-
Journal of Clinical Microbiology Nov 2021Diarrhea is a leading cause of death in children under five. Molecular methods exist for the rapid detection of enteric pathogens; however, the logistical costs of...
Diarrhea is a leading cause of death in children under five. Molecular methods exist for the rapid detection of enteric pathogens; however, the logistical costs of storing stool specimens limit applicability. We sought to demonstrate that dried specimens preserved using filter paper can be used to identify diarrheal diseases causing significant morbidity among children in resource-constrained countries. A substudy was nested into cholera surveillance in Cameroon. Enrollment criteria included enrollment between 1 August 2016 and 1 October 2018, age of <18 years, availability of a stool specimen, and having three or more loose stools within 24 h with the presence of dehydration and/or blood. A total of 7,227 persons were enrolled, of whom 2,746 met enrollment criteria and 337 were included in this analysis using the enteric TaqMan array card. Bacterial pathogens were compared to severity of diarrhea, age, and sex, among other variables. One hundred seven were positive for enterotoxigenic Escherichia coli, of which 40.2% ( = 43) had heat-labile enterotoxin (LT) and the heat-stable enterotoxin STh, 19.6% ( = 21) had LT and the heat-stable enterotoxin STp, and 49.5% ( = 53) had LT only. Major colonization factors (CFs) were present in 43.9% of enterotoxigenic E. coli (ETEC)-positive patients. Ninety-six were positive for , of whom 14 (14.6%) reported dysentery. Model-derived quantitative cutoffs identified 116 (34.4%) with one highly diarrhea-associated pathogen and 16 (4.7%) with two or more. and rotavirus were most strongly associated with diarrhea in children with mixed infections. Dried-filter-paper-preserved specimens eliminate the need for frozen stool specimens and will facilitate enteric surveillance and contribute to the understanding of disease burden, which is needed to guide vaccine development and introduction. This study confirms rotavirus, , and ETEC as major contributors to pediatric diarrheal disease in two regions of Cameroon.
Topics: Adolescent; Child; Diarrhea; Enterotoxigenic Escherichia coli; Enterotoxins; Escherichia coli Infections; Humans
PubMed: 34524885
DOI: 10.1128/JCM.01703-21 -
PloS One 2019Blood collection, transportation and storage remain a problem in countries where infrastructure, laboratory facilities and skilled manpower are scarce. This limits...
BACKGROUND
Blood collection, transportation and storage remain a problem in countries where infrastructure, laboratory facilities and skilled manpower are scarce. This limits evaluation of immune responses in natