Come in Out of the Cold: Alternatives to Freezing for Microbial Biorepositories

Clinical Microbiology N e w s l e t

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Vol. 39, No. 4 February 15, 2017 www.cmnewsletter.com I n Th is

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27 Come in Out of the Cold: Alternatives to Freezing for Microbial Biorepositories

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Come in Out of the Cold: Alternatives to Freezing for Microbial Biorepositories Erica Isaacs,1 Monika Schmelz,2 1McCormick School of Engineering, Northwestern University, Evanston, IL, 2 Department of Pathology, University of Arizona, Tucson, Arizona

Abstract Biorepositories are “libraries” in which biospecimens, bacteria, or DNA and RNA extracts are stored for either clinical or research purposes. Such specimens enable modern molecular-based research and could support method verification, validation, quality control, and, in some cases, proficiency testing in clinical laboratories. Cryopreservation of extracted nucleic acids ensures the stability and longevity of DNA and RNA from patient samples, with the most common methods used for long-term storage of samples being the use of -80°C freezers or liquid nitrogen. Frozen biospecimens are crucial for translational research as they contain well-preserved nucleic acids and protein; however, traditional -80°C freezers consume both energy and space, with costs of maintenance and repairs reaching thousands of dollars annually to freeze and protect biospecimens. Additionally, liquid nitrogen is hazardous to work with, and failure to maintain adequate levels in storage containers can result in loss of specimens. Recently, new room temperature, or “green,” technology has been developed for dry storage of nucleic acids, ultimately reducing costs in terms of energy output and carbon footprint. This review compares and contrasts the use of dry-storage infrastructure with that of freezing samples, in terms of its use in clinical microbiology and highlights considerations to be made if implementing the technologies. Storage alternatives to freezers that equal or exceed their performance with regard to sample preservation and protection against degradation while at the same time reducing space requirements, costs, and energy consumption could be a financial and operational benefit if properly deployed and characterized. Perhaps it is time for laboratories to consider getting molecular quality control material and remnant extracted samples in from the cold and to evaluate the use of dry, room temperature storage technology for use in our microbial biorepositories.

Introduction

Corresponding author: Monika Schmelz, Ph.D., Department of Pathology, University of Arizona, 1501 N. Campbell Ave., P.O. Box 245043, Tucson, AZ 85724-5043. Tel.: 520626-8864. Fax: 520-626-8893. E-mail: schmelz@pathology. arizona.edu.

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For both clinical and research purposes, the storage of DNA and RNA samples in biorepositories is essential and enables modern molecular-based diagnostics and research. Scientists know that the generation of quality data correlates closely with the sample quality and integrity, yet longterm frozen storage of DNA and the freeze-thaw process that occurs when using frozen samples repeatedly may degrade samples. Degraded samples can limit the likelihood of obtaining meaningful and significant research results. Due to its chemical instability, RNA stability is even more uncertain [1-4].

Currently, the most common method of longterm storage of samples is the use of cold-storage equipment (i.e., -80°C and -20°C freezers and liquid nitrogen containers). Cold-storage equipment, like freezers, rank among the highest energy consumers of all laboratory equipment, contributing to the global greenhouse effect and weighing heavily on an organization’s infrastructure. Excessive energy use imposes a disproportionate financial strain to support utility costs. Facility space, often limited, must be allocated to cold storage. In summary, there is a critical need for technologies that provide alternative to

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Figure 1. Example of a Whatman filter paper card. To use this card for sample preservation, sample(s) is applied to the spots and the card is air dried. Then the card can be folded to close the card and stored or shipped at room temperature. To extract DNA, paper punches are removed from the card and used with the chosen DNA extraction process.

