Molecular Epidemiological Survey Research Articles

Molecular Identification and Subtype Analysis of Blastocystis sp. Isolates from Wild Mussels (Mytilus edulis) in Northern France.

Blastocystis sp. is the most common single-celled eukaryote colonizing the human gastrointestinal tract worldwide. Because of the proven zoonotic potential of this protozoan, sustained research is therefore focused on identifying various reservoirs of transmission to humans, and in particular animal sources. Numerous groups of animals are considered to be such reservoirs due to their handling or consumption. However, some of them, including mollusks, remain underexplored. Therefore, a molecular epidemiological survey conducted in wild mussels was carried out in Northern France (Hauts-de-France region) to evaluate the frequency and subtypes (STs) distribution of Blastocystis sp. in these bivalve mollusks. For this purpose, 100 mussels (Mytilus edulis) were randomly collected in two sampling sites (Wimereux and Dannes) located in the vicinity of Boulogne-sur-Mer. The gills and gastrointestinal tract of each mussel were screened for the presence of Blastocystis sp. by real-time polymerase chain reaction (qPCR) assay followed by direct sequencing of positive PCR products and subtyping through phylogenetic analysis. In parallel, sequences of potential representative Blastocystis sp. isolates that were previously obtained from temporal surveys of seawater samples at marine stations offshore of Wimereux were integrated in the present analysis. By taking into account the qPCR results from all mussels, the overall prevalence of the parasite was shown to reach 62.0%. In total, more than 55% of the positive samples presented mixed infections. In the remaining mussel samples with a single sequence, various STs including ST3, ST7, ST14, ST23, ST26 and ST44 were reported with varying frequencies. Such distribution of STs coupled with the absence of a predominant ST specific to these bivalves strongly suggested that mussels might not be natural hosts of Blastocystis sp. and might rather be carriers of parasite isolates from both human and animal (bovid and birds) waste. These data from mussels together with the molecular identification of isolates from marine stations were subsequently discussed along with the local geographical context in order to clarify the circulation of this protozoan in this area. The identification of human and animal STs of Blastocystis sp. in mussels emphasized the active circulation of this protozoan in mollusks and suggested a significant environmental contamination of fecal origin. This study has provided new insights into the host/carrier range and transmission of Blastocystis sp. and emphasized its potential as an effective sentinel species for water quality and environmental contamination.

Expansion and transmission dynamics of high risk carbapenem-resistant Klebsiella pneumoniae subclones in China: An epidemiological, spatial, genomic analysis

AimsCarbapenem-resistant Klebsiella pneumonia (CRKP) is a global threat that varies by region. The global distribution, evolution, and clinical implications of the ST11 CRKP clone remain obscure. MethodsWe conducted a multicenter molecular epidemiological survey using isolates obtained from 28 provinces and municipalities across China between 2011 and 2021. We integrated sequences from public databases and performed genetic epidemiology analysis of ST11 CRKP. ResultsAmong ST11 CRKP, KL64 serotypes exhibited considerable expansion, increasing from 1.54% to 46.08% between 2011 and 2021. Combining our data with public databases, the phylogenetic and phylogeography analyses indicated that ST11 CRKP appeared in the Americas in 1996 and spread worldwide, with key clones progressing from China’s southeastern coast to the inland by 2010. Global phylogenetic analysis showed that ST11 KL64 CRKP has evolved to a virulent, resistant clade with notable regional spread. Single-nucleotide polymorphism (SNP) analysis identified BMPPS (bmr3, mltC, pyrB, ppsC, and sdaC) as a key marker for this clade. The BMPPS SNP clade is associated with high mortality and has strong anti-phagocytic and competitive traits in vitro. ConclusionsThe high-risk ST11 KL64 CRKP subclone showed strong expansion potential and survival advantages, probably owing to genetic factors.

Distinct genetic clusters in HIV-1 CRF01_AE-infected patients induced variable degrees of CD4+ T-cell loss.

Retroviruses swiftly adapt, employing error-prone enzymes for genetic and phenotypic evolution, optimizing survival strategies, and enhancing virulence levels. HIV-1 CRF01_AE has persistently undergone adaptive selection, and cluster 1 and 2 infections display lower counts and fast loss of CD4+ T cells than other HIV-1 sub-types and CRF01_AE clusters. Its mechanisms are associated with increased CXCR4 tropism due to an envelope structure change favoring a tropism shift from CCR5 to CXCR4, thereby shaping viral phenotype features and impacting pathogenicity. This underscores the significance of consistently monitoring HIV-1 genetic evolution and phenotypic transfer to see whether selection bias across risk groups alters the delicate balance of transmissible versus toxic trade-offs, since virulent strains such as CRF01_AE clusters 1 and 2 could seriously compromise the efficacy of antiviral treatment. Only through such early warning and diagnostic services can precise antiviral treatments be administered to those infected with more virulent HIV-1 strains.

Characterization of an imported HIV-1 A1/A7/G recombinant in China

BackgroundInternational migration has accelerated the HIV-1 spread across national borders, gradually reducing the restrictions on the geographical distribution of HIV-1 subtypes. Subtypes A and G are globally recognized as the third and sixth most dominant HIV-1 genotypes, mainly prevalent in Africa, but rarely detected in China. Here we reported an imported HIV-1 recombinant which was composed of sub-subtypes A1 and A7 of subtype A and subtype G genes in a Chinese female. This virus was the first HIV-1 recombinant including A7 genes reported in the world.Case presentationThe near full-length genome (NFLG) was obtained from the plasma sample of the female in an HIV-1 molecular epidemiological survey with 853 participants in China. Phylogenetic analyses showed that this NFLG sequence contains three A7 segments, four G segments and one A1 segment with seven breakpoints, and all these segments were closely related to HIV-1 references circulating in Africa. The evidence from epidemiological investigation indicated that this female participant had a more-than-two-years heterosexual contact history with a fixed partner from Nigeria, a country in west Africa, which further supported the results of phylogenetic analyses. By the Bayesian phylogenetic analyses, the times of most recent common ancestors (tMRCA) of the partial pol gene (nt2308-3284, A7 region) and full-length vpr-vpu plus partial env gene (nt5534-6858, G region) were estimated around 1989 and 1984, respectively.ConclusionsIn this study, by using the NFLG sequencing, we identified an imported HIV-1 A1/A7/G recombinant which was estimated to originate around 1980s in Africa and introduced into China with international migration. This study highlighted the complexity of the global HIV-1 epidemic, the necessity of using genome sequences to determine HIV-1 genotypes and the importance of real-time monitoring of HIV-1 infection among international migrants and travelers.

Multi-locus sequencing typing reveals geographically related intraspecies variability of Sporothrix brasiliensis

Sporotrichosis is a subcutaneous mycosis caused by pathogenic Sporothrix species. Among them, Sporothrix brasiliensis is the main species associated with endemic regions in South America, especially Brazil. It is highly virulent and can be spread through zoonotic transmission. Molecular epidemiological surveys are needed to determine the extent of genetic variation, to investigate outbreaks, and to identify genotypes associated with antifungal resistance and susceptibility. This study investigated the sequence variation of different constitutive genes and established a novel multilocus sequence typing (MLST) scheme for S. brasiliensis. Specific primers were designed for 16 genes using Primer-BLAST software based on the genome sequences of three S. brasiliensis strains (ATCC MYA-4823, A001 and A005). Ninety-one human, animal, and environmental S. brasiliensis isolates from different Brazilian geographic regions (South, Southeast, Midwest and Northeast) andtwo isolates from Paraguay were sequenced. The loci that presented the highest nucleotide diversity (π) were selected for the MLST scheme. Among the 16 studied genetic loci, four presented increased π value and were able to distinguish all S. brasiliensis isolates into seven distinct haplotypes. The PCR conditions were standardized for four loci. Some of the obtained haplotypes were associated with the geographic origin of the strains. This study presents an important advance in the understanding of this important agent of sporotrichosis in Brazil. It significantly increased the discriminatory power for genotyping of S. brasiliensis isolates, and enabled new contributions to the epidemiological studies of this human and animal pathogen in Brazil and in other countries.

Using the Molecular Transmission Networks to Analyze the Epidemic Characteristics of HIV-1 CRF08_BC in Kunming, Yunnan.

HIV-1CRF08_BC is the most prevalent epidemic subtype among heterosexual (HET) and intravenous drug users (IDUs) in Kunming, Yunnan. Using the pol region of gene sequences derived from molecular epidemiological surveys, we developed a molecular transmission network for the purpose of analyzing its epidemiological characteristics, assessing its epidemiological trends, identifying its potential transmission relationships, and developing targeted interventions. HyPhy 2.2.4 was used to calculate pairwise genetic distances between sequences; GraphPad-Prism 8.0 was employed to determine the standard genetic distance; and Cytoscope 3.7.2 was applied to visualize the network. We used the network analysis tools to investigate network characteristics and the Molecular Complex Detection (MCODE) tool to observe the growth of the network. We utilized a logistic regression model to examine the factors influencing clustering and a zero-inflated Poisson model to investigate the factors influencing potential transmission links. At the standard genetic distance threshold of 0.008, 406 out of 858 study participants were clustered in 132 dissemination networks with a total network linkage of 868, and the number of links per sequence ranged from 1 to 19. The MCODE analysis identified three significant modular clusters in the networks, with network scores ranging from 4.9 to 7. In models of logistic regression, HET, middle-aged and elderly individuals, and residents of northern and southeastern Kunming were more likely to enter the transmission network. According to the zero-inflated Poisson model, age, transmission category, sampling year, marital status, and CD4+ T level had a significant effect on the size of links. The molecular clusters in Kunming's molecular transmission network are specific and aggregate to a certain extent. HIV-1 molecular network analysis provided information on local transmission characteristics, and these findings helped to determine the priority of transmission-reduction interventions.

Temporal changes in antibiotic resistance and population structure of methicillin-resistant Staphylococcus pseudintermedius between 2010 and 2021 in the United States

The aim of this study was to perform a phenotypic and molecular epidemiological survey to determine temporal changes in the antimicrobial resistance and population structure of methicillin-resistant Staphylococcus pseudintermedius (MRSP) in the United States. Samples from 200 S. pseudintermedius isolates were obtained from veterinary diagnostic facilities located in geographic regions sampled approximately ten years ago and compared to samples obtained in 2021. Kirby-Bauer disk diffusion was used to determine antimicrobial susceptibility. geoBURST analysis and MrBayes were used to infer relationships of isolates using MLST data. Almost all MRSP isolates (98%) in 2021 were multidrug-resistant with 21% of these isolates resistant to more than 16 antimicrobials. In 2010, 190 S. pseudintermedius isolates were collected and 141 of them were MRSP. From 2010–2021 there was a significant increase in resistance observed to all antibiotics tested except cephalothin and sulfonamides. Whereas ten years previously multilocus sequence types (ST) ST68 (35.7%), ST71 (10%), and ST84 (17.4%) predominated, these strains have been supplanted by other STs, notably ST45 (n = 14), ST155 (n = 9), ST181 (n = 13), ST496 (n = 9) and ST551 (n = 9). The newly prevalent STs are distantly related to ST68, ST71 and ST84 and most likely do not share any recent common ancestors. The population structure of MRSP is far more elastic than expected with new, highly resistant strains replacing the ones that predominated in the United States a decade ago. Antibiotic use may play a role in selection; however, the strains that were replaced were also multidrug-resistant and other factors are likely involved.

Molecular epidemiological survey of pathogenic trypanosomes in naturally infected cattle in northern Côte d'ivoire.

Bovine trypanosomiasis is a significant health concern for livestock intensification in Côte d'Ivoire. This study aimed to determine the prevalence and distribution of pathogenic trypanosomes and identify the most infected cattle breed in northern Côte d'Ivoire. We examined 700 cattle and found that polymerase chain reaction (PCR) was more sensitive (12.3%) than microscopic observation (5.6%). Among the trypanosome species detected in naturally infected cattle, Trypanosoma vivax was 7.3%, Trypanosoma simiae tsavo was 6.7%, and Trypanosoma congolense was 0.4%. The overall prevalence of trypanosome infection in all cattle breeds was 12.3%, while the prevalence in individual breeds was 14.8%, 7.3%, 10.6%, and 12.3% for N'Dama, Baoule, Zebu, and Mere breed, respectively. The infected animals had low packed cell volume, influencing the prevalence. Our findings indicate that bovine trypanosomes are prevalent in Côte d'Ivoire, and their prevalence varies by region and breed. These pathogens include T. vivax, T. simiae tsavo, and T. congolense.

Genomic epidemiology and transmission characteristics of mcr1-positive colistin-resistant Escherichia coli strains circulating at natural environment

MCR-positive Escherichia coli (MCRPEC) have been reported in humans worldwide. The high prevalence of mcr-1 poses clinical and environmental risks due to its diverse genetic mechanisms. Given the vital role of animals and the environment in the spread of antibiotic resistance, a “One Health” perspective should be taken when addressing antimicrobial resistance issues. This study conducted a prospective study in six farms (located in Jiaxing City, Zhejiang province, China) in 2019. MCRPEC strains were screened from samples of different sources. The molecular epidemiological surveys and transmission potential were investigated by whole-genome sequencing and phylogenetic analysis. MCRPEC were detected in different farms with various sources. Sequence type complex 10 was dominant and distributed widely in multiple sources. Core-genome multilocus sequence type (cgMLST) analysis indicated that clonal transmission could occur within and between farms. In addition, mcr-1 genes with different locations showed different transmission tendencies. The study indicated that interspecies and cross-regional transmission of MCRPEC could occur between different sectors in farms. Further surveillance and research of non-clinical MCRPEC strains are necessary to reduce the threat of MCRPEC.

First Epidemiological Survey on the Prevalence and Subtypes Distribution of the Enteric Parasite Blastocystis sp. in Vietnam.

Although Blastocystis sp. is the most common enteric protozoan in human stools worldwide, various geographical areas remain to be investigated regarding the frequency and circulation of this parasite. Such is the case of some developing countries in Southeast Asia that exhibit a higher risk for parasitic infections due to unsanitary conditions. While several epidemiological surveys have been conducted, for instance, in Thailand, little or no data are available from neighboring countries, such as Vietnam. Therefore, in order to determine the prevalence and subtype (ST) distribution of Blastocystis sp. and to clarify the transmission of the parasite, the first molecular epidemiological survey ever conducted in this country was performed. For this purpose, a total of 310 stool specimens were collected from patients enrolled at the Family Hospital of Da Nang and then tested for the presence of Blastocystis sp. by real-time Polymerase Chain Reaction (qPCR), followed by subtyping of the isolates. The overall prevalence of the parasite reached 34.5% in this Vietnamese cohort. No significant association was found between parasite infection and gender, age, symptomatic status, contact with animals or source of drinking water. Out of the 107 positive patients, nearly half presented mixed infections. Therefore, some of the corresponding samples were reanalyzed by end-point PCR, followed by PCR products cloning and sequencing. Of the 88 total subtyped isolates, ST3 was predominant, followed by ST10, ST14, ST7, ST1, ST4, ST6 and ST8. Our study was, thus, the first to report ST8, ST10 and ST14 in the Southeast Asian population. The predominance of ST3 within this Vietnamese cohort, coupled with its low intra-ST genetic variability, reflected a large inter-human transmission, while ST1 transmission was suggested to be not only anthroponotic, but also likely correlated to animal or environmental sources. Strikingly, isolates considered of animal origin (ST6-ST8, ST10 and ST14) accounted for more than 50% of the subtyped isolates. These findings improved our knowledge of the epidemiology and circulation of Blastocystis sp. in Southeast Asia, and in particular, in Vietnam, and highlighted both a major burden of the parasite in this country and a high risk of zoonotic transmission, mainly from poultry and livestock.

Molecular Detection and Genetic Characterization of Japanese Encephalitis Virus in Animals from 11 Provinces in China.

