hematological and clinical findings. Hb H percentage and serum ferritin levels were significantly higher in non-deletional patients in comparison to the deletional group (p < 0.05). 12 (11.9%) and 40 (39.6%) out of 101 patients were on regular and occasional transfusions, respectively. 83% of those with regular transfusion belonged to the non-deletional group. Among transfusion-dependent patients, –MED/αCSα and α20 .5/-α5NT were the most common genotypes. In this study, two patients with -α20 .5/αCSα and –MED/α−5NT genotypes experienced thrombotic events. This study indicated that although non-deletional genotypes of Hb H disease were responsible for more clinical severity of the disease, due to the presence of severe phenotypes even in deletional types, no definite correlation was found between genotype and phenotype.

Artificial Intelligence-Based Portable Bioelectronics Platform for SARS-CoV-2 Diagnosis with Multi-nucleotide Probe Assay for Clinical Decisions.

In the context of the recent pandemic, the necessity of inexpensive and easily accessible rapid-test kits is well understood and need not be stressed further. In light of this, we report a multi-nucleotide probe-based diagnosis of SARS-CoV-2 using a bioelectronics platform, comprising low-cost chemiresistive biochips, a portable electronic readout, and an Android application for data acquisition with machine-learning-based decision making. The platform performs the desired diagnosis from standard nasopharyngeal and/or oral swabs (both on extracted and non-extracted RNA samples) without amplifying the viral load. Being a reverse transcription polymerase chain reaction-free hybridization assay, the proposed approach offers inexpensive, fast (time-to-result: ≤ 30 min), and early diagnosis, as opposed to most of the existing SARS-CoV-2 diagnosis protocols recommended by the WHO. For the extracted RNA samples, the assay accounts for 87 and 95.2% test accuracies, using a heuristic approach and a machine-learning-based classification method, respectively. In case of the non-extracted RNA samples, 95.6% decision accuracy is achieved using the heuristic approach, with the machine-learning-based best-fit model producing 100% accuracy. Furthermore, the availability of the handheld readout and the Android application-based simple user interface facilitates easy accessibility and portable applications. Besides, by eliminating viral RNA extraction from samples as a pre-requisite for specific detection, the proposed approach presents itself as an ideal candidate for point-of-care SARS-CoV-2 diagnosis.

Association between serum amyloid A1 genotype and age of onset restricts to M694 homozygote familial Mediterranean fever patients in Armenia.

Familial Mediterranean fever (FMF) is an autosomal-recessive, inflammatory disorder characterised by short, recurrent attacks of fever, accompanied by pain in the abdomen, chest, or joints and complications of amyloidosis. Recently, we observed a significant association between the serum amyloid A1 (SAA1) β/β genotype and a delayed disease onset in 386 M694V homozygous FMF patients. This follow-up study was conducted to additionally analyse MEFV genotypes other than M694V/M694V for a possible influence of the SAA1 genotype on the age of disease onset. A total of 700 Armenian patients diagnosed with FMF based on the Tel-Hashomer criteria and carrying two MEFV mutant alleles were included in this study. Patients were divided into three MEFV genotypic subgroups: M694V homozygotes (M694V/M694V), M694V compound heterozygotes (M694V/Other), and patients with genotypes excluding M694V (Other/Other). MEFV and SAA1 analyses were performed by a commercial reverse-hybridisation assay, and resulting genotypes were matched against the demographic and clinical characteristics of the patients. Within the subgroup of M694/M694 homozygotes, SAA1 genotype β/β could be identified in 115 (34.43%) and 32 (61.54%) patients with an age of onset <20 and ≥20 years, respectively(p<0.001). However, no such relationship could be observed for MEFV genotypic subgroups M694V/Other (p=0.465) and Other/Other (p=0.697). Our data suggest, that the influence of SAA1 genotypic variation on the age of disease onset restricts to FMF patients homozygous for MEFV mutation M694V.

