Nucleic Acid Preservation Research Articles

The Preservation of HBV, HCV, HIV Viral Nucleic Acids in Plasma by Dry Spot Method and the Duration of Preservation

To establish a method for preserving viral nucleic acids in plasma using a blood collection card based on the dry spot method, to predict the duration of nucleic acid preservation by establishing the Arrhenius equation, and to demonstrate the feasibility of this preservation method for the re-testing of nucleic acids in blood samples retained by blood banks. Plasma samples positive for HBV, HCV, and HIV nucleic acids were prepared into preservation cards in the form of dry plasma spots for storage. The prepared preservation cards were placed under accelerated storage conditions at 37, 45, 50, and 55 ℃. The preservation cards were periodically retrieved from each temperature condition for nucleic acid extraction, and the nucleic acid samples were purified for subsequent PCR testing, with the recorded CT values. An Arrhenius equation model was established between the expiration time and the storage temperature, thereby predicting the validity period of nucleic acid preservation in blood collection cards under specified storage temperature conditions. For the plasma samples positive for HBV, HCV, and HIV nucleic acids preserved using the dry spot method, the regression equations for the duration with temperature were as follows: y=-11546 x + 31.74 for HBV, y=-12949x + 36.88 for HCV, and y=-12204x + 34.48 for HIV, with the correlation coefficient r greater than 0.98 for all. It was predicted that at a storage temperature of 4 ℃, the preservation periods for HBV, HCV, and HIV viral nucleic acids using the dry spot method would be 20 792 days, 19 289 days, and 14 285 days, respectively. At a storage temperature of 20 ℃, the preservation periods would be 2 135 days 1 502 days, and 1 289 days, respectively. The nucleic acids of the three common viral pathogens in blood samples, when preserved using the dry spot method, conform to a first-order reaction pattern in the accelerated degradation experiment. The relationship between the rate of nucleic acid degradation and the absolute temperature of storage is consistent with the Arrhenius equation. Based on the calculations using this equation, the stability and validity period of plasma nucleic acid samples preserved using the dry spot method can reach a minimum of 3.5 years under storage conditions not exceeding 20 ℃, which essentially meets the requirements for the preservation period of blood samples retained by blood banks.

Cryo-X-Ray Phase Contrast Imaging Enables Combined 3D Structural Quantification and Nucleic Acid Analysis of Myocardial Biopsies.

Snap-frozen biopsies serve as a valuable clinical resource of archival material for disease research, as they enable a comprehensive array of downstream analyses to be performed, including extraction and sequencing of nucleic acids. Obtaining three-dimensional (3D) structural information before multi-omics is more challenging but can potentially allow for better characterization of tissues and targeting of clinically relevant cells. Conventional histological techniques are limited in this regard due to their destructive nature and the reconstruction artifacts produced by sectioning, dehydration, and chemical processing. These limitations are particularly notable in soft tissues such as the heart. In this study, the feasibility of using synchrotron-based cryo-X-ray phase contrast imaging (cryo-X-PCI) of snap-frozen myocardial biopsies is assessed and 3D structure tensor analysis of aggregated myocytes, followed by nucleic acid (DNA and RNA) extraction and analysis. It is shown that optimal sample preparation is the key driver for successful structural and nucleic acid preservation which is unaffected by the process of cryo-X-PCI. It is proposed that cryo-X-PCI has clinical value for 3D tissue analysis of cardiac and potentially non-cardiac soft tissue biopsies before nucleic acid investigation.

Reversible Nucleic Acid Storage in Deconstructable Glassy Polymer Networks.