Japanese encephalitis virus (JEV), which uses a mosquito primary vector and swine as a reservoir host, poses a significant risk to human and animal health. JEV can be detected in cattle, goats and dogs. A molecular epidemiological survey of JEV was conducted in 3105 mammals from five species, swine, fox, racoon dog, yak and goat, and 17,300 mosquitoes from 11 Chinese provinces. JEV was detected in pigs from Heilongjiang (12/328, 3.66%), Jilin (17/642, 2.65%), Shandong (14/832, 1.68%), Guangxi (8/278, 2.88%) and Inner Mongolia (9/952, 0.94%); in goats (1/51, 1.96%) from Tibet; and mosquitoes (6/131, 4.58%) from Yunnan. A total of 13 JEV envelope (E) gene sequences were amplified in pigs from Heilongjiang (5/13), Jilin (2/13) and Guangxi (6/13). Swine had the highest JEV infection rate of any animal species, and the highest infection rates were found in Heilongjiang. Phylogenetic analysis indicated that the predominant strain in Northern China was genotype I. Mutations were found at residues 76, 95, 123, 138, 244, 474 and 475 of E protein but all sequences had predicted glycosylation sites at 'N154. Three strains lacked the threonine 76 phosphorylation site from non-specific (unsp) and protein kinase G (PKG) site predictions; one lacked the threonine 186 phosphorylation site from protein kinase II (CKII) prediction; and one lacked the tyrosine 90 phosphorylation site from epidermal growth factor receptor (EGFR) prediction. The aim of the current study was to contribute to JEV prevention and control through the characterization of its molecular epidemiology and prediction of functional changes due to E-protein mutations.

Discovery of novel Mamastroviruses in Bactrian camels and dromedaries reveals complex recombination history.

Virus emergence may occur through interspecies transmission and recombination of viruses coinfecting a host, with potential to pair novel and adaptive gene combinations. Camels are known to harbor diverse ribonucleic acid viruses with zoonotic and epizootic potential. Among them, astroviruses are of particular interest due to their cross-species transmission potential and endemicity in diverse host species, including humans. We conducted a molecular epidemiological survey of astroviruses in dromedaries from Saudi Arabia and Bactrian camels from Inner Mongolia, China. Herein, we deployed a hybrid sequencing approach coupling deep sequencing with rapid amplification of complementary deoxyribonucleic acid ends to characterize two novel Bactrian and eight dromedary camel astroviruses, including both partial and complete genomes. Our reported sequences expand the known diversity of dromedary camel astroviruses, highlighting potential recombination events among the astroviruses of camelids and other host species. In Bactrian camels, we detected partially conserved gene regions bearing resemblance to human astrovirus types 1, 4, and 8 although we were unable to recover complete reading frames from these samples. Continued surveillance of astroviruses in camelids, particularly Bactrian species and associated livestock, is highly recommended to identify patterns of cross-species transmission and to determine any epizootic threats and zoonotic risks posed to humans. Phylogenomic approaches are needed to investigate complex patterns of recombination among the astroviruses and to infer their evolutionary history across diverse host species.

Multilocus Microsatellite Analysis of the Molecular Epidemiology of Microsporum canis Isolated in Japan.

Microsporum canis, a major causative agent of zoonotic dermatophytosis, has become prevalent in Japan. Molecular epidemiological surveys using multilocus microsatellite typing (MLMT), a sensitive genotyping tool for fungi, have been conducted to reveal intraspecies polymorphisms of M. canis.The present study utilized MLMT optimized for Japanese strains of M. canis to analyze epidemic trends of fungal infection. Six individual loci were targeted; namely, MS1, 2, 4, 5, 6, and 7. Analysis of data from 1974 through 2022 identified 416 strains, which were sorted into 60 genotypes by MLMT.The major genotypes showed changes in dominance during this period-changes that may reflect historical increases and decreases in the numbers of patients infected with M. canis patients. The main origins of infection included animal breeders and pet stores, as well as stray cat communities. Forty-nine episodes of familial outbreaks and cohabitant animal infections were recorded, and genotypes responsible for each episode were determined. MLMT analysis is not only a robust tool to understand population structures, but likely the most suitable method for tracking M. canis infections.

