Circular Whole-Transcriptome Amplification (cWTA) and mNGS Screening Enhanced by a Group Testing Algorithm (mEGA) Enable High-Throughput and Comprehensive Virus Identification.

Metagenomic next-generation sequencing (mNGS) offers a hypothesis-free approach for pathogen detection, but its applicability in clinical diagnosis, in addition to other factors, remains limited due to complicated library construction. The present study describes a PCR-free isothermal workflow for mNGS targeting RNA, based on a multiple displacement amplification, termed circular whole-transcriptome amplification (cWTA), as the template is circularized before amplification. The cWTA approach was validated with clinical samples and nanopore sequencing. Reads homologous to dengue virus 2 and chikungunya virus were detected in clinical samples from Bangladesh and Brazil, respectively. In addition, the practicality of a high-throughput detection system that combines mNGS and a group testing algorithm termed mNGS screening enhanced by a group testing algorithm (mEGA) was established. This approach enabled significant library size reduction while permitting trackability between samples and diagnostic results. Serum samples of patients with undifferentiated febrile illnesses from Vietnam (n = 43) were also amplified with cWTA, divided into 11 pools, processed for library construction, and sequenced. Dengue virus 2, hepatitis B virus, and parvovirus B19 were successfully detected without prior knowledge of their existence. Collectively, cWTA with the nanopore platform opens the possibility of hypothesis-free on-site comprehensive pathogen diagnosis, while mEGA contributes to the scaling up of sample throughput. IMPORTANCE Given the breadth of pathogens that cause infections, a single approach that can detect a wide range of pathogens is ideal but is impractical due to the available tests being highly specific to a certain pathogen. Recent developments in sequencing technology have introduced mNGS as an alternative that provides detection of a wide-range of pathogens by detecting the presence of their nucleic acids in the sample. However, sequencing library preparation is still a bottleneck, as it is complicated, costly, and time-consuming. In our studies, alternative approaches to optimize library construction for mNGS were developed. This included isothermal nucleic acid amplification and expansion of sample throughput with a group testing algorithm. These methods can improve the utilization of mNGS as a diagnostic tool and can serve as a high-throughput screening system aiding infectious disease surveillance.

Correlating biodegradation kinetics of 2,3,7,8-tetrachlorodibenzo-p-dioxin to the dynamics of microbial communities originating from soil in Vietnam contaminated with herbicides and dioxins.

We studied the succession of bacterial communities during the biodegradation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). The communities originated from a mesocosm with soil from Bien Hoa airbase in Vietnam heavily contaminated with herbicides and dioxins. They were grown in defined media with different carbon and Gibbs energy sources and 2,3,7,8-TCDD. Cultures with dimethyl sulfoxide (DMSO) as the sole carbon and energy source degraded about 95% of 2,3,7,8-TCDD within 60 days of cultivation. Those with an additional 1 mM of vanillin did that in roughly 90 days. Further 16S rRNA gene amplicon sequencing showed that the increase in relative abundance of members belonging to the genera Bordetella, Sphingomonas, Proteiniphilum, and Rhizobium correlated to increased biodegradation of 2,3,7,8-TCDD in these cultures. A higher concentration of vanillin slowed down the biodegradation rate. Addition of alternative carbon and Gibbs energy sources, such as amino acids, sodium lactate and sodium acetate, even stopped the degradation of 2,3,7,8-TCDD completely. Bacteria from the genera Bordetella, Achromobacter, Sphingomonas and Pseudomonas dominated most of the cultures, but the microbial profiles also significantly differed between cultures as judged by non-metric multidimensional scaling (NMDS) analyses. Our study indicates that 2,3,7,8-TCDD degradation may be stimulated by bacterial communities preadapted to a certain degree of starvation with respect to the carbon and energy source. It also reveals the succession and abundance of defined bacterial genera in the degradation process.

Degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) by fungi originating from Vietnam.

