Suboptimal Storage Conditions Research Articles

A validated in-house assay for HIV drug resistance mutation surveillance from dried blood spot specimens

Despite increasing scale-up of antiretroviral therapy (ART) coverage, challenges related to adherence and HIV drug resistance (HIVDR) remain. The high cost of HIVDR surveillance is a persistent challenge with implementation in resource-constrained settings. Dried blood spot (DBS) specimens have been demonstrated to be a feasible alternative to plasma or serum for HIVDR genotyping and are more suitable for lower resource settings. There is a need for affordable HIVDR genotyping assays which can amplify HIV-1 sequences from DBS specimens, particularly those with low viral loads, at a low cost. Here, we present an in-house assay capable of reliably amplifying HIV-1 protease and partial reverse transcriptase genes from DBS specimens, which covers the complete World Health Organization 2009 list of drug resistance mutations under surveillance. DBS specimens were prepared using whole blood spiked with HIV-1 at concentrations of 10,000, 5000, 1000, and 500 copies/mL (n=30 for each concentration). Specimens were tested in triplicate. A two-step approach was used consisting of cDNA synthesis followed by nested PCR. The limit of detection of the assay was calculated to be approximately 5000 (95% CI: 3200–10,700) copies/mL for the protease gene and 3600 (95% CI: 2200–10,000) copies/mL for reverse transcriptase. The assay was observed to be most sensitive with higher viral load specimens (97.8% [95% CI: 92.2–99.7]) for both protease and reverse transcriptase at 10,000 copies/mL with performance decreasing with the use of specimens with lower viral loads (46.7% [36.1–57.5] and 60.0% [49.1–70.2] at 500 copies/mL for protease and reverse transcriptase, respectively). Ultimately, this assay presents a promising opportunity for use in resource-constrained settings. Future work should involve validation under field conditions including sub-optimal storage conditions and preparation of DBS with fingerprick blood in order to accurately reflect real-world collection scenarios.

Diagnostic Guidance for C. difficile Infections.

Diagnosis of Clostridioides difficile infection (CDI) can be challenging. First of all, there has been debate on which of the two reference assays, cell cytotoxicity neutralization assay (CCNA) or toxigenic culture (TC), should be considered the gold standard for CDI detection. Although the CCNA suffers most from suboptimal storage conditions and subsequent toxin degradation, TC is reported to falsely increase CDI detection rates as it cannot differentiate CDI patients from patients asymptomatically colonised by toxigenic C. difficile. Several rapid assays are available for CDI detection and fall into three broad categories: (1) enzyme immunoassays for glutamate dehydrogenase, (2) enzyme immunoassays or single-molecule array assays for toxins A/B and (3) nucleic acid amplification tests detecting toxin genes. All three categories have their own limitations, being suboptimal specificity and/or sensitivity or the inability to discern colonised patients from CDI patients. In light of these limitations, multi-step algorithmic testing has been advocated by international guidelines (IDSA/SHEA and ESCMID) in order to optimize diagnostic accuracy. As a result, a survey performed in 2018-2019 in Europe revealed that most of all hospital sites reported using more than one test to diagnose CDI. CDI incidence rates are also influenced by sample selection criteria, as several studies have shown that if not all unformed stool samples are tested for CDI, many cases may be missed due to an absence of clinical suspicion. Since methods for diagnosing CDI remain imperfect, there has been a growing interest in alternative testing strategies like faecal microbiota biomarkers, immune modulating interleukins, cytokines and imaging methods. At the moment, these alternative methods might play an adjunctive role, but they are not suitable to replace conventional CDI testing strategies.

Human Umbilical Cord Blood Endothelial Progenitor Cell-Derived Extracellular Vesicles Control Important Endothelial Cell Functions.

Circulating endothelial progenitor cells (EPCs) play a pivotal role in the repair of diseases in which angiogenesis is required. Although they are a potentially valuable cell therapy tool, their clinical use remains limited due to suboptimal storage conditions and, especially, long-term immune rejection. EPC-derived extracellular vesicles (EPC-EVs) may be an alternative to EPCs given their key role in cell-cell communication and expression of the same parental markers. Here, we investigated the regenerative effects of umbilical cord blood (CB) EPC-EVs on CB-EPCs in vitro. After amplification, EPCs were cultured in a medium containing an EVs-depleted serum (EV-free medium). Then, EVs were isolated from the conditioned medium with tangential flow filtration (TFF). The regenerative effects of EVs on cells were investigated by analyzing cell migration, wound healing, and tube formation. We also analyzed their effects on endothelial cell inflammation and Nitric Oxide (NO) production. We showed that adding different doses of EPC-EVs on EPCs does not alter the basal expression of the endothelial cell markers nor change their proliferative potential and NO production level. Furthermore, we demonstrated that EPC-EVs, when used at a higher dose than the physiological dose, create a mild inflammatory condition that activates EPCs and boosts their regenerative features. Our results reveal for the first time that EPC-EVs, when used at a high dose, enhance EPC regenerative functions without altering their endothelial identity.