cryopreservation of biological samples that are used in clinical and research laboratories across the globe. One solution to the limitations of cold storage is dry, room temperature storage. Several dry-storage methods exist and have been used successfully in research, allowing samples to be moved from remote regions of the globe to academic research laboratories for testing. The technology has also been used in forensic applications, allowing specimens to be stored for up to 1 to 2 years [5,6], but dry storage has fewer publications describing its use for microbiology samples. We know that desiccated tissues of mummies and dinosaurs have been shown to yield DNA after thousands of years. Similarly, removing moisture from biospecimens slows enzymatic activity, including DNA and RNA nuclease activity, and room temperature dry-storage methods leverage desiccation to replace the costly yet vulnerable frozen storage that is so common in clinical laboratories today. To some extent, desiccated chemical matrices, used in dry storage technology, mimic the natural extremophile biology, in which organisms, such as tardigrades or brine shrimp (“SeaMonkeys”), are able to protect cellular systems in a dried state and later revive via rehydration—a process known as anhydrobiosis [7]. As these new dry-storage technologies may benefit clinical microbiology and other molecular laboratories, publications that describe dry storage of infectious agents are reviewed here.

Using Paper Filters for Nucleic Acid Storage Drying DNA using Whatman filter paper goes back to at least 1980, when scientists used DEAE-cellulose Whatman filters for hybridization of bacterial DNA from strains of Enterobacteriaceae [8]. Since that time, dry storage of microbial DNA on paper has aided researchers around the globe, enabling room temperature transport of both microbial isolates and clinical specimens. Whatman Flinders Technology Associates (FTA) cards and the FTA Elute Sample Collection Cards are commercially available from a variety of vendors and are used for collection, transportation, and storage of biological samples and processing of nucleic acids at room temperature (Fig. 1). The sample is applied to the FTA card or FTA Elute card (FTA Elute contains chemicals that lyse cells, denature proteins, and protect nucleic acids from nucleases), and the released nucleic acids are entrapped and preserved in the fibers of the matrix. Following simple purification steps, DNA remains bound to the matrix of FTA cards, which can then be used directly for downstream molecular applications. For FTA Elute cards, DNA is eluted into water after purification. The eluted DNA can be used for quantitative DNA analysis, but produces variable results. While results reviewed in here are promising, further studies are required prior to routine use of dry storage in clinical laboratories to assess the impact of paper type, different extraction methods, humidity, length of storage, sample type, and other variables. Today, there are several approaches to binding DNA for clinical applications [9] and long-term storage. Safe retrieval of DNA from microorganisms have been achieved with various paper matrices [10]. Success has been achieved for bacteria, viruses, fungi, and

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blood parasites; the reports are summarized here for microbes and for clinical specimens.

CSF specimens For cerebrospinal fluid (CSF), FTA Elute Micro Card filter paper (Whatman, Maidstone, United Kingdom) has been successfully used for drying samples and for downstream PCR for diagnosis of common causes of bacterial meningitis. Elliot et al. used mock CSF to optimize filter paper varieties, paper punch sizes, elution volumes, and quantities of DNA template [11]. In this setting, they found that reliable, consistent, and sensitive bacterial DNA detection was achieved from filter paper specimens. Following optimization, the lower limit of detection for Streptococcus pneumoniae from dried mock CSF spots was 14 genomic equivalents (GE)/µl [11]. Furthermore, a prospective clinical evaluation of S. pneumoniae and Neisseria meningitidis tested both culture and PCR on fresh liquid CSF and from dried CSF spots for patients admitted with a clinical diagnosis of meningitis (n = 73). Four of five fresh PCR-positive CSF samples also tested PCR positive from dried CSF spots, with one false negative result [11]. Finally, in a retrospective study of S. pneumoniae samples (n = 20), the median interquartile range for CSF S. pneumoniae bacterial load was 1.1 × 104 GE/µl. Utilizing the optimized methodology, the authors estimated an extrapolated sensitivity of 90%, based on the range of bacterial genome equivalents found in patients within their geographic region. While not as optimal as fresh-sample testing, dried CSF filter paper spots could potentially help confirm diagnosis in remote geographical regions [11].