First genetic detection and characterization of canine parvovirus type 2 (Carnivore protoparvovirus 1) in southwestern Ethiopia

The species Carnivore protoparvovirus 1 includes viruses, e.g. canine parvovirus (CPV-2) and feline panleukopenia virus (FPV), which are among the most relevant for pets, causing extremely severe clinical signs and high fatality rate in dogs and cats. Moreover, a broad range of wild hosts, including endangered ones, were proven to be susceptible. Currently, no data on CPV-2 molecular epidemiology and strain characterization are available in Ethiopia, also considering the frequent contacts between domestic and wild populations. In the present study, a molecular epidemiology survey was performed on 92 fecal samples collected from domestic (n = 84) and stray (n = 8) dogs in southwestern Ethiopia in 2021. Approximately, 10% of the samples tested positive and the complete VP2 sequences of 5 strains were obtained, classified within the CPV-2a (n = 1) and CPV-2c (n = 4) antigenic variants. In most instances, the closest genetic relatives were strains of Chinese origin, which is fully compatible with the intense relationships that have been developing between the two countries, involving human being travels and their pets as well. Considering the clinical relevance of this infection and the risk it poses to local domestic and wild carnivore populations, especially endangered ones, much stronger monitoring and surveillance activity on foreign incoming animals should be performed. More strict constraints on animal introduction, e.g. compulsory vaccination, should also be considered.

Molecular Epidemiological Survey for Degenerative Myelopathy in German Shepherd Dogs in Japan: Allele Frequency and Clinical Progression Rate.

Simple SummaryCanine degenerative myelopathy (DM) is an adult-onset, chronic, progressive neurodegenerative disease caused by a DM-associated mutation (SOD1:c.118G>A, p.E40K) that commonly occurs in German Shepherd Dogs (GSDs). This study aimed to determine the mutant allele frequency in the Japanese GSD population and to analyze the clinical progression rate among GSDs with the homozygous mutant A/A genotype. The survey found 330 G/G dogs (61%), 184 G/A dogs (34%), and 27 A/A dogs (5%) among the 541 dogs examined, indicating that the mutant allele frequency was 0.220; the Japanese GSD population can be considered to be in Hardy–Weinberg equilibrium. Clinical analysis revealed that the clinical progression rate was particularly high (100%) among A/A dogs aged >10 years. Appropriate mating management is crucial for the management and prevention of DM in the Japanese GSD population.Canine degenerative myelopathy (DM) is an adult-onset, chronic, progressive neurodegenerative disease reported in multiple canine breeds, including the German Shepherd Dog (GSD). Clinical signs include progressive motor neuron paralysis, which begins in the pelvic limbs and eventually leads to respiratory distress, which may necessitate euthanasia. A common DM-associated mutation is a single nucleotide substitution that causes an amino acid substitution (c.118G>A, p.E40K) in the canine SOD1 gene. This SOD1 mutation and the clinical progression rate of A/A risk genotype in the Japanese GSD population have not been analyzed before. Therefore, the aim of this study was to determine the frequency of the mutated allele and analyze the clinical progression rate in the Japanese GSD population. We studied 541 GSDs registered with the Japanese German Shepherd Dog Registration Society between 2000 and 2019. Genotyping was performed using real-time PCR with DNA extracted from the hair roots of each dog. The study revealed 330 G/G dogs (61%), 184 G/A dogs (34%), and 27 A/A dogs (5%), indicating a frequency of the mutant allele of 0.220, which are in Hardy–Weinberg equilibrium. We analyzed the clinical signs in A/A dogs with an age limit of 10 years based on information obtained from the dogs’ owners. Of the seven A/A dogs older than 10 years, owners reported DM-related clinical signs, indicating a clinical progression rate of 100%. These results, further genotyping, and thorough clinical examinations of SOD1 A/A risk genotype will help control and prevent DM in the Japanese GSD population.

Molecular Typing and Rapid Identification of Human Adenoviruses Associated With Respiratory Diseases Using Universal PCR and Sequencing Primers for the Three Major Capsid Genes: Penton Base, Hexon, and Fiber