Abstract 3665: Umbilical cord blood endothelial progenitor cell-derived extracellular vesicles control important endothelial cell functions

Abstract Background: Circulating endothelial progenitor cells (EPCs) play a pivotal role in vascular regeneration and the repair of diseases in which angiogenesis is required, including ischemia and tissue transplantation. Although they are a potentially valuable cell therapy tool, their clinical use remains limited due to suboptimal storage conditions and especially, immune rejection. Even if these cells have been shown to be tolerated by the immune system, their in vivo long-term effects still remain to be elucidated. EPC-derived extracellular vesicles (EPC-EVs) may be an attractive alternative to EPCs given their key role in cell-cell communication and expression of the same parental markers. In this study, we investigated the regenerative effects of umbilical cord blood EPC-EVs on EPCs in vitro. Methods: EPCs were obtained from human umbilical cord blood (CB) and characterized by flow cytometry. After amplification, EPCs were cultured in a medium containing an EVs-depleted serum (EV-free medium). Then the conditioned medium was used to isolate the EVs with Tangential Flow Filtration (TFF). The EVs were characterized by a series of tests defining their size, number, and protein content. The regenerative effects of EPC-EVs on endothelial cells were investigated by analyzing cell migration, wound healing, and tube formation. We also analyzed the effect of EPC-EVs on endothelial cell inflammation and NO production. Results: We showed that adding different doses of EPC-EVs on EPCs does not alter the basal expression of the endothelial cell markers CD31, CD144, and KDR, nor change their proliferative potential and NO production level. Furthermore, we demonstrated that EPC-EVs, when used at a higher than physiologic dose, create a mild inflammatory condition that activates EPCs and boosts their regenerative features like migration and tube formation functions. Conclusions: Our results reveal for the first time that EPC-EVs, when used at a high dose, could enhance EPC regenerative functions without altering their endothelial identity. Citation Format: Sawssen Ben Fraj, Sina Naserian, Bileyle Lorenzini, Sylvie Goulinet, Philippe Mauduit, Georges Uzan, Houda Haouas. Umbilical cord blood endothelial progenitor cell-derived extracellular vesicles control important endothelial cell functions. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3665.

Using Dried Blood Spots for a Sero-Surveillance Study of Maternally Derived Antibody against Group B Streptococcus.

Vaccination during pregnancy could protect women and their infants from invasive Group B Streptococcus (GBS) disease. To understand if neonatal dried blood spots (DBS) can be used to determine the amount of maternally derived antibody that protects infants against invasive GBS disease, a retrospective case-control study was conducted in England between 1 April 2014 and 30 April 2015. The DBS of cases with invasive GBS disease (n = 61) were matched with healthy controls (n = 125). The haematocrit, DBS storage temperature, freeze-thaw cycle, and paired serum/DBS studies were set up to optimise the antibody assessment. The samples were analysed using a multiplex immunoassay, and the results were assessed using parametric and nonparametric tests. Antibody concentrations were stable at haematocrits of up to 50% but declined at 75%. DBS storage at room temperature was stable for three months compared with storage from collection at -20 °C and rapidly degraded thereafter. Total IgG levels measured in DBS and paired serum showed a good correlation (r2 = 0.99). However, due to suboptimal storage conditions, no difference was found in the GBS IgG levels between DBS samples from cases and controls. We have demonstrated a proof of concept that assays utilising DBS for assessing GBS serotype-specific antibodies in infants is viable. This method could be used to facilitate future large sero-correlate studies, but DBS samples must be stored at -20 °C for long term preservation of antibody.