The rapid decline in DNA sequencing costs has fueled the demand for nucleic acid collection to unravel genomic information, develop treatments for genetic diseases, and track emerging biological threats. Current approaches to maintaining these nucleic acid collections hinge on continuous electricity for maintaining low-temperature and intricate cold-chain logistics. Inspired by the millennia-long preservation of fossilized biological specimens in calcified minerals or glassy amber, we present Thermoset-REinforced Xeropreservation (T-REX): a method for storing DNA in deconstructable glassy polymer networks. Key to T-REX is the development of polyplexes for nucleic acid encapsulation, streamlining the transfer of DNA from aqueous to organic phases, replete with initiators, monomers, cross-linkers, and thionolactone-based cleavable comonomers required to form the polymer networks. This process successfully encapsulates DNA that spans different length scales, from tens of bases to gigabases, in a matter of hours compared to days with traditional silica-based encapsulation. Further, T-REX permits the extraction of DNA using comparatively benign reagents, unlike the hazardous hydrofluoric acid required for recovery from silica. T-REX provides a path toward low-cost, time-efficient, and long-term nucleic acid preservation for synthetic biology, genomics, and digital information storage, potentially overcoming traditional low-temperature storage challenges.

Cold-chain free nucleic acid preservation using porous super-absorbent polymer (PSAP) beads to facilitate wastewater surveillance

The instability of viral targets including SARS-CoV-2 in sewage is an important challenge in wastewater monitoring projects. The unrecognized interruptions in the ‘cold-chain’ transport from the sample collection to RNA quantification in the laboratory may undermine the accurate quantification of the virus. In this study, bovine serum albumin (BSA)-modified porous superabsorbent polymer (PSAP) beads were applied to absorb raw sewage samples as a simple method for viral RNA preservation. The preservation efficiency for SARS-CoV-2 and pepper mild mottle virus (PMMoV) RNA were examined during storage for 14 days at 4 °C or room temperature against the control (no beads applied). While a non-significant difference was observed at 4 °C (∼80 % retention for both control and PSAP-treated sewage), the reduction of SARS-CoV-2 RNA concentrations was significantly lower in sewage retrieved from PSAP beads (25–40 % reduction) compared to control (>60 % reduction) at room temperature. On the other hand, the recovery of PMMoV, known for its high persistence in raw sewage, from PSAP beads or controls were consistently above 85 %, regardless of the storage temperature. Our results demonstrate the applicability of PSAP beads to wastewater-based epidemiology (WBE) projects for preservation of SARS-CoV-2 RNA in sewage, especially in remote settings with no refrigeration capabilities.

Evaluation of long-term preservation methods for viral RNA in mosquitoes at room temperature

Mosquitoes are important vectors of various pathogenic viruses. Almost all viruses transmitted by mosquitoes are RNA viruses. Therefore, to detect viral genes, mosquito samples must be kept at low temperatures to prevent RNA degradation. However, prolonged transport from the field to laboratory can pose challenges for temperature control. The aim of this study was to evaluate methods for preserving viral RNA in mosquito bodies at room temperature. Virus-infected mosquito samples were immersed in ethanol, propylene glycol, and a commercially available nucleic acid preservation reagent at room temperature, and viral RNA stability was compared. As a result, for the two RNA viruses (San Gabriel mononegavirus and dengue virus 1) subjected to this experiment, no significant decrease in the viral RNA was observed for at least eight weeks after immersion in the reagents, and the amount of RNA remaining was equivalent to that of samples stored at − 80 °C. These results indicate that immersion storage in these reagents used in this study is effective in preserving viral RNA in mosquitoes under room temperature conditions and is expected to be implemented in epidemiologic surveillance that is not limited by the cold chain from the field to the laboratory.

Zinc-based fixative as a novel approach for histological preservation: A comparative study with formalin-based fixatives.

Formalin is a widely used histological fixative despite its carcinogenic properties, inadequate nucleic acid preservation, and prolonged fixation time. The study intended to prepare a novel zinc-based fixative that is, formalin-free, and cost-effective, provides optimal preservation, with rapid penetration rates, and enhanced Hematoxylin/Eosin-stained sections. Liver, kidney, brain, and small intestine specimens were collected from 10 adult laboratory rats (Rattus norvegicus) and preserved in formalin 4%, formalin 10%, neutral buffered formalin (NBF), and the experimental EZAT solution. The penetration depth (discolored edges) was measured at 6, 12, 24, and 48 hours. The shrinkage ratio was calculated by measuring the width and length of the samples before and after 48 hours of fixation. The histological evaluation, included staining affinity, cellular outline, cytoplasmic and nuclear attributes, and overall tissue structure, was conducted by a panel of academic experts and rated using a scale of poor (1), fair (2), good (3), very good (4), and excellent (5) grades. The data were later statistically analyzed to determine the significant differences among the tested fixative types. A higher penetration rate was observed with the EZAT solution at 6 and 12 hours' time and the samples reached optimal fixation after 24 hours; with an accelerated diffusion coefficient, and a minimal shrinking effect compared to formalin 10% and NBF. The microscopic evaluation of hematoxylin/eosin-stained sections revealed better staining affinity, refined histological details, and enhanced cytoplasmic and nuclear properties. The overall structural evaluation revealed an excellent microscopic appearance with the EZAT solution compared to formalin-based fixatives. The EZAT fixative should be considered as an everyday preservative in histology and histopathology laboratories. Future studies should be focused on the potential of EZAT in cellular histochemistry and immunohistochemistry practices.

Comparison of different DNA preservation solutions for oral cytological samples

ObjectiveThe objective of this study was to compare the DNA preservation capacity of buccal mucosa exfoliated cells when stored in different solutions under varying time and temperature conditions. DesignDNA preservation solutions, including Dimethyl sulphoxide disodium-EDTA-saturated NaCl (DESS), Tris-EDTA-NaCl-Tween20 buffer (TENT), Nucleic Acid Preservation Buffer (NAP), and phosphate-buffered saline (PBS), were prepared. Buccal mucosa cells from a single patient were collected, dispensed into these solutions, and stored at room temperature (RT) and 4 °C for 24 h, 72 h, 30 days, 90 days, and 180 days. DNA was extracted using the salting-out method and the QIAamp DNA Mini Kit. DNA concentration and purity were determined using the QuBit device and NanoDrop, while DNA integrity was assessed using the Agilent 4200 TapeStation system. The ability to amplify the IFNA primer was also evaluated by PCR. ResultsThe salting-out method yielded better concentration and purity results, with PBS, TENT, and DESS buffers demonstrating superior concentration values when stored at 4 °C, resulting in mean values exceeding 10 ng/μL for up to 30 days. DESS consistently exhibited the best integrity values over time for both temperature conditions. Amplification capacity was enhanced when samples were stored at 4 °C. When stored at RT, PBS achieved 100% amplification within 24 h. NAP yielded the poorest results. ConclusionIn the context of long-term preservation, the DESS buffer emerges as the most effective solution, maintaining requisite DNA quality and quantity standards for up to 30 days at RT and up to 3 months at 4 °C.

Homemade Nucleic Acid Preservation Buffer Proves Effective in Preserving the Equine Faecal Microbiota over Time at Ambient Temperatures.

The equine faecal microbiota is often assessed as a proxy of the microbial community in the distal colon, where the microbiome has been linked to states of health and disease in the horse. However, the microbial community structure may change over time if samples are not adequately preserved. This study stored equine faecal samples from n = 10 horses in four preservation treatments at room temperature for up to 150 h and assessed the resulting impact on microbial diversity and the differential abundance of taxa. Treatments included "COLD" (samples packaged with a cool pack), "CLX" (2% chlorhexidine digluconate solution), "NAP" (nucleic acid preservation buffer), and "FTA" (Whatman FTA™ cards). The samples were assessed using 16S rRNA gene sequencing after storage for 0, 24, 72, and 150 h at room temperature under the different treatments. The results showed effective preservation of diversity and community structure with NAP buffer but lower diversity (p = 0.001) and the under-representation of Fibrobacterota in the FTA card samples. The NAP treatment inhibited the overgrowth of bloom taxa that occurred by 72 h at room temperature. The COLD, CLX, and NAP treatments were effective in preserving the faecal microbiota for up to 24 h at room temperature, and the CLX and NAP treatments improved the yield of Patescibacteria and Fibrobacterota in some cases. The cold and CLX treatments were ineffective in preventing community shifts that occurred by 72 h at room temperature. These findings demonstrate the suitability of the COLD, NAP, and CLX treatments for the room temperature storage of equine faeces for up to 24 h and of NAP buffer for up to 150 h prior to processing.

Detecting human cytomegalovirus in urine, vagina and saliva: Impact of biological fluids and storage durations and temperatures on CMV DNA recovery.

Sample collection, transport and storage conditions vary in the human cytomegalovirus (CMV) shedding literature. Currently, limited data exist on the impact of biological fluids and pre-analytical sample handling on the detection of CMV DNA. To evaluate CMV DNA recovery from urine, vaginal fluid and saliva stored in different conditions, adult urine, vaginal and saliva fluids and swabs, stored with or without selected nucleic acid preservation media at various durations and temperatures, was compared by polymerase chain reaction (PCR) quantitation of spiked samples and self-collected urine (n = 45) and vaginal swabs (n = 58) from CMV seropositive pregnant women. There was a time-dependent reduction in CMV DNA recovery from urine, urine diluted in phosphate-buffered saline, and saliva stored at 2-8°C, but not from urine preserved in cobas® PCR transport media (CPM) (urine/CPM). For vaginal fluid, a reduction in recovery was evident after 7 days storage at 2-8°C. CMV DNA recovery over 91 days was similar between -80°C and -20°C storage for urine and vaginal swabs preserved in CPM, and saliva swabs preserved in eNAT® PCR transport media. A statistically significant change in CMV DNA recovery after 25 months storage (median) at -80°C was not observed for self-collected urine/CPM and vaginal swab/CPM from pregnant women. Taken together, recovery of CMV DNA is dependent on fluid type and storage conditions. To improve the validity and reliability of detection at different storage durations and temperatures, the use of nucleic acid preserving transport media at the point of collection for urine, vaginal fluid and saliva may be essential.

Genotype heterogeneity of high-risk human papillomavirus infection in Ethiopia.

Cervical cancer is a vaccine-preventable sexually transmitted disease. In the year 2020, there were an estimated 604,000 new cases and 342,000 deaths worldwide. Although its incidence is global, it is much higher in sub-Saharan African countries. In Ethiopia, there is a scarcity of data about the prevalence of high-risk HPV infection and its association with cytological profiles. Therefore, this study was conducted to fill this information gap. A hospital-based cross-sectional study was conducted from April 26 to August 28, 2021, and enrolled 901 sexually active women. Socio-demographic and other relevant bio-behavioral and clinical data were collected using a standardized questionnaire. Visual inspection with acetic acid [VIA] was done as an initial screening method for cervical cancer. The cervical swab was then collected using L-Shaped FLOQSwabs in eNAT nucleic acid preservation and transportation medium. A Pap test was done to determine the cytological profile. Nucleic acid was extracted using STARMag 96 ProPrep Kit on SEEPREP32. A Real-time multiplex assay was performed to amplify and detect the HPV L1 gene used for genotyping. The data were entered into Epi data version 3.1 software and exported to STATA version 14 for analysis. A total of 901 (age range from 30 to 60 years, mean age = 34.8 years, and SD± 5.8) women were screened for cervical cancer using VIA and 832 women had a valid co-testing (Pap test and HPV DNA testing) results for further process. The overall prevalence of hr HPV infection was 13.1%. Out of 832 women, 88% of them had normal and 12% had abnormal Pap test results. The proportion of high risk HPV was significantly higher among women with abnormal cytology (X 2 = 688.446, p < 0.001) and younger age (X 2 = 15.3408, p = 0.018). Among 110 women with hr HPV, 14 genotypes (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, and -68) were identified while HPV-16, -31, -52, -58, and -35 genotypes were highly prevalent. The high risk HPV infection continues to be a significant public health problem among women 30-35 years old. The presence of high-risk HPV irrespective of genotypes is highly correlated with cervical cell abnormalities. Genotype heterogeneity is observed suggesting the importance of periodic geospatial genotyping surveillance for vaccine effectiveness.

Nucleic Acid Preservation Card Surveillance Is Effective for Monitoring Arbovirus Transmission on Crocodile Farms and Provides a One Health Benefit to Northern Australia

The Kunjin strain of West Nile virus (WNVKUN) is a mosquito-transmitted flavivirus that can infect farmed saltwater crocodiles in Australia and cause skin lesions that devalue the hides of harvested animals. We implemented a surveillance system using honey-baited nucleic acid preservation cards to monitor WNVKUN and another endemic flavivirus pathogen, Murray Valley encephalitis virus (MVEV), on crocodile farms in northern Australia. The traps were set between February 2018 and July 2020 on three crocodile farms in Darwin (Northern Territory) and one in Cairns (North Queensland) at fortnightly intervals with reduced trapping during the winter months. WNVKUN RNA was detected on all three crocodile farms near Darwin, predominantly between March and May of each year. Two of the NT crocodile farms also yielded the detection of MVE viral RNA sporadically spread between April and November in 2018 and 2020. In contrast, no viral RNA was detected on crocodile farms in Cairns during the entire trapping period. The detection of WNVKUN and MVEV transmission by FTATM cards on farms in the Northern Territory generally correlated with the detection of their transmission to sentinel chicken flocks in nearby localities around Darwin as part of a separate public health surveillance program. While no isolates of WNVKUN or MVEV were obtained from mosquitoes collected on Darwin crocodile farms immediately following the FTATM card detections, we did isolate another flavivirus, Kokobera virus (KOKV), from Culex annulirostris mosquitoes. Our studies support the use of the FTATM card system as a sensitive and accurate method to monitor the transmission of WNVKUN and other arboviruses on crocodile farms to enable the timely implementation of mosquito control measures. Our detection of MVEV transmission and isolation of KOKV from mosquitoes also warrants further investigation of their potential role in causing diseases in crocodiles and highlights a “One Health” issue concerning arbovirus transmission to crocodile farm workers. In this context, the introduction of FTATM cards onto crocodile farms appears to provide an additional surveillance tool to detect arbovirus transmission in the Darwin region, allowing for a more timely intervention of vector control by relevant authorities.

Biosafety risk assessment and risk control of clinical laboratory in designated hospitals for treating COVID-19 in Chongqing, China

BackgroundIf a nucleic acid preservation solution containing viral inactivators is used, the biosafety risk in the process of detecting the nucleic acid of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will be low. Patients infected with SARS-CoV-2 are sent to designated hospitals for treatment in China, except for detecting nucleic acid of SARS-CoV-2, other laboratory tests such as bacterial culture may also be carried out while the patients are being treated. However, in addition to nucleic acid testing, biosafety risks in the testing of these items for patients with coronavirus disease 2019 (COVID-19) might be ignored. Therefore, we identified and evaluated risks in these detection processes and formulated appropriate, but not excessive control measures for biosafety risk, to improve the work efficiency and prevent biosafety accidents. MethodsBiosafety risks in all laboratory tests for COVID-19 patients were identified and evaluated according to the risk severity and occurrence probability. Subsequently, the corresponding control measures for biosafety risk were formulated according to the identified risk. Hereafter, risk monitoring was carried out. ResultsMore than 32 risks in the entire laboratory testing process were identified and evaluated, and the residual risk after the implementation of the control measures was acceptable. ConclusionsThe biosafety risk assessment of laboratories in designated hospitals for treating COVID-19 should be re-implemented before testing specimens for COVID-19 patients. Risk management by risk monitoring is even more important, as it can prevent the occurrence of biosafety incidents and can continuously improve risk management.

Molecular Evidence of Orthomyxovirus Presence in Colombian Neotropical Bats.

The Orthomyxoviridae family includes the genera Influenzavirus, Isavirus, Quaranjavirus, and Thogotovirus. In turn, Influenzavirus can be classified into four types: α, β, γ, and δ (Formerly A, B, C, and D), from which Alphainfluenzavirus (AIV) has the broadest host range, including birds, mammals, reptiles, and amphibians. Additionally, AIV has shown global epidemiological relevance owing to its pandemic potential. The epidemiological relevance of Chiropteran due to its multiple functional characteristics makes them ideal reservoirs for many viral agents. Recently, new influenza-like subtypes have been reported in Neotropical bats, but little is known about the relevance of bats as natural reservoirs of influenza viruses. Therefore, the current study aimed to determine the presence of AIV and new influenza-like subtypes in South American bats. For a better understanding of the drivers and interactions between AIV and bats, we used molecular assays with different gene targets (i.e., M, NP, and PB1) to identify AIV in New World bats. A housekeeping gene (CytB) PCR was used to check for nucleic acid preservation and to demonstrate the bat-origin of the samples. A total of 87 free-living bats belonging to 25 different species of the families Phyllostomidae and Vespertilionidae were collected in Casanare, Colombia. As a result, this study found seven AIV-positive bat species, three of them reported for the first time as AIV prone hosts. Neither of the AIV-like analyzed samples were positive for H17N10/H18/N11 subtypes. Although additional information is needed, the presence of a completely new or divergent AIV subtype in neotropical bats cannot be discarded. Collectively, the results presented here expand the epidemiological knowledge and distribution of AIV in neotropical free-ranging bats and emphasize the need to continue studying these viruses to establish the role they could play as a threat to animal and public health.

Deep Learning Predicts EBV Status in Gastric Cancer Based on Spatial Patterns of Lymphocyte Infiltration.

Simple SummaryEBV infection represents a distinct subtype in gastric cancer, so determining infection status is important in guiding treatment decisions. Currently, EBV infection in gastric cancer is most often determined using PCR and in situ hybridization, which requires multiple steps and nucleic acid preservation. On the other hand, histopathology images are widely available and included in the course of diagnosis for patients. Thus, our development of an approach to determine EBV status from these histopathology images could save costs and time associated with making EBV diagnoses for gastric cancer patients or independently validate the results from traditional methods. Additionally, our model’s predictions are able to classify patients into EBV infection categories that are significantly correlated with prognosis. This may serve to better inform clinicians’ decisions in prescribing immunotherapy, as both EBV infection status and prognosis are critical factors in whether immunotherapy is effective or worth the costs and side effects.EBV infection occurs in around 10% of gastric cancer cases and represents a distinct subtype, characterized by a unique mutation profile, hypermethylation, and overexpression of PD-L1. Moreover, EBV positive gastric cancer tends to have higher immune infiltration and a better prognosis. EBV infection status in gastric cancer is most commonly determined using PCR and in situ hybridization, but such a method requires good nucleic acid preservation. Detection of EBV status with histopathology images may complement PCR and in situ hybridization as a first step of EBV infection assessment. Here, we developed a deep learning-based algorithm to directly predict EBV infection in gastric cancer from H&E stained histopathology slides. Our model can not only predict EBV infection in gastric cancers from tumor regions but also from normal regions with potential changes induced by adjacent EBV+ regions within each H&E slide. Furthermore, in cohorts with zero EBV abundances, a significant difference of immune infiltration between high and low EBV score samples was observed, consistent with the immune infiltration difference observed between EBV positive and negative samples. Therefore, we hypothesized that our model’s prediction of EBV infection is partially driven by the spatial information of immune cell composition, which was supported by mostly positive local correlations between the EBV score and immune infiltration in both tumor and normal regions across all H&E slides. Finally, EBV scores calculated from our model were found to be significantly associated with prognosis. This framework can be readily applied to develop interpretable models for prediction of virus infection across cancers.

Comparing sediment preservation methods for genomic biomonitoring of coastal marine ecosystems

To avoid loss of genetic information in environmental DNA (eDNA) field samples, the preservation of nucleic acids during field sampling is a critical step. In the development of standard operating procedures (SOPs) for eDNA-based compliance monitoring, the effect of different routinely used sediment preservations on biological community structures serving as bioindicators has gone untested. We compared eDNA metabarcoding results of marine bacterial communities from sample aliquots that were treated with a nucleic acid preservation solution (treated samples) and aliquots that were frozen without further treatment (non-treated samples). Sediment samples were obtained from coastal locations subjected to different stressors (aquaculture, urbanization, industry). DNA extraction efficiency, bacterial community profiles, and measures of alpha- and beta-diversity were highly congruent between treated and non-treated samples. As both preservation methods provide the same relevant information to environmental managers and regulators, we recommend the inclusion of both methods into SOPs for biomonitoring in marine coastal environments.

A Sanger sequencing protocol for SARS-CoV-2 S-gene.

We describe a Sanger sequencing protocol for SARS‐CoV‐2 S‐gene the Spike (S)‐glycoprotein product of which, composed of receptor‐binding (S1) and membrane fusion (S2) segments, is the target of vaccines used to combat COVID‐19. The protocol can be used in laboratories with basic Sanger sequencing capabilities and allows rapid “at source” screening for SARS‐CoV‐2 variants, notably those of concern. The protocol has been applied for surveillance, with clinical specimens collected in either nucleic acid preservation lysis‐mix or virus transport medium, and research involving cultured viruses, and can yield data of public health importance in a timely manner.

Fecal sample collection methods and time of day impact microbiome composition and short chain fatty acid concentrations

Associations between the human gut microbiome and health outcomes continues to be of great interest, although fecal sample collection methods which impact microbiome studies are sometimes neglected. Here, we expand on previous work in sample optimization, to promote high quality microbiome data. To compare fecal sample collection methods, amplicons from the bacterial 16S rRNA gene (V4) and fungal (ITS2) region, as well as short chain fatty acid (SCFA) concentrations were determined in fecal material over three timepoints. We demonstrated that spot sampling of stool results in variable detection of some microbial members, and inconsistent levels of SCFA; therefore, sample homogenization prior to subsequent analysis or subsampling is recommended. We also identify a trend in microbial and metabolite composition that shifts over two consecutive stool collections less than 25 h apart. Lastly, we show significant differences in bacterial composition that result from collecting stool samples in OMNIgene·Gut tube (DNA Genotec) or Stool Nucleic Acid Collection and Preservation Tube (NORGEN) compared to immediate freezing. To assist with planning fecal sample collection and storage procedures for microbiome investigations with multiple analyses, we recommend participants to collect the first full bowel movement of the day and freeze the sample immediately after collection.

Microbiota identified from preserved Anopheles

BackgroundMosquito species from the Anopheles gambiae complex and the Anopheles funestus group are dominant African malaria vectors. Mosquito microbiota play vital roles in physiology and vector competence. Recent research has focused on investigating the mosquito microbiota, especially in wild populations. Wild mosquitoes are preserved and transported to a laboratory for analyses. Thus far, microbial characterization post-preservation has been investigated in only Aedes vexans and Culex pipiens. Investigating the efficacy of cost-effective preservatives has also been limited to AllProtect reagent, ethanol and nucleic acid preservation buffer. This study characterized the microbiota of African Anopheles vectors: Anopheles arabiensis (member of the An. gambiae complex) and An. funestus (member of the An. funestus group), preserved on silica desiccant and RNAlater® solution.MethodsMicrobial composition and diversity were characterized using culture-dependent (midgut dissections, culturomics, MALDI-TOF MS) and culture-independent techniques (abdominal dissections, DNA extraction, next-generation sequencing) from laboratory (colonized) and field-collected mosquitoes. Colonized mosquitoes were either fresh (non-preserved) or preserved for 4 and 12 weeks on silica or in RNAlater®. Microbiota were also characterized from field-collected An. arabiensis preserved on silica for 8, 12 and 16 weeks.ResultsElizabethkingia anophelis and Serratia oryzae were common between both vector species, while Enterobacter cloacae and Staphylococcus epidermidis were specific to females and males, respectively. Microbial diversity was not influenced by sex, condition (fresh or preserved), preservative, or preservation time-period; however, the type of bacterial identification technique affected all microbial diversity indices.ConclusionsThis study broadly characterized the microbiota of An. arabiensis and An. funestus. Silica- and RNAlater®-preservation were appropriate when paired with culture-dependent and culture-independent techniques, respectively. These results broaden the selection of cost-effective methods available for handling vector samples for downstream microbial analyses.

Stool microbiota profiling of patients with muscle invasive bladder cancer receiving neoadjuvant pembrolizumab.

4533 Background: Immune checkpoint inhibitors (ICIs) showed efficacy in metastatic urothelial carcinoma (UC) and promising activity in muscle-invasive and non-muscle invasive UC of the bladder. Recent studies revealed the immunomodulatory effect of the gut microbiota on ICIs efficacy across several malignancies, identifying microbial “signatures” associated with response to therapy and effective antitumoral T-cell activity. In our study, we aimed to study the stool microbiota in patients undergoing neoadjuvant immunotherapy (IO) for muscle-invasive UC. Methods: Pre-IO stools were available for analysis from 42 patients enrolled in the PURE-01 trial (NCT02736266), testing 3x200mg flat-dose pembrolizumab every 21 days before radical cystectomy (RC). All samples were collected using Stool Nucleic Acid Collection and Preservation Tubes (Norgen) and extracted using the Stool DNA Isolation Kit (Norgen), according to the manufacturer’s protocol. 16s sequencing was performed using standardized protocols at the internal facility, using mock communities and DNA standards (ZymoBIOMICS) to control for extraction and sequencing contaminations. A QIIME-based bioinformatic pipeline was used for microbiome analyses. Complete response (CR) to neoadjuvant IO was defined as ypT0N0 at pathologic examination on radical cystectomy specimens, while partial response (PR) was defined as &lt; ypT2N0. Concomitant antibiotic therapy (ABT) was defined as any ATB between 30 days prior to the first pembrolizumab dose and the planned RC. Results: In our study sample, 23 patients responded to IO (21 CR + 2 PR). Overall median age was 68.5 years. Among responders, 20 (87%) patients had a smoking history (vs. 15 (79%) in non-responders) and 4 (17%) underwent concomitant ABT (vs. 6 (32%) in non-responders). Alpha-diversity assessed by richness (ACE index) was higher in responders vs. non-responders (p = 0.05), while no significant diversity was found. Beta-diversity did not show clear clustering of responders vs. non-responders. LEfSe identified 16 bacterial taxa with a linear discriminant analysis (LDA) score ≥2.5 that were differently enriched between responders and non-responders. Among them, we identified the genus Sutterella enriched in responders (p = 0.02), while the species Ruminococcus bromii was enriched in non-responders (p = 0.02). Conclusions: Our analyses showed an association between response to neoadjuvant-IO and microbiome composition in an intention-to-cure population with muscle invasive UC. We found bacterial taxa specifically enriched in responders or non-responders using pre-therapy stool specimens. The identified taxa may be tested in future studies as potential indicators of therapy outcomes, alone or in combination with other IO biomarkers. These results may also inspire new strategies of gut microbiota modulation to promote response in immunotherapy-refractory patients.

Comparison of methods for preservation of activated sludge samples for high-throughput nucleic acid sequencing and bacterial diversity analysis

Preservation of environmental samples is an important step in maintaining the original microbial community until the nucleic acid extraction in the laboratory. Here, we collected activated sludge samples to both immediately extract nucleic acids (control) and submit to different storage/preservation methods for 48 h before nucleic acids extraction: room temperature storage, storage on ice, direct storage at −20 °C, rapid freezing in liquid nitrogen followed by storage at −20 °C, and preservation with TRIzol®, RNAlater®, and Nucleic Acid Preservation (NAP) buffers. Bacterial community was compared by high-throughput 16S rRNA gene sequencing from total DNA and RNA. Among the evaluated methods, TRIzol® and rapid freezing with liquid nitrogen were the least indicated, since they led to significant changes in the microbial community structure and abundance of functional groups involved in ammonia metabolism. Compared to control, storage on ice and NAP preserved more than 80% and 75% of genera at the DNA and RNA level respectively, indicating that these methods are the most suitable for storage/preservation of activated sludge samples intended for nucleic acids sequencing.