Human adenoviruses (HAdVs) within species B, C, and E are responsible for highly contagious and potentially severe respiratory disease infections. The traditional method to type these pathogens was based on virus neutralization and hemagglutination assays, which are both time-consuming and difficult, particularly due to the nonavailability of reagents. Subsequent molecular typing based on the partial characterization of the hexon gene and/or the restriction enzyme analysis (REA) of the genomes is inadequate, particularly in identifying recombinants. Here, a rapid, simple, and cost-effective method for molecular typing HAdV respiratory pathogens is presented. This incorporates three pairs of universal PCR primers that target the variable regions of the three major capsid genes, i.e., hexon, penton base, and fiber genes, that span the genome. The protocol enables typing and characterization of genotypes within species B, C, and E, as well as of some genotypes within species D and F. To validate this method, we surveyed 100 children with HAdV-associated acute respiratory infections identified by direct immunofluorescence (Hong Kong; July through October, 2014). Throat swab specimens were collected and analyzed by PCR amplification and sequencing; these sequences were characterized by BLAST. HAdVs were detected in 98 out of 100 (98%) samples, distributing as follows: 74 HAdV-B3 (74%); 10 HAdV-E4 (10%); 7 HAdV-C2 (7%); 2 HAdV-C6 (2%); 1 HAdV-B7 (1%); 1 HAdV-C1 (1%); 2 co-infection (2%); and 1 novel recombinant (1%). This study is the first detailed molecular epidemiological survey of HAdVs in Hong Kong. The developed method allows for the rapid identification of HAdV respiratory pathogens, including recombinants, and bypasses the need for whole genome sequencing for real-time surveillance of circulating adenovirus strains in outbreaks and populations by clinical virologists, public health officials, and epidemiologists.

Using molecular network analysis to explore the characteristics of HIV-1 transmission in a China-Myanmar border area.

BackgroundThe China-Myanmar border area is considered a hot spot of active HIV-1 recombination in Southeast Asia. To better understand the characteristics of HIV-1 transmission in this area, a cross-sectional HIV-1 molecular epidemiological survey was conducted in Baoshan Prefecture of Yunnan Province.MethodsIn total, 708 newly reported HIV-1 cases in Baoshan Prefecture from 2019 to 2020 were included in this study. HIV-1 gag, pol and env genes were sequenced, and the spatial and demographic distributions of HIV-1 genotypes were analyzed. The characteristics of HIV-1 transmission were investigated using the HIV-1 molecular network method.ResultsIn the 497 samples with genotyping results, 19 HIV-1 genotypes were found, with URFs being the predominant strains (30.2%, 150/497). The main circulating HIV-1 strains were mostly distributed in the northern area of Baoshan. URFs were more likely identified in Burmese individuals, intravenous drug users and those younger than 50 years old. CRF08_BC was more likely detected in farmers and those of Han ethnicity, CRF01_AE in the young and those of Han ethnicity, and CRF07_BC in the subpopulation with junior middle school education and higher. Moreover, CRF118_BC and CRF64_BC were more likely found in the subpopulation aged ≥40 years and ≥50 years, respectively. Among 480 individuals with pol sequence detection, 179 (37.3%) were grouped into 78 clusters, with Baoshan natives being more likely to be in the network. The proportion of the linked individuals showed significant differences when stratified by the regional origin, marital status, age and county of case reporting. In the molecular network, recent infections were more likely to occur among nonfarmers and individuals aged below 30 years.ConclusionsHIV-1 genetics has become complex in Baoshan. HIV-1 molecular network analysis provided transmission characteristics in the local area, and these findings provided information to prioritize transmission-reduction interventions.

Molecular epidemiological survey, genetic characterization and phylogenetic analysis of Anaplasma ovis infecting sheep in Northern Egypt

Anaplasma ovis is the most common etiologic agent of ovine anaplasmosis, mainly transmitted by ticks. The present study aimed to determine the molecular prevalence of A. ovis in sheep from Egypt and assessed the associated risk factors. The study was conducted, between January and December 2020, in four governorates situated in Northern Egypt. Blood samples from 355 asymptomatic sheep were collected and examined by the use of PCR specific to A. ovis. Diversity analysis and phylogenetic study based on partial msp4 gene sequence were performed on revealed A. ovis DNA. Overall, the molecular prevalence rate of A. ovis was 15.5% and the highest rate was observed in Kafr ElSheikh governorate (16.8%). Statistical analysis revealed that A. ovis infection was significantly related to sheep gender and to tick infestation. The risk factors that were found to be associated with A. ovis infection in exposed sheep were: female sex (OR=2.6, 95%CI: 1.13–6.12), and infestation with ticks (OR=2.1, 95%CI: 1.11–3.79). The analysis of A. ovis msp4 sequences revealed two different genotypes classified in the Old World sub-cluster with other Egyptian isolates. Investigation on prevalence, risk factors and genetic variability of A. ovis in sheep reported in this study is important for the implementation of control programs. Further studies are needed to determine the vectors and reservoirs of A. ovis in Egyptian small ruminants and to identify the real economic impact of A. ovis infection on the country.