Effect of Laser Biostimulation on Germination of Sub-Optimally Stored Flaxseeds (Linum usitatissimum)

Sub-optimal storage of grains could deteriorate seed germination and plant viability. Recent research studies have established that laser biostimulation of seeds could be used as a safe and sustainable alternative to chemical treatment for improving crop germination and growth. Herein, the efficacy of this novel technique is evaluated to see if poor germinability caused by sub-optimal storage of flaxseeds (Linum usitatissimum) could be reversed using laser biostimulation. Healthy flaxseeds were first subjected to sub-optimal storage conditions (30 °C for ten weeks) to degrade their germinability. Two low-cost lasers, including a single-wavelength red laser (659 nm) and a dual-wavelength green/infrared laser (531 and 810 nm (ratio ~10:1)) were then used on two groups viz. healthy (properly stored) and sub-optimally stored (artificially degraded (AD)) seeds and irradiated for 0 (control), 5, 10, and 15 min using total power densities of 7.8 and 6.2 mW/cm2, respectively. In the case of AD seeds, 5-min dual-wavelength laser treatment was found to be the most efficient setting as it improved the mean germination percentage, mean germination time, germination speed, germination rate index, wet weight, and dry weight by 29.3, 16.8, 24.2, 24.2, 15.7, and 20.6%, respectively, with respect to control samples. In the case of healthy seeds, dual-wavelength laser treatment could induce significant enhancement in seeds’ root length, wet weight, and dry weight (improved by 26, 23, and 8%, respectively) under 10 min of irradiation. On the other hand, the effect of applied red laser treatment was not very promising as it could only induce significant enhancement in the mean germination time of AD seeds (improved by 17%). Overall, this study demonstrates the potential of laser biostimulation in reversing the adverse effect of poor crop storage. We believe these findings could spur the development of a physical tool for manipulating seed germination and plant growth.

Inline nondestructive internal disorder detection in pear fruit using explainable deep anomaly detection on X-ray images

To preserve the quality of fresh fruit after harvest and to meet the year-round demand for high-quality fruit, pears are stored under a controlled atmosphere. However, due to preharvest events or suboptimal storage conditions, internal disorders might develop resulting in severe quality loss. Examples include internal browning and cavities, which are invisible externally. Here, X-ray radiography is investigated as a technique for internal quality inspection. The detection of defect fruit is approached as an anomaly detection (AD) problem, in which a model is constructed using nominal data and an anomaly score is used to identify defect fruit. In this work, multiple deep AD methods are shown to be effective to detect pears with internal cavity and browning disorders using X-ray radiographs (mean area under the receiver operating characteristic curve (AUC) up to 0.962). The best performing methods were found on par with a state-of-the-art multisensor disorder detection method (mean AUC up to 0.966). By investigating AD performance in function of internal disorder severity, it was shown that defect fruit with a cavity volume percentage > 1.0% could be detected 100% accurate using inline X-ray imaging. For lower cavity area percentages, the accuracy depended on the internal browning severity. Additionally, the explainability of the deep AD methods, i.e., how well human interpretable insight can be provided from each method’s predictions, were qualitatively evaluated using anomaly heatmaps, which provided useful insight in the execution of the deep learning algorithms.

A Cystatin C Cleavage ELISA Assay as a Quality Control Tool for Determining Sub-Optimal Storage Conditions of Cerebrospinal Fluid Samples in Alzheimer's Disease Research.

Background:An N-terminal octapeptide cleavage of the cystatin C protein was discovered by mass spectrometry when cerebrospinal fluid (CSF) was stored at –20°C for 3 months, which did not occur when CSF was stored at –80°C.Objective:The aim was to develop an immunoassay as quality assessment tool to detect this –20°C cleavage of cystatin C in CSF and support Alzheimer’s disease research.Methods:A specific monoclonal antibody and a double indirect sandwich ELISA were developed: one assay quantifies the octapeptide uncleaved protein specifically and the other quantifies the total cystatin C present in the biological fluid (both cleaved and uncleaved forms). The ratio of these concentrations was calculated to assess the extent of cleavage of cystatin C. The novel ELISA was validated and applied in a short-term (up to 4 weeks) and mid-term (up to one year) stability study of CSF stored at 4°C, –20°C, –80°C, and liquid nitrogen. Impact of freeze-thaw cycles, adsorption, and protease inhibitors were tested.Results:The ratio of truncated protein was modified following –20°C storage and seemed to reach a plateau after 6 months. The ratio was impacted neither by freeze-thaw cycles nor adsorption. The –20°C specific cleavage was found to be protease related.Conclusion:Using this novel double indirect sandwich ELISA, absolute levels of the total and uncleaved cystatin C and the ratio of truncated cystatin C can be measured. This assay is an easily applicable tool which can be used to confirm that CSF biospecimen are fit-for-purpose for Alzheimer’s disease research.