Stool specimens Stool may seem like an unlikely sample type for assessment of dry storage, yet due to diarrheal illness across the globe, there are several publications that explore its use. In one study, detection of enteropathogens by hybridization techniques was performed for 416 children with diarrhea by spotting specimens to Whatman 541 filters [12]. Enterotoxigenic Escherichia coli was detected in 38 patients by examining colony blots, in 52 patients by examining replicate blots, and in 45 of 52 patients by examining direct stool blots. Shiga-like toxin-producing E. coli was detected in 0 of 12 patients by stool blots. Shigella spp. were identified in dried stool blots in 35 of 82 culture-confirmed samples [12]. While examining replicate blots with specific probes identified more enterotoxigenic E. coli (P < 0.005) and Shiga-like toxin-producing E. coli (P

< 0.005) infections, overall, more Shigella spp. and enteroinvasive E. coli infections were identified by standard bacteriological methods (P < 0.001), making the Whatman method less than optimal for detection of the two pathogens [12]. Of note, blots are generally less sensitive than PCR, which could have contributed to the study’s results.

Miscellaneous specimens

In a cohort of human immunodeficiency virus (HIV)-positive patients, Sokolova et al. reported that microsporidial DNA was successfully amplified in 6 of 50 stool samples that were applied to FTA cards (Whatman Inc., Florham Park, NJ). Amplicons were directly sequenced and identified as common microsporidial species (Encephalitozoon intestinalis, Encephalitozoon cuniculi, Encephalitozoon. hellem, and Enterocytozoon bieneusi) [13].

Bacterial isolates in broth. Broth from cultivation of bacterial isolates has been used to assess the accuracy of PCR and DNA sequencing from samples stored on FTA cards [17]. Cultivated Campylobacter species of poultry origin in Brucella broth were adjusted to a 0.5 McFarland turbidity standard and added 110 µl of the cell suspension to one circle on Whatman FTA classic cards. The samples were then covered and allowed to dry at room temperature. After 3 months of storage at room temperature and shipping from Brazil to Arkansas, amplification of the Campylobacter gyrA gene for ciprofloxacin resistance was successful for all 50 samples preserved on FTA cards. Mutations present in each gene were confirmed by DNA sequencing [17].

Urine specimens Lodh et al. collected approximately 40-ml urine specimens from 125 test and control individuals living in northern Argentina [14]. Fresh stool specimens were also processed using three different methods. Urine specimens were filtered in the field through a 12.5-cm Whatman no. 3 filter, which was then dried and packed individually in sealable plastic bags with desiccant and shipped for testing. DNA was recovered from the filter and PCR-amplified genetic regions specific to a dispersed repetitive sequence. The prevalence of Strongyloides stercoralis infection by stool culture and direct examination was 35/125 (28%). In contrast, PCR-based detection of parasite-specific trans-renal DNA in urine indicated that 56/125 individuals (44.8%) carried the parasite. Of the patients who tested positive for urine-based parasite DNA, approximately half also tested positive in their stool specimens. There were a small number (6.4%) of cases where parasite larvae were seen in the stool but no DNA was amplified from the urine [14].

Ocular specimens Since techniques for specimen collection and DNA extraction before PCR may interfere with test sensitivity of ocular infections, Menassa et al. investigated the use of DNA-stabilizing FTA filter paper (Whatman, Maidstone, United Kingdom) for specimen collection without DNA extraction in a single-step, non-nested PCR [15]. In this study, specimens were collected from ocular surfaces with FTA filter discs, which were directly used in single-step PCRs to detect fungal DNA in cases of fungal keratitis. Test sensitivity was evaluated with serial dilutions of Candida albicans, Fusarium oxysporum, and Aspergillus fumigatus cultures. In this study, 15 microbial keratitis patients were tested, along with 196 and 155 healthy individuals from Switzerland and Egypt, respectively. In healthy volunteers, fungal PCR was positive in 1.0% and 8.4% of eyes from Switzerland and Egypt, respectively, representing results that may vary by region. Fungal PCR remained negative in 10 cases of culture-proven bacterial keratitis, became positive in 4 cases of fungal keratitis, and missed 1 case of culture-proven A. fumigatus keratitis. Despite these limitations, authors concluded that FTA filter paper for specimen collection, combined with direct PCR, may be a promising method of detecting fungal keratitis [15].