First report of Theileria buffeli/Theileria orientalis group and identification of piroplasms via Nested PCR-based RLB Hybridization assay in zebu cattle in the Western Highlands of Cameroon

Piroplasms infections are tick-borne diseases caused by haemoparasite of the genus Theileria or Babesia. They have a great impact on livestock production, especially cattle in sub-Saharan countries. However, data on the prevalence of bovine piroplasms and their genetic diversity are scanty in Cameroon. This study was aimed at highlight the species composition and determine the prevalence of piroplasms infecting cattle in the Western Highlands of Cameroon. To achieve this aim, blood samples from a total of 162 cattle were collected and examined using Reverse Line Blot hybridization (RLB) assay. The amplified hypervariable V4 region of the 18S rRNA gene of bovine piroplasms species, including Theileria parva, T. annulata, T mutans, T. velifera, T. buffeli/T. orientalis, T. taurotragi, Theileria sp (buffalo), Babesia bovis, B. bigemina, B. divergens, B. major and B. occultans was hybridized against species-specific probes. RLB hybridization assay revealed the presence of four piroplasms species with the overall prevalence of infection of 82.1%. Theileria velifera (71.6%) was the most prevalent species followed by Theileria mutans (43.21%), Theileria buffeli/T. orientalis (5.55%) and Babesia bigemina (3.7%). However, the study provided the first molecular evidence for the presence of T. buffeli/T. orientalis group species in cattle in Cameroon. Higher overall prevalence of infection of tick-borne pathogens was observed in this study area as well as the increase in prevalence and widespread of T. velifera and the observance of a new species of piroplasms. These results are an indication that special attention should be given to epizootiological investigations alongside well-adopted control programs.

Hepatitis C virus subtyping in Uttarakhand, India: a comparative study.

The online version contains supplementary material available at 10.1007/s13337-021-00729-9.

Ticks on wild boar in the metropolitan area of Barcelona (Spain) are infected with spotted fever group rickettsiae.

Tick-borne pathogens (TBPs) constitute an emerging public health concern favoured by multidimensional global changes. Amongst these, increase and spread of wild boar (Sus scrofa) populations are of special concern since this species can act as a reservoir of zoonotic pathogens and promote tick abundance. Thus, we aimed to make a first assessment of the risk by TBPs resulting from wild boar and ticks in the vicinity of a highly populated area. Between 2014 and 2016, we collected spleen samples and 2256 ticks from 261 wild boars (out of 438 inspected) in the metropolitan area of Barcelona (MAB; northeast Spain). We morphologically identified four tick species: Hyalomma lusitanicum (infestation prevalence: 33.6%), Dermacentor marginatus (26.9%), Rhipicephalus sanguineus sensu lato (18.9%) and R. bursa (0.2%). Ticks were pooled according to species and individual host. A total of 180 tick pools and 167 spleen samples were screened by real-time PCR and/or reverse line blot hybridization assay for Ehrlichia sp., Anaplasma sp., Babesia sp., Rickettsia sp., Borrelia burgdorferi sensu lato and Coxiella burnetii. Seventy-two out of the 180 tick pools were positive to Rickettsia spp. (minimum prevalence of 8.7%), including Rickettsia massiliae, R. slovaca and R. raoultii. We did not detect Rickettsia spp. in wild boar spleens nor other TBPs in ticks or wild boars. Since the ticks identified can bite humans, and the recorded spotted fever group (SFG) rickettsiae are zoonotic pathogens, there is a risk of SFG rickettsiae transmission for MAB inhabitants. Our results suggest a broader distribution of H. lusitanicum, competent vector for the Crimean-Congo haemorrhagic fever virus than previously known. Wild boar is not a Rickettsia spp. reservoir according to the spleen negative results. However, its abundance could favour tick life cycle and abundance, and its proximity to humans could promote the infection risk by Rickettsia spp.

Cross border transhumance involvement in ticks and tick-borne pathogens dissemination and first evidence of Anaplasma centrale in Burkina Faso