Covering Norway spruce container seedlings with reflective shading cloth during field storage affects seedling post-planting growth

The field storage of container seedlings is often a necessity after the seedlings have been transported from nurseries close to the regeneration area. Suboptimal storage conditions can lead to reduced growth or even seedling mortality. Supporting favourable conditions for the seedlings during storage requires labour, especially for the watering of seedlings, and therefore increases the costs of forest regeneration. In this study, we examined the effects of covering Norway spruce container seedlings with a reflective shading cloth and neglecting the watering during field storage. This was done by measuring the drying of the seedlings during field storage, the rooting of the seedlings when planting, and the subsequent survival and performance in the field two and three growing seasons after planting. The study comprised one-year-old Norway spruce container seedlings covered with reflective shading cloth and seedlings with no cover (subject to rainfall) in 2012 and 2013; additionally, in 2013, replicates from both treatments were stored in conditions where rainfall was impeded. The stem water potential was measured, a rooting test was carried out, and the seedlings were planted in the test field weekly over 42 days of field storage without watering. As a novel finding, the results indicate that covering the seedlings with a reflective shading cloth during the field storage increased the risk of grey mould in summer storage and led to negative effects regarding post planting seedling development. The application of a shading cloth directly on the seedlings cannot be recommended to prolong the watering intervals during field storage in the current climatic conditions in Southern Finland, where the risk of grey mould is high.

Non-destructive internal disorder detection of Conference pears by semantic segmentation of X-ray CT scans using deep learning

Long term storage is required to deliver high quality pear fruit year-round. Under suboptimal storage conditions, internal disorders, such as internal browning and cavity formation, can develop and are often invisible from the outside. We present a non-destructive inspection method to quantify internal disorders in X-ray CT scans of pear fruit using a deep neural network for semantic segmentation. Herein, a U-net based model was trained to automatically indicate healthy tissue, core and regions affected by internal disorders, i.e., cavity formation and internal browning. The quantitative data resulting from the segmentations was used to measure the severity of internal disorders. Excellent classification accuracies of 99.4 and 92.2% were obtained for the classification of “consumable” vs “non-consumable” fruit on the one hand and “healthy” vs “defect but consumable” vs “non-consumable” fruit on the other hand. The identification of “defect but consumable” fruit showed to be the most difficult.

Nondestructive internal quality inspection of pear fruit by X-ray CT using machine learning

To preserve the quality of fresh pear fruit after harvest and deliver quality fruit year-round a controlled supply chain and long-term storage are applied. During storage, however, internal disorders can develop due to suboptimal storage conditions that may not cause externally visible symptoms. This makes them impossible to be detected by current commercial quality grading systems in a reliable and non-destructive way. A combination of a Support Vector Machine coupled with a feature extraction algorithm and X-ray Computed Tomography is proposed to successfully detect internal disorders in ‘Conference’ and ‘Cepuna’ pear fruit nondestructively. Classifiers were able to distinguish defective from sound fruit with classification accuracies ranging between 90.2 and 95.1% depending on the cultivar and number of used features. Moreover, low false positive and negative rates were obtained, respectively ranging between 0.0 and 6.7%, and 5.7 and 13.3%. Classifiers trained on ‘Conference’ data were transferred effectively to the ‘Cepuna’ cultivar, suggesting generalizability to other cultivars as well. With continuing developments in both hardware and software to increase inspection speed and reduce equipment costs, the method can be implemented in industrial applications, e.g., inline translational X-ray CT.

Quantitative evaluation of bioaerosols in different particle size fractions in dust collected on the International Space Station (ISS).