Sputum. A virulent clonal strain of Pseudomonas aeruginosa (Australian epidemic strain I) that is widespread in Australian cystic fibrosis patients was identified from Whatman FTA Elute cards with PCR-based assays to rapidly detect the presence of P. aeruginosa strains in cystic fibrosis sputum [16].

Debes at al. evaluated samples preserved on Whatman filter paper for DNA extraction prior to molecular characterization using multi-locus variable-number tandem-repeat analysis for differentiation of Vibrio cholerae genotypes [18]. Forty-seven V. cholerae isolates and 18 enriched clinical specimens (e.g., stool specimens after enrichment in broth) from cholera outbreaks in Cameroon were analyzed. Eighty-seven DNAs were successfully analyzed, including 16 paired samples (both a cultured isolate and the matching enriched broth specimen from which the isolate was cultivated). Genotypic results were identical between 15 enriched specimens and their culture isolates; one pair differed at only a single locus. In this study, two closely related, but distinct, clonal complexes were identified [18].

Bacterial genotyping Since rapid, standardized methods for high-resolution genotyping of archaea are not widely available, Cleland et al. evaluated the ability of Whatman FTA paper to recover DNA from 79 strains representing 27 genera of Crenarchaeota and Euryarchaeota compared to that of chemical extraction [19]. Organisms were analyzed by the repetitive sequence-based PCR method (rep-PCR) using an archaeal primer set. All the organisms could be successfully genotyped, and the results were reproducible. No differences in rep-PCR profiles were detected between DNA extracted using the chemical extraction kit and FTA paper. Based on their results, rep-PCR was a useful tool for the genotyping and strain identification of archaea and could be performed from DNA stored on Whatman paper [19].

Fungal isolates Conventional methods for purifying PCR grade fungal genomic DNA typically require cell disruption (either physical or enzymatic) coupled with laborious organic extraction and precipitation stages, or expensive column-based technologies. In contrast, Borman et al. reported an easy and extremely rapid method of preparing genomic DNAs from yeast and mold cultures using Whatman FTA filters [20]. The study represents an inexpensive and rapid

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method of fungal genomic-DNA preparation and also potentially represents a powerful fungal DNA archiving and storage system [20]. In this study, aqueous suspensions of yeast cells or hyphal fragments and conidia were applied directly (or after freeze-thawing) to dry FTA filters. The inoculated filters were then subjected to brief microwave treatment to dry the filters and to inactivate the organisms. Punched discs of filter paper were then removed, washed rapidly, dried, and placed directly into PCR mixtures. This study showed that the microwave procedure inactivated all of the 38 yeast and 75 mold species tested while generating PCR grade DNA preparations in approximately 15 minutes. A total of 218 of 226 fungal isolates tested produced amplifiable DNA after application to FTA filters. Detection limits with yeast cultures were approximately 10 CFU per punch. The authors concluded that Whatman FTA technology offered an ultra-rapid method of fungal genomic-DNA preparation, and it also potentially represents a powerful fungal DNA archiving and storage system [20]. In 2010, the same research team evaluated the feasibility of pyrosequencing analysis of a portion of the internal transcribed spacer 2 (ITS2) region for identification of pathogenic yeasts (n = 477 clinical isolates) encompassing 43 different fungal species [21]. The molecular identifications produced by pyrosequencing were compared with those obtained using conventional biochemical tests (AUXACOLOR2). PCR amplification and sequencing of the D1-D2 portion of the nuclear 28S large rRNA gene resulted in over 98% (469/477) of isolates and 40 of the 43 fungal species tested being correctly identified by pyrosequencing of 35 bp of ITS2. When pyrosequencing was combined with the Whatman FTA paper technology for the rapid extraction of fungal genomic DNA, molecular identification could be accomplished within 6 h from the time of starting from pure cultures [21]. Later in 2010, the authors published an improved version of the extraction of fungal genomic DNA from pure cultures using Whatman FTA filter paper matrix technology, which automatically lyses collected cells and stabilizes nucleic acids. This modified protocol was significantly more cost-effective than the original method and substantially reduced the problem of potential cross-contamination between sequential filters [22]. As one of the few studies performed using samples in a clinical mycology laboratory, the feasibility and sufficiency of DNA extraction by Whatman FTA filter matrix technology and DNA sequencing of the D1-D2 region of the large ribosomal subunit gene for identification of clinical isolates of 21 yeasts and 160 molds was assessed [23]. Yeast isolates were identified at the species level with 100% homology. Mold identification was less efficient, with only 102 (63.75%) clinically important mold isolates identified at the species level and 56 (35%) isolates at the genus level. Two (1.25%) isolates could not be identified [23]. Besides PCR methods, FTA filters can be processed and used in loop-mediated isothermal amplification (LAMP) assays. When LAMP was combined with a simplified DNA extraction method using an FTA filter, identification to the species level was achieved within 2 h for Scedosporium species [24].