In West Africa, cross-border transhumance, also called seasonal migration, is known to be a very important animal production strategy, as it involves about 70 to 90% of cattle. In spite of the cattle movements, some strategic areas of transhumance remain poorly explored regarding ticks and their associated pathogens investigations. The purpose of this study is to evaluate the involvement of transhumance in the spread of cattle ticks and associated pathogens in Burkina Faso (BF) and Benin (BN), in a context of speedy invasion of West African livestock by Rhipicephalus microplus. A longitudinal survey was performed on 210 cattle from BF, monitored for ticks and tick-borne pathogens (TBP) during one seasonal transhumance. The first sampling coded “T0BF” took place in eastern BF, at the transhumance departure. A second sampling “T1BN” was carried out in northern BN, the transhumance arrival zone. A third sampling “T2BF” was done at the return of cattle in eastern BF. Ticks were morphologically identified and TBP detected with reverse line blot hybridization (RLB) assay. A total of 1027 ticks (7 species), 1006 ticks (11 species) and 1211 ticks (9 species) were respectively found at T0BF, T1BN and T2BF. Some species were collected at the three times of sampling without any significant difference in their relative abundances. However, other tick species appeared only at T1BN and/or T2BF. The TBP species found at the three points surveyed were Theileria annulata, Theileria mutans, Theileria velifera, Babesia bigemina and Anaplasma marginale. The most prevalent was T. mutans with 166/210 (79%), 159/210 (75.7%) and 78/210 (37%) cattle positive respectively at T0BF, T1BN and T2BF. Anaplasma centrale was evidenced with 0.5% and 0.9% respectively at T0BF and T2BF. To our knowledge, this represents its first report in the study area.Overall, the TBP prevalences were significantly lower at T2BF, highlighting the effect of tick populations changes induced by transhumance combined with the seasonal variation influence.

Novel Protozoans in Austria Revealed through the Use of Dogs as Sentinels for Ticks and Tick-Borne Pathogens.

We previously isolated and cultivated the novel Rickettsia raoultii strain Jongejan. This prompted us to ask whether this strain is unique or more widely present in Austria. To assess this issue, we retrospectively screened ticks collected from dogs in 2008. Of these collected ticks, we randomly selected 75 (47 females and 28 males) Dermacentor reticulatus, 44 (21 females, 7 males, and 16 nymphs) Haemaphysalis concinna, and 55 (52 females and 3 males) ticks of the Ixodes ricinus complex. Subsequently, these ticks were individually screened for the presence of tick-borne pathogens using the reverse line blot hybridization assay. In our current study, we detected DNA from the following microbes in D. reticulatus: Anaplasma phagocytophilum, Borrelia lusitaniae, Borrelia spielmanii, Borrelia valaisiana, and R. raoultii, all of which were R. raoultii strain Jongejan. In H. concinna, we found DNA of a Babesia sp., Rickettsia helvetica, and an organism closely related to Theileria capreoli. Lastly, I. ricinus was positive for Anaplasma phagocytophilum, Borrelia afzelii, Borrelia burgdorferi sensu stricto, Borrelia garinii/Borrelia bavariensis, B. lusitaniae, B. spielmanii, B. valaisiana, Candidatus Neoehrlichia mikurensis, Rickettsia helvetica, Rickettsia monacensis, and Theileria (Babesia) microti DNA. The detection of DNA of the Babesia sp. and an organism closely related to Theileria capreoli, both found in H. concinna ticks, is novel for Austria.

Genotyping and Drug Susceptibility Patterns of M. Tuberculosis Isolated from HIV Seropositive Patients in Tehran Iran

to investigate the prevalence and drug-resistance M. tuberculosis isolated from HIV seropositive individuals in Tehran, Iran. Human immunodeficiency virus (HIV) is one of the most important risk factors for developing active tuberculosis (TB). To determine the rate of transmission and drug resistant M. tuberculosis (MTB) strains isolated from HIV seropositive patients in Tehran province, Iran. This study consecutively enrolled 217 TB/HIV coinfected patients from April 2018 to August 2019 at Emam Khomeini referral hospital and 5 other health centers in Tehran province. The isolates were genotyped using 15 loci Mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTR). Minimum inhibitory concentration (MIC) was determined for 6 drugs. In addition, mutations were assessed in rpoB, katG, inhA, and ahpC genes using Reverse Blot Hybridization Assay System. A 20 (9.2%) patients were culture-positive for M. tuberculosis and typed by MIRU-VNTR, 13 (65%) strains formed 5 clusters, but 6 (30%) isolates had a unique pattern. The total Hunter-Gaston discrimination index (HGDI) for all 15 loci was 0.846, and the cluster size was 2 to 4 patients. The estimated proportion of recent transmission was 45%. The mutation was identified in 1 isolate, lost inhAW1 and mutation in MT1 loci, which was resistant to isoniazid (INH). Moreover, 1 (5%) and 3 (15%) isolates were resistant to INH and ethambutol (EMB), respectively, of which 1 was resistant to INH and EMB. The transmission rate of TB in HIV patients was relatively high; however, the prevalence of drug-resistant strains and TB infection in females was insignificant in this study (p < 0.05); none of the isolates was MDR strains.