Exposure to bioaerosols can adversely influence human health through respiratory tract, eye, and skin irritation. Bioaerosol composition is unique on the International Space Station (ISS), where the size distribution of particles in the air differs from those on Earth. This is due to the lack of gravitational settling and sources of biological particles. However, we do not understand how microbes are influenced by particle size in this environment. We analyzed two types of samples from the ISS: (1) vacuum bag debris which had been sieved into five different size fractions and (2) passively collected particles on a tape substrate with a passive aerosol sampler. Using quantitative polymerase chain reaction (qPCR), the highest concentration of fungal spores was found in the 106-150 μm-sized sieved dust particles, while the highest concentration of bacterial cells was found in the 150-250 μm-sized sieved dust particles. Illumina MiSeq DNA sequencing revealed that particle size was associated with bacterial and fungal communities and statistically significant (p = 0.035, p = 0.036 respectively). Similar fungal and bacterial species were found within the passive aerosol sample and the sieved dust samples. The most abundant fungal species identified in the aerosol and sieved samples are commonly found in food and plant material. Abundant bacterial species were most associated with the oral microbiome and human upper respiratory tract. One limitation to this study was the suboptimal storage conditions of the sieved samples prior to analysis. Overall, our results indicate that microbial exposure in space may depend on particle size. This has implications for ventilation and filtration system design for future space vehicles and habitats.

Storage and Transportation of "HIV" RNA in Plasma Samples up to 45°C in a Lyophilized Stabilizer.

Blood or plasma samples from rural areas are often transported under suboptimal conditions to central laboratories. The negative influence of different storage temperatures during transportation as well as long transportation times on the stability of unprotected HIV RNA is well known. Therefore, the correct and reliable quantification of HIV RNA might be very difficult. A stabilization solution for the storage and transportation of plasma samples was developed which stabilizes RNA for seven days up to 45°C without viral load changes. Blood samples from HIV positive individuals were collected into EDTA containing tubes. The isolated plasma samples in Germany were pipetted into pre-prepared RNA stabilization tubes and incubated for seven days at 45°C. HIV-1 RNA quantification was performed on a HIV-1 LCx m 2000 system from Abbott and a Qiagen/Artus HI Virus-1 RG RT-PCR Kit on a Rotor-Gene Q PCR machine. In addition, plasma samples were collected and tested using existing SOP for storage and transportation in Nigeria. Plasma samples were treated with and without stabilization solution and the AMPLICOR HIV-1 MONITORTM test was used to determine viral load. Seventy-four stabilized plasma samples were tested in Germany and results were compared to those tested unprotected within two hours. No significant changes of viral load were detected up to seven days and 45°C in case of stabilized samples. In contrast RNA of the same unprotected samples was no longer detectable after one day at 45°C. Additionally, 22 plasma samples were investigated on day zero and under field conditions in Nigeria without changes of the viral load after seven days under given temperature conditions. No cooling chain is necessary for the storage and/or transportation of plasma samples treated with the new RNA stabilization solution for up to seven days. The use of this solution to preserve plasma RNA will be very helpful in countries where the environmental temperature is higher than 30°C, thus addressing the problem of unreliable viral load results due to suboptimal storage or transportation conditions. Further, the costs of storage and transportation of samples for viral load quantification could be significantly reduced.

Non-destructive and rapid discrimination of hard-to-cook beans using hyperspectral imaging

Dry beans stored under sub-optimal conditions tend to develop hard-to-cook (HTC) defect, which extends the cooking time making them less palatable while reducing their nutritional value. The current methods of identifying HTC beans are time-consuming, destructive, and unreliable. A rapid non-destructive inspection technique for pre-screening beans could help identify and discard HTC beans prior to processing. To this end, the potential of hyperspectral imaging technique covering the entire visible to near infrared (NIR) spectral range (400‒2500 nm) was evaluated for rapid and non-destructive identification of HTC beans. The HTC phenomenon was artificially induced in healthy white beans using two different combinations of suboptimal storage conditions of temperature and relative humidity (35℃, 75% RH for 45 days and 60℃, 75% RH for 10 days). Subsequently, the beans were cooked for specified durations and their hardness measured using a texture analyzer. The HTC and control (i.e. easy-to-cook (ETC)) beans were scanned with push-broom hyperspectral imaging systems. Results indicate that both sets of storage conditions rendered the beans HTC but the phenomenon induced by the two different methods was detected in different spectral ranges using hyperspectral imaging. Wavelengths across the entire visible and NIR ranges of electromagnetic spectrum were found useful in detecting HTC as beans stored at 35℃ and 75% RH for 45 days were identified mainly in the 1000‒2500 nm range and those stored at 60℃ and 75% RH for 10 days were identified in the 400‒1000 nm region. The degree of HTC defect could not be ascertained using this technique and requires further investigation.

Orientation and Training of New Biobank Personnel.

The personnel who operate a biomedical biobank should function as a unit to efficiently manage the numerous types of biospecimens that are to be utilized for both clinical and research purposes. Therefore, new staff must be appropriately trained before becoming fully integrated into the work environment. This chapter focuses on several key aspects to this training that should be completed by all personnel. This first step is an orientation where the new trainee is provided with the priorities and expectations of the biobank. The next and perhaps most important step is training on the various safety precautions. The trainee should learn how to protect patient privacy if human biospecimens are involved. They should gain a basic understanding of different types of biospecimens and their vulnerabilities to suboptimal storage conditions. The trainee must learn the various aspects of the day to day work which encompasses the methods and equipment needed for procuring, labeling, handling, tracking, storing, disbursing, and shipping biospecimens. They should become familiar with aspects of quality assurance.

Platelet storage properties are associated with donor age: in vitro quality of platelets from young donors and older donors with and without Type 2 diabetes.

Previously it has been shown that platelet (PLT) storage performance is consistent by donor. Differences involved metabolic activity, which might be caused by mitochondrial (dys)function, associated with age and age-related diseases like Type 2 diabetes (T2D). We aimed to test PLTs from young donors in comparison with PLTs from older donors with or without diagnosis for T2D. Fifteen whole blood donors <30year were selected, and single-donor platelet concentrates (sPC) were prepared from buffy coats (BC) and plasma. Also, 2×11 sPC were prepared from matched donors >45years with and without T2D. The sPC were stored for 8days and analysed at regular intervals for invitro quality. Donors were 24±3, 60±7 (without T2D) and 59±8 (with T2D) years old. All sPC groups had comparable volume and PLT content. On Day 8, sPC from young donors showed higher pH37°C than sPC from older donors (6.84±0.15 vs. 6.40±0.48, P<0.01), due to lower lactate production. Also, CD62P expression (22.9±7.4 vs. 48.8±24.0%, P<0.01) and HSR reflected better invitro quality. PLT storage properties of sPC obtained from T2D donors (pH=6.51±0.35) were not different from sPC of matched donors (pH=6.40±0.48). No differences in mitochondrial membrane potential were detected between the groups. Platelets from young donors exhibited the best storage conditions. On average, PLTs from older donors showed poorer invitro quality but, considering the sub-optimal storage conditions, the implications for the daily blood bank routine is probably small. No association with T2D was found.

Diagnostic Guidance for C. difficile Infections.

Diagnosis of Clostridium difficile infection (CDI) can be challenging. First of all, there has been debate on which of the two reference assays, cell cytotoxicity neutralization assay (CCNA) or toxigenic culture (TC) should be considered the gold standard for CDI detection. Although the CCNA suffers most from suboptimal storage conditions and subsequent toxin degradation, TC is reported to falsely increase CDI detection rates as it cannot differentiate CDI patients from patients asymptomatically colonised by toxigenic C. difficile. Several rapid assays are available for CDI detection and fall into three broad categories: (1) enzyme immunoassays for glutamate dehydrogenase, (2) enzyme immunoassays for toxins A/B and (3) nucleic acid amplification tests detecting toxin genes. All three categories have their own limitations, being suboptimal specificity and/or sensitivity or the inability to discern colonised patients from CDI patients. In light of these limitations, multi-step algorithmic testing has now been advocated by international guidelines in order to optimize diagnostic accuracy. Despite these recommendations, testing methods between hospitals vary widely, which impacts CDI incidence rates. CDI incidence rates are also influenced by sample selection criteria, as several studies have shown that if not all unformed stool samples are tested for CDI, many cases may be missed due to an absence of clinical suspicion. Since methods for diagnosing CDI remain imperfect, there has been a growing interest in alternative testing strategies like faecal biomarkers, immune modulating interleukins, cytokines and imaging methods. At the moment, these alternative methods might play an adjunctive role, but they are not suitable to replace conventional CDI testing strategies.

Nondestructive Apple Ripening Stage Determination Using the Delta Absorbance Meter at Harvest and after Storage