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Viruses HIV. The collection of dried blood spots (DBS) on Whatman 903 cards has been used in the detection of HIV-1 DNA in infants by PCR as early as 4 to 6 weeks after birth in resource-limited settings. As other methods became available, scientists performed testing at the Centers for Disease Control and Prevention and in four laboratories in Africa [25]. They evaluated the qualitative detection of HIV-1 DNA by PCR performed from DBS prepared and stored under a variety of conditions on three commercially available blood collection cards: Ahlstrom grade 226, Munktell TFN, and Whatman 903. DBS were stored at ambient room temperature, at 37°C with high humidity, and at -20°C for varying lengths of time. The presence of HIV-1 DNA was detected using Roche Amplicor HIV-1 DNA (v 1.5). Testing was performed weekly for 4 weeks and then again at 8 and 12 weeks for room temperature and 37°C. Testing was also performed at weeks 4, 8, and 18 for storage at -20°C. Samples were also tested after international shipment at room temperature. In addition, after nearly 3 years of storage at -20°C, DBS were also evaluated independently using the COBAS Ampliprep/TaqMan HIV-1 Qual and Abbott RealTime HIV-1 Qualitative tests. In all cases, HIV-1 DNA was detected equally well on the three blood collection cards regardless of storage conditions and PCR assay. The authors concluded that Ahlstrom 226 and Munktell TFN papers were comparable to Whatman 903 for qualitative HIV-1 DNA detection and could be considered optional blood collection devices in resource-limited countries [25]. Hepatitis A virus. Desbois et al. assessed the feasibility of using Whatman filter paper for the serological and molecular detection of hepatitis A virus (HAV) infection [26]. Sixty-eight sera were spotted onto filter papers (Whatman, United Kingdom) and were used for detection of total anti-HAV antibodies, and 64 sera were used for detection of immunoglobulin M antibody to HAV. Dried serum spots were stored at 4°C, room temperature, and 37°C for 1, 2, and 4 weeks. The sensitivity and specificity of the serological assays were 100% regardless of temperature and storage duration [26]. In the same study, the stability of HAV RNA was assessed by using qualitative and quantitative reverse transcription (RT)-PCR performed with human plasma spiked with serial dilutions of cultured HAV spotted on Whatman FTA filter paper cards. The filters were stored at room temperature and processed for RT-PCR assays. No reduction of the viral load was observed after 5, 15, and 30 days of storage. A slight reduction in detection (nearly 10-fold) was attributable to a smaller sample input in dried samples. This method was further evaluated using 35 frozen sera, from which an HAV RNA amplification assay showed 100% specificity and 92.3% sensitivity, and sequence analysis was performed from dried serum spots [26]. Human papilloma virus. By collecting two cervical specimens from 396 women—one sample stored using a liquid preservative solution and the other applied to a Whatman Indicating FTA Elute card—human papilloma virus (HPV) testing methods were compared [27]. All the specimens were processed using three HPV testing methods: Hybrid capture 2 (HC2) (Qiagen, Germany),