A clinical and molecular epidemiological survey of hepatitis C in Blantyre, Malawi, suggests a historic mechanism of transmission.

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. There are no previous representative community HCV prevalence studies from Southern Africa, and limited genotypic data. Epidemiological data are required to inform an effective public health response. We conducted a household census‐based random sampling serological survey, and a prospective hospital‐based study of patients with cirrhosis and hepatocellular carcinoma (HCC) in Blantyre, Malawi. We tested participants with an HCV antigen/antibody ELISA (Monolisa, Bio‐Rad), confirmed with PCR (GeneXpert, Cepheid) and used line immunoassay (Inno‐LIA, Fujiribio) for RNA‐negative participants. We did target‐enrichment whole‐genome HCV sequencing (NextSeq, Illumina). Among 96,386 censused individuals, we randomly selected 1661 people aged ≥16 years. Population‐standardized HCV RNA prevalence was 0.2% (95% CI 0.1–0.5). Among 236 patients with cirrhosis and HCC, HCV RNA prevalence was 1.9% and 5.0%, respectively. Mapping showed that HCV RNA+ patients were from peri‐urban areas surrounding Blantyre. Community and hospital HCV RNA+ participants were older than comparator HCV RNA‐negative populations (median 53 vs 30 years for community, p = 0.01 and 68 vs 40 years for cirrhosis/HCC, p < 0.001). Endemic HCV genotypes (n = 10) were 4v (50%), 4r (30%) and 4w (10%). In this first census‐based community serological study in Southern Africa, HCV was uncommon in the general population, was centred on peri‐urban regions and was attributable for <5% of liver disease. HCV infection was observed only among older people, suggesting a historic mechanism of transmission. Genotype 4r, which has been associated with treatment failure with ledipasvir and daclatasvir, is endemic.

Molecular Detection and Characterization of Blastocystis sp. and Enterocytozoon bieneusi in Cattle in Northern Spain.

Some enteric parasites causing zoonotic diseases in livestock have been poorly studied or even neglected. This is the case in stramenopile Blastocystis sp. and the microsporidia Enterocytozoon bieneusi in Spain. This transversal molecular epidemiological survey aims to estimate the prevalence and molecular diversity of Blastocystis sp. and E. bieneusi in cattle faecal samples (n = 336) in the province of Álava, Northern Spain. Initial detection of Blastocystis and E. bieneusi was carried out by polymerase chain reaction (PCR) and Sanger sequencing of the small subunit (ssu) rRNA gene and internal transcribed spacer (ITS) region, respectively. Intra-host Blastocystis subtype diversity was further investigated by next generation amplicon sequencing (NGS) of the ssu rRNA gene in those samples that tested positive by conventional PCR. Amplicons compatible with Blastocystis sp. and E. bieneusi were observed in 32.1% (108/336, 95% CI: 27.2–37.4%) and 0.6% (2/336, 95% CI: 0.0–1.4%) of the cattle faecal samples examined, respectively. Sanger sequencing produced ambiguous/unreadable sequence data for most of the Blastocystis isolates sequenced. NGS allowed the identification of 10 Blastocystis subtypes including ST1, ST3, ST5, ST10, ST14, ST21, ST23, ST24, ST25, and ST26. All Blastocystis-positive isolates involved mixed infections of 2–8 STs in a total of 31 different combinations. The two E. bieneusi sequences were confirmed as potentially zoonotic genotype BEB4. Our data demonstrate that Blastocystis mixed subtype infections are extremely frequent in cattle in the study area. NGS was particularly suited to discern underrepresented subtypes or mixed subtype infections that were undetectable or unreadable by Sanger sequencing. The presence of zoonotic Blastocystis ST1, ST3, and ST5, and E. bieneusi BEB4 suggest cross-species transmission and a potential risk of human infection/colonization.