The delta absorbance (DA) meter is a handheld instrument which noninvasively measures the chlorophyll content in fruits. In the present work, it was used to monitor the ripening process linked to the climacteric phase in apple (Malus ×domestica). The results [index of absorbance difference (IAD)] were correlated to quality attributes at harvest and after commercial scale storage at different conditions. Two cultivars (Red Delicious, Golden Delicious) were analyzed in two different seasons, whereas Morgenduft and Gala were analyzed only in the first and second seasons, respectively. In general, a linear reduction of the IAD values was observed in all apple cultivars along with the progression of ripening and ethylene biosynthesis. When ethylene production was inhibited by 1-methylcyclopropene (1-MCP) treatment, the decrease of IAD values was markedly reduced. IAD threshold values for each cultivar were identified, delineating the central phase of the ethylene climacteric rise. Predictive models were built by correlating IAD index to the soluble solids concentration (SSC), titratable acidity (TA), and firmness measured at harvest and after removal from different storage regimes. The best model was developed for SSC prediction on ‘Red Delicious’ apple [ratio performance deviation (RPD) = 1.88] and for firmess evaluation in ‘Golden Delicious’ apple (RPD = 1.84). Moreover, IAD values were consistently associated with the differences in fruit quality as affected by optimal and suboptimal storage conditions. The IAD, due to its acceptable accuracy and speed of assessment, can be a promising tool for assisting in sorting apples before and after storage in warehouses or commercial packing lines. IAD cannot totally replace standard ripening indices, but can effectively supplement data for these parameters.

Toward Optimal Cryopreservation and Storage for Achievement of High Cell Recovery and Maintenance of Cell Viability and T Cell Functionality.

Cryopreservation of biological materials such as cells, tissues, and organs is a prevailing topic of high importance. It is employed not only in many research fields but also in the clinical area. Cryopreservation is of great importance for reproductive medicine and clinical studies, as well as for the development of vaccines. Peripheral blood mononuclear cells (PBMCs) are commonly used in vaccine research where comparable and reliable results between different research institutions and laboratories are of high importance. Whereas freezing and thawing processes are well studied, controlled, and standardized, storage conditions are often disregarded. To close this gap, we investigated the influence of suboptimal storage conditions during low-temperature storage on PBMC viability, recovery, and T cell functionality. For this purpose, PBMCs were isolated and exposed with help of a robotic system in a low-temperature environment from 0 up to 350 temperature fluctuation cycles in steps of 50 cycles to simulate storage conditions in large biorepositories with sample storage, removal, and sorting functions. After the simulation, the viability, recovery, and T cell functionality were analyzed to determine the number of temperature rises, which ultimately lead to significant cell damage. All studied parameters decreased with increasing number of temperature cycles. Sometimes after as little as only 50 temperature cycles, a significant effect was observed. These results are very important for all fields in which cell cryopreservation is employed, particularly for clinical and multicenter studies wherein the comparability and reproducibility of results play a crucial role. To obtain reliable results and to maintain the quality of the cells, not only the freezing and thawing processes but also the storage conditions should be controlled and standardized, and any deviations should be documented.

Initial Accuracy of HIV Rapid Test Kits Stored in Suboptimal Conditions and Validity of Delayed Reading of Oral Fluid Tests.

ObjectivesTo evaluate the effect of storing commonly used rapid diagnostic tests above manufacturer-recommended temperature (at 37°C), and the accuracy of delayed reading of oral fluid kits with relevance to HIV self-testing programmes.DesignA quality assurance study of OraQuick (OraSure), Determine HIV 1/2™ (Alere) and Uni-Gold™ (Recombigen®).MethodsConsecutive adults (≥18y) attending Ndirande Health Centre in urban Blantyre, Malawi in January to April 2012 underwent HIV testing with two of each of the three rapid diagnostic test kits stored for 28 days at either 18°C (optimally-stored) or at 37°C (pre-incubated). Used OraQuick test kits were stored in a laboratory for delayed day 1 and subsequent monthly re-reading was undertaken for one year.ResultsOf 378 individuals who underwent parallel testing, 5 (1.3%) were dropped from the final analysis due to discordant or missing reference standard results (optimally-stored Determine and Uni-Gold). Compared to the diagnostic reference standard, OraQuick had a sensitivity of 97.2% (95% CI: 93.6–99.6). There were 7 false negative results among all test kits stored at 37°C and three false negatives among optimally stored kits. Excellent agreement between pre-incubated tests and optimally-stored tests with Kappa values of 1.00 for Determine and Uni-Gold; and 0.97 (95% CI: 0.95; 1.00) for OraQuick were observed. There was high visual stability on re-reading of OraQuick, with only 1/375 pre-incubated and 1/371 optimally-stored OraQuick kits changing from the initial result over 12 months.ConclusionErroneous results observed during HIV testing in low income settings are likely to be due to factors other than suboptimal storage conditions. Re-reading returned OraQuick kits may offer a convenient and accurate quality assurance approach, including in HIV self-testing programmes.