careHPV (Qiagen, Germany), and Cobas 4800 (Roche Molecular, Indianapolis, IN) tests. Compared to a liquid-based carrier, the FTA card demonstrated comparable sensitivity for detecting high-grade cervical intraepithelial neoplasia using HC2 (91.7 %), careHPV (83.3 %), and Cobas 4800 (91.7 %). Moreover, the FTA card showed higher specificity than the liquid-based carrier for HC2 (79.5% vs. 71.6%; P = 0.015) and comparable specificity for careHPV (78.1% vs. 73.0%; P > 0.05) but lower specificity for the Cobas 4800 test (62.4% vs. 69.9%; P = 0.032), indicating paper methods may work better with some downstream applications than others [27]. In another study, a simple paper smear method with a piece of Whatman filter paper cut to the size of a microscope glass slide for dry collection and storage of cervical specimens was used. Multiplex PCR was used for simultaneous detection of generic HPVs, as well as typing of the high-risk HPV-16 and -18 [28]. DNA was eluted by boiling a single paper punch in a single tube. Sixty HPV-positive biopsy specimens and corresponding paper specimens from cervical cancer patients, as well as cervical smears from 100 healthy women with or without abnormal cytology, were collected both as paper smears and in PBS. Detection of HPV DNA from cervical biopsy specimens collected in PBS and corresponding cervical scrapes on a paper smear or in PBS by conventional and multiplex PCR showed concordance of 100% and adequacy of 93%. A similar comparative study of cervical scrapings from healthy women also revealed 100% concordance. The technique was validated in a multicenter study at four different national laboratories. Paper smears collected by different centers showed variable adequacy (73 to 82%), but the use of multiple paper smear discs for DNA extraction significantly increased the adequacy. Integration of paper smears with multiplex PCR for the detection and typing of HPVs is a highly convenient, efficient, simple, and cost-effective method for large-scale epidemiological studies in resource-poor settings [28]. Gustavvson et al. developed a method for dry collection of cervical epithelial cell samples for subsequent PCR-based HPV testing [29]. They optimized their methods by comparing the amounts of DNA present in a cytobrush and from paper filters; they then assessed 1,040 samples applied to the FTA Elute microcard. The agreement in HPV positivity between the cytobrush and FTA samples was high (94%; kappa = 0.88; 95% confidence interval, 0.748 to 1). All 1,040 samples on the FTA card had sufficient amounts of genomic DNA (>10 copies of a single-copy gene) to be suitable for HPV typing for the most common genotypes [29].

Blood parasites Whatman 903 specimen collection papers were used to preserve samples prior to LAMP testing, Oriero et al. collected blood from 341 subjects and screened for malaria by three methods: the RDT SD Bioline Malaria Antigen P.f., thick blood films for microscopy, and DBS collected on Whatman 903 cards. DNA was extracted from the dried sample using a crude (methanol/heating) extraction method [30]. A laboratory-based PCR amplification using DNA extracted with the Qiagen kit was used as the reference method.

Plasmodium falciparum malaria prevalence was 37% (127/341) as detected by LAMP, 30% (104/341) by microscopy, and 37% (126/341) by rapid diagnostic test (RDT). Compared to the reference PCR method, the sensitivity of the assay from Whatman 903 was 92% for LAMP, 78% for microscopy, and 76% for antigen testing; the specificity was 97% for LAMP, 99% for microscopy, and 88% for antigen [30]. Whatman 3MM filter papers were used to assess the presence and prevalence of asymptomatic P. falciparum and Plasmodium vivax infections (n = 1,094) [31]. Samples were analyzed by blood film preparation for microscopy and RDT and by PCR from dried blood samples. The prevalence of asymptomatic carriage for P. falciparum, P. vivax, and mixed species was 5.0% (55/1,094) as determined by microscopy, while the prevalence as determined using RDT was 8.2% (90/1,094). PCR was able to be performed on 47 of 55 microscopy-confirmed and on 79 of 90 RDT-confirmed samples, with parasite DNA found in 89.4% (42/47) of the microscopy-positive samples and in 77.2% (61/79) of the RDTpositive samples [31]. It was not possible to determine if the limited DNA detection was due to the storage method.