Three different fungi were tested for their ability to degrade 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid and for the role of laccases and cytochromes P450-type in this process. We studied a white-rot fungus Rigidoporus sp. FMD21, which has a high laccase activity, for its efficiency to degrade these herbicides. A positive correlation was found between its laccase activity and the corresponding herbicide degradation rate. Even more, the doubling of the enzyme activity in this phase corresponded with a doubling of the herbicide degradation rate. It is, therefore, tempting to speculate that laccase is the most dominant enzyme in the degradation of 2,4-D and 2,4,5-T under these conditions. In addition, it was shown that Rigidoporus sp. FMD21 partly relies on cytochromes P450-type for the breakdown of the herbicides as well. Two filamentous fungi were isolated from soil contaminated with herbicides and dioxins located at Bien Hoa airbase. They belong to genera Fusarium and Verticillium of the phylum Ascomycota as judged by their 18S rRNA gene sequences. Both isolated fungi were able to degrade the herbicides but with different rates. Their laccase activity, however, was very low and did not correlate with the rate of breakdown of the herbicides. These data indicate that the white-rot fungus most likely synthesizes laccase and cytochromes P450-type for the breakdown of the herbicides, while the types of enzyme used for the breakdown of the herbicides by the two Ascomycota remain unclear.

Antimicrobial Activity of Quercetin: An Approach to Its Mechanistic Principle.

Quercetin, an essential plant flavonoid, possesses a variety of pharmacological activities. Extensive literature investigates its antimicrobial activity and possible mechanism of action. Quercetin has been shown to inhibit the growth of different Gram-positive and Gram-negative bacteria as well as fungi and viruses. The mechanism of its antimicrobial action includes cell membrane damage, change of membrane permeability, inhibition of synthesis of nucleic acids and proteins, reduction of expression of virulence factors, mitochondrial dysfunction, and preventing biofilm formation. Quercetin has also been shown to inhibit the growth of various drug-resistant microorganisms, thereby suggesting its use as a potent antimicrobial agent against drug-resistant strains. Furthermore, certain structural modifications of quercetin have sometimes been shown to enhance its antimicrobial activity compared to that of the parent molecule. In this review, we have summarized the antimicrobial activity of quercetin with a special focus on its mechanistic principle. Therefore, this review will provide further insights into the scientific understanding of quercetin's mechanism of action, and the implications for its use as a clinically relevant antimicrobial agent.

Global research alliance in infectious disease: a collaborative effort to combat infectious diseases through dissemination of portable sequencing.

OBJECTIVE: To disseminate the portable sequencer MinION in developing countries for the main purpose of battling infectious diseases, we found a consortium called Global Research Alliance in Infectious Diseases (GRAID). By holding and inviting researchers both from developed and developing countries, we aim to train the participants with MinION's operations and foster a collaboration in infectious diseases researches. As a real-life example in which resources are limited, we describe here a result from a training course, a metagenomics analysis from two blood samples collected from a routine cattle surveillance in Kulan Progo District, Yogyakarta Province, Indonesia in 2019. RESULTS: One of the samples was successfully sequenced with enough sequencing yield for further analysis. After depleting the reads mapped to host DNA, the remaining reads were shown to map to Theileria orientalis using BLAST and OneCodex. Although the reads were also mapped to Clostridium botulinum, those were found to be artifacts derived from the cow genome. An effort to construct a consensus sequence was successful using a reference-based approach with Pomoxis. Hence, we concluded that the asymptomatic cow might be infected with T. orientalis and showed the usefulness of sequencing technology, specifically the MinION platform, in a developing country.

A targeted approach with nanopore sequencing for the universal detection and identification of flaviviruses.

Nucleic acid test (NAT), most typically quantitative PCR, is one of the standard methods for species specific flavivirus diagnosis. Semi-comprehensive NATs such as pan-flavivirus PCR which covers genus Flavivirus are also available; however, further specification by sequencing is required for species level differentiation. In this study, a semi-comprehensive detection system that allows species differentiation of flaviviruses was developed by integration of the pan-flavivirus PCR and Nanopore sequencing. In addition, a multiplexing method was established by adding index sequences through the PCR with a streamlined bioinformatics pipeline. This enables defining cut-off values for observed read counts. In the laboratory setting, this approach allowed the detection of up to nine different flaviviruses. Using clinical samples collected in Vietnam and Brazil, seven different flaviviruses were also detected. When compared to a commercial NAT, the sensitivity and specificity of our system were 66.7% and 95.4%, respectively. Conversely, when compared to our system, the sensitivity and specificity of the commercial NAT were 57.1% and 96.9%, respectively. In addition, Nanopore sequencing detected more positive samples (n = 8) compared to the commercial NAT (n = 6). Collectively, our study has established a semi-comprehensive sequencing-based diagnostic system for the detection of flaviviruses at extremely affordable costs, considerable sensitivity, and only requires simple experimental methods.

Antimicrobial use in household, semi-industrialized, and industrialized pig and poultry farms in Viet Nam.

The use and misuse of antimicrobials in livestock production contributes to increasing antimicrobial resistance (AMR). Antimicrobial use (AMU), has been identified as a problem in Viet Nam. There were many identified drivers of AMU in Viet Nam such as lack of access to veterinary services, easy access to cheap over-the-counter antimicrobials, and insufficient farm biosecurity. This study included chicken farmers (n = 540) and pig farmers (n = 540) from household, semi-industrialized, and industrialized farms in the North, Central, and South of Viet Nam. The objective of this study was to determine farmers rationale behind AMU on their farms and their usage patterns. On pig farms, 98.1% of the farmers reported use of antimicrobials in their production. On chicken farms, 87.9% reported use of antimicrobials in their production. The results of the survey showed that the three main purposes of AMU were treatment of sick animals, disease prevention, and weight gain. Treatment accounted for 81.3% in pig farming and 62.1% in chicken farming. The main reason to start antimicrobial therapy in pig and chicken production was observation of the first clinical signs of disease (73.9% of the pig farmers and 74.9% of chicken farmers). The proportion of industrial pig farms performing diagnostic tests before using antimicrobials was singnificantly (p < 0.05) higher than household farms (OR = 45.3). The proportion of chicken farmers who used diagnostic tests before using antimicrobials on semi-industrial (OR = 4.1) and industrial farms (OR = 26.7) were significantly higher compared with household farms. Through encouraging the prudent use of antimicrobials in animal husbandry we can reduce the use of antimicrobials at the primary production level and thereby lowering the risk of AMR.

CD8+ T cell-based strong selective pressure on multiple simian immunodeficiency virus targets in macaques possessing a protective MHC class I haplotype.

In human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections, host major histocompatibility complex class I (MHC-I) genotypes have a great impact on viral replication and MHC-I-associated viral genome mutations are selected under CD8+ T-cell pressure. Association of MHC-I genotypes with HIV/SIV control has been investigated at MHC-I allele levels but not fully at haplotype levels. We previously established groups of rhesus macaques sharing individual MHC-I haplotypes. In the present study, we compared viral genome diversification after SIV infection in macaques possessing a protective MHC-I haplotype, 90-010-Id, with those possessing a non-protective MHC-I haplotype, 90-010-Ie. These two MHC-I haplotypes are associated with immunodominant CD8+ T-cell responses targeting similar regions of viral Nef antigen. Analyses of viral genome sequences and antigen-specific T-cell responses showed four and two candidates of viral CD8+ T-cell targets associated with 90-010-Id and 90-010-Ie, respectively, in addition to the Nef targets. In these CD8+ T-cell target regions, higher numbers of mutations were detected at the setpoint after SIV infection in macaques possessing 90-010-Id than those possessing 90-010-Ie. These results indicate higher selective pressure on overall CD8+ T-cell targets associated with the protective MHC-I haplotype, suggesting a pattern of HIV/SIV control by multiple target-specific CD8+ T-cell responses.

Human leukocyte antigen-associated gag and nef polymorphisms in HIV-1 subtype A/E-infected individuals in Vietnam.

Numbers of HLA-associated polymorphisms have been reported on HIV-1 subtypes B and C, but few on other subtypes. Here, we analyzed HLA-associated gag and nef polymorphisms in HIV-1 subtype A/E prevalent in Vietnam. We determined HLA-A, B and C genotypes in 179 HIV-1-infected Vietnamese by next generation sequencing and analyzed proviral genome sequences in 144 of them, showing that 142 of the 144 were subtype A/E. Analysis revealed HLA-associated subtype A/E gag and nef polymorphisms at nineteen residues including those newly determined. Accumulation of these data would contribute to our understanding of HIV-1 subtype A/E and host immune interaction.

Nanopore sequencing of drug-resistance-associated genes in malaria parasites, Plasmodium falciparum.

Here, we report the application of a portable sequencer, MinION, for genotyping the malaria parasite Plasmodium falciparum. In the present study, an amplicon mixture of nine representative genes causing resistance to anti-malaria drugs is diagnosed. First, we developed the procedure for four laboratory strains (3D7, Dd2, 7G8, and K1), and then applied the developed procedure to ten clinical samples. We sequenced and re-sequenced the samples using the obsolete flow cell R7.3 and the most recent flow cell R9.4. Although the average base-call accuracy of the MinION sequencer was 74.3%, performing >50 reads at a given position improves the accuracy of the SNP call, yielding a precision and recall rate of 0.92 and 0.8, respectively, with flow cell R7.3. These numbers increased significantly with flow cell R9.4, in which the precision and recall are 1 and 0.97, respectively. Based on the SNP information, the drug resistance status in ten clinical samples was inferred. We also analyzed K13 gene mutations from 54 additional clinical samples as a proof of concept. We found that a novel amino-acid changing variation is dominant in this area. In addition, we performed a small population-based analysis using 3 and 5 cases (K13) and 10 and 5 cases (PfCRT) from Thailand and Vietnam, respectively. We identified distinct genotypes from the respective regions. This approach will change the standard methodology for the sequencing diagnosis of malaria parasites, especially in developing countries.

Prevention and control of fish-borne zoonotic trematodes in fish nurseries, Vietnam.

Worldwide, >18 million persons were infected with fish-borne zoonotic trematodes in 2002. To evaluate the effectiveness of interventions for reducing prevalence and intensity of fish-borne zoonotic trematode infections in juvenile fish, we compared transmission rates at nurseries in the Red River Delta, northern Vietnam. Rates were significantly lower for nurseries that reduced snail populations and trematode egg contamination in ponds than for nurseries that did not. These interventions can be used in the development of programs for sustained control of zoonotic trematodes in farmed fish.

Hepatitis C virus in Vietnam: high prevalence of infection in dialysis and multi-transfused patients involving diverse and novel virus variants.

Hepatitis C virus (HCV) is a genetically diverse pathogen infecting approximately 2-3% of the world's population. Herein, we describe results of a large, multicentre serological and molecular epidemiological study cataloguing the prevalence and genetic diversity of HCV in five regions of Vietnam; Ha Noi, Hai Phong, Da Nang, Khanh Hoa and Can Tho. Individuals (n=8654) with varying risk factors for infection were analysed for the presence of HCV Ab/Ag and, in a subset of positive specimens, for HCV RNA levels (n=475) and genotype (n=282). In lower risk individuals, including voluntary blood donors, military recruits and pregnant women, the prevalence of infection was 0.5% (n=26/5250). Prevalence rates were significantly higher (p<0.001) in intravenous drug users (IDUs; 55.6%, n=556/1000), dialysis patients (26.6%, n=153/575) commercial sex workers (CSWs; 8.7%, n=87/1000), and recipients of multiple blood transfusions (6.0%, n=32/529). The prevalence of HCV in dialysis patients varied but remained high in all regions (11-43%) and was associated with the receipt of blood transfusions [OR: 2.08 (1.85-2.34), p=0.001], time from first transfusion [OR: 1.07 (1.01-1.13), p=0.023], duration of dialysis [OR: 1.31 (1.19-1.43), p<0.001] and male gender [OR: 1.60 (1.06-2.41), p=0.026]. Phylogenetic analysis revealed high genetic diversity, particularly amongst dialysis and multi-transfused patients, identifying subtypes 1a (33%), 1b (27%), 2a (0.4%), 3a (0.7%), 3b (1.1%), 6a (18.8%), 6e (6.0%), 6h (4.6%), 6l (6.4%) and 2 clusters of novel genotype 6 variants (2.1%). HCV genotype 1 predominated in Vietnam (60%, n=169/282) but the proportion of infections attributable to genotype 1 varied between regions and risk groups and, in the Southern part of Vietnam, genotype 6 viruses dominated in dialysis and multi-transfused patients (73.9%). This study confirms a high prevalence of HCV infection in Vietnamese IDUs and, notably, reveals high levels of HCV infection associated with dialysis and blood transfusion.

HIV type 1 coreceptor tropism, CCR5 genotype, and integrase inhibitor resistance profiles in Vietnam: implications for the introduction of new antiretroviral regimens.

In Vietnam, where an estimated 280,000 people will be HIV-positive by 2012, recommended antiretroviral regimens do not include more recently developed therapeutics, such as Integrase inhibitors (INI) and coreceptor antagonists. This study examined HIV-1 coreceptor tropism and INI drug resistance profiles, in parallel with CCR5 genotypes, in a cohort of 60 HIV-positive individuals from different regions of Vietnam. No evidence of INI resistance was detected. Some 40% of individuals had X4-tropic HIV-1, making them unsuitable for treatment with CCR5 antagonists. We identified a novel CCR5 variant-S272P-along with other, previously reported variants: G106R, C178R, W153C, R223Q, and S336I. Interestingly, CCR5 variants known to affect HIV-1 infectivity were observed only in individuals harboring X4-tropic virus. Together, this study presents valuable baseline information on HIV-1 INI resistance, coreceptor tropism, and CCR5 variants in HIV-positive individuals in Vietnam. This should help inform policy on the future use of novel antiretrovirals in Vietnam.

Prevalence of HIV type 1 antiretroviral drug resistance mutations in Vietnam: a multicenter study.

The prevalence of HIV-1 drug resistance mutations (DRMs) was determined for a cross-section of individuals (n=8654) in five centers across Vietnam (Hanoi, Hai Phong, Da Nang, Khanh Hoa, and Can Tho) between 2008 and 2009. Following serological screening for HIV infection, HIV-1 viral load was determined, using an in-house real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay. Samples with quantifiable viral loads [all either commercial sex workers (CSW) or intravenous drug users (IDU)] underwent DRM analysis. Sequences were obtained for 92 treatment-naive individuals, the majority of whom were infected with HIV-1 CRF01_AE (99%), with one instance of subtype A1 also detected. DRMs were detected in seven treatment-naive individuals (7.6%). The most common DRMs observed were M184V, V75A/M, M41L, and K65R (NRTI) and K103N, G190A, and Y181C (NNRTI). Overall, the data from this first multicenter survey of DRMs in Vietnam indicate that the problem of transmitted drug resistance is of major concern in the highest-risk groups of IDU and CSW.

Prevalence and risks for fishborne zoonotic trematode infections in domestic animals in a highly endemic area of North Vietnam.

Fishborne zoonotic trematodes (FZT) are endemic in humans and cultured fish in Vietnam but little is known about FZT in domestic animals. A study was designed to determine FZT prevalence and species diversity, and risk factors for infection, in dogs, cats and pigs. Faecal samples from 186 dogs, 94 cats and 168 pigs belonging to 132 households in Nghia Hung district, Nam Dinh province, were examined for small trematode eggs; those were trematode eggs with length less than 50 microm. Prevalence of FZT varied significantly between cats (70.2%), dogs (56.9%) and pigs (7.7%). Forty-nine of the egg-positive animals (25 dogs, 20 cats and 4 pigs) were necropsied to obtain adult trematodes for identification. The liver fluke, Clonorchis sinensis, and 11 species of intestinal flukes including Haplorchis, Stellantchasmus, Stictodora and Centrocestus were recovered from the infected animals. The practice of feeding raw fish to the animals was a significant risk factor for infection; this risk was reduced if the animals were periodically treated with anthelmintics. Based on the high prevalence of FZT and certain risky husbandry practices, domestic animals are likely to be major contributors of FZT eggs to the environment. Therefore, education of farmers to avoid feeding raw fish and to perform regular anthelmintic treatment of dogs, cats and pigs is needed in integrated FZT control programs.