Biomatrica Dry Storage Biomatrica’s room temperature dry-storage cabinets (now commercially available as Qiagen QIASafe) employ desiccant beads to ensure a controlled low-humidity environment. Dry-storage cabinets require no energy input and help to save money required for electricity. Two key products can be used in combination with the room temperature dry-storage cabinets. Biomatrica’s DNAStable and RNAStable were developed by Biomatrica (San Diego, CA) and designed to preserve DNA and RNA samples at room temperature using a synthetic storage medium. These products are based on the natural principles of anhydrobiosis, a process used by certain organisms to survive in arid conditions for more than 100 years by protecting their DNA, RNA, proteins, membranes, and cellular systems for survival in a dry state. Revival can occur by rehydration. DNAStable stabilizes purified DNA samples (e.g., genomic and plasmid DNA, oligonucleotides, and PCR products) at room temperature without degradation when stored in dry storage cabinets. DNAStable technology was designed by combining extremophile biology that enables long-term survival of organisms in extremely dry environments with synthetic chemistry. RNAStable preserves and stores RNA at room temperature by utilizing a similar synthetic chemistry as that of DNAStable. RNAStable protects purified total RNA and poly(A) RNA samples from degradation during dry storage in room temperature storage cabinets for extended periods (i.e., months). Purified RNA samples are applied to RNAStable and then dried at room temperature prior to storage. While several publications describe the use of these technologies for human nucleic acid [5,32-36], there are fewer publications that describe its use with microbes. Several notable publications are reviewed here.

Blood parasites In 2012, researchers used the Biomatrica technology in the diagnosis of Plasmodium ovale, which was determined by microscopy

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and confirmed by PCR [37]. In this study, white blood cells were removed from the blood by passing through a non-woven fabric filter [38], and genomic DNA was extracted using a QIAamp DNA blood minikit (Qiagen, Germany) according to the manufacturer’s instructions. Samples were then stored in a DNAStable tube until use [37]. Genome sequencing of several Plasmodium species and genome assembly were successful using this approach [39].

Biothreats Rare studies assessed the feasibility to used controls prepared in dry-stabilized format using DNAStable blood and RNAStable reagents. One such study successfully used inactivated yellow fever virus for controls used in a downstream recombinase polymerase amplification assay [40]. Importantly, during the Ebola virus disease outbreak in Guinea, Liberia, Sierra Leone, and Nigeria, scientists used Biomatrica’s technology to store and sequence 99 Ebola virus genomes from 78 patients in Sierra Leone to approximately 2,000× coverage [41]. Other researchers leveraged Biomatrica’s CloneStable and RNAStable to stabilize samples of Yersinia pestis and Venezuelan equine encephalitis virus. When heated to 95°C in RNAStable, Y. pestis had 13-fold improvement in detection via real-time PCR compared to heated samples in buffer alone. In addition, CloneStable preserved Y. pestis antigens for 7 days. Venezuelan equine encephalitis virus in RNAStable had approximately 10-foldimproved detection versus heated samples in buffer alone [42], supporting their advantage over storage in high-salt buffers.

Planning for Evaluation of Dry-Storage Products As one can surmise from review of the literature, dry-storage technology is clearly useful in remote and low-resource settings, allowing subsequent advanced molecular characterization of pathogens. Furthermore, since freezer space is always limited in clinical laboratories, dry-storage technology could be useful and shows promise for storage of quality control material. Development of robust long-term room temperature biospecimen tissue storage technology could potentially reduce costs for the laboratory community in the face of decreasing budgets [43], but much more research is needed for documentation of utility. So, why has widespread use of dry-storage technology not yet occurred in clinical laboratories? First, the commercially available Biomatrica technology, QIASafe DNA tubes and QIASafe DNA 96-well plates, are labeled for research use only, and not for use in diagnostic procedures. It is also clear that different genetic targets and even different methods must be evaluated and tested with the product concerned to ensure the feasibility of long-term dry storage of purified RNA and DNA samples and to determine if the product satisfies specimen storage needs and quality criteria in clinical laboratories. Rigorous criteria must be used to assess recovery of nucleic acid samples and ensure that the products maintain sample integrity and protect against degradation during the period of room temperature storage as compared to sample recovery and protection when stored using the traditional cryopreservation methods.

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Such evaluation would be a costly and lengthy task for clinical laboratories to undertake. If it were to be undertaken, a genomic analysis would need to be performed as a comparative baseline immediately after DNA/RNA extraction from fresh samples, without any storage. Then, aliquots of the remaining samples would need to be stored a -80°C in a freezer and at room temperature using the new dry-storage system. Samples would need to be stored for various periods, for example, 3, 6, 12, and 24 months, at a minimum. At each storage time, samples would be retrieved from the freezer, and the dry-storage alternative and the integrity of the samples would be tested by molecular methods, such as real-time PCR, to assess the target amplification and amplification reproducibility. The results would need to be performed in at least triplicate in order to compute and compare the means of quantifiable measurements, such as cycle thresholds (CT). The means and standard deviations of the CT values would need to be compared to the baseline measurements. Secondly, in contrast to extremophiles, which inclusively preserve cellular structure, nucleic acids, and protein, current commercial chemical matrices permit the long-term dry storage of DNA or RNA alone, not both simultaneously. For most clinical laboratories, it might not be cost-effective to prospectively extract DNA and RNA from biospecimens when only a subset of specimens may eventually be used. Rather, the dry-storage matrices may be best suited for remnant post-extraction samples to store excess extracted nucleic acids, high-demand or rare samples, or back-up samples of key frozen biospecimens. Another challenge is that there is little independent data, particularly long-term data, regarding most of the commercial matrices, and there is virtually no data that describes long-term results in clinical laboratories. Most of the dry-storage matrices are proprietary, and only limited information as to their precise composition is available. Another impediment is that for dry storage, nucleic acid samples are typically added to the storage matrix and then dried and stored, which is a detriment for use in clinical laboratories, where safety cabinets may be in use 24 hours a day and where risk of contamination with control materials would be viewed as a limitation. Clearly, the technology would best be used in well-controlled environments, perhaps in facilities that manufacture control material already. Finally, according to College of American Pathologists guidelines, changes in biospecimen processing that can impact testing must be validated; therefore, if quality control were to change, the new controls would need to be evaluated for a large number of molecular assays.

Summary Green, dry-storage technology has potential to be used by numerous downstream methodologies, especially when working in remote areas and in developing countries, where access to laboratory facilities and equipment is limited. Theoretical benefits for clinical laboratories are easy to envision, yet routine use of the technology in clinical laboratories is limited. Nevertheless, the

technology holds opportunities for cost saving and warrants further investigation. Over the long term, room temperature storage is less expensive in terms of lower electricity, maintenance, labor, and environmental costs. Room temperature storage requires minimal startup costs, space, and overhead for maintaining samples, while the up-front costs of freezers are substantial and include room air conditioning to offset the heat that is generated by the freezers and installing circuitry connected to back-up generators, monitors, and dedicated electrical lines for each freezer. In addition, the freezer life span is limited, and they must be periodically replaced, perhaps every 10 to 15 years, with freezer warranties, maintenance, electricity, back-up power sources, back-up carbon dioxide, or liquid nitrogen costs accumulating over the long term. Perhaps it is time for microbiology laboratories to consider vetting this technology for molecular quality control material. Based on experience with FTA paper and accelerated aging studies of newer matrices, it is possible that room temperature stability could be achieved, potentially for decades, for PCR, as well as a broad range of testing, including next-generation sequencing. Perhaps it is time to take our specimens out of the cold.

Acknowledgements This review was funded by the Green Fund Committee of the University of Arizona (GF 12.30) (http:/studentaffairs.arizona. edu/green/fund/). There are no conflicts of interest to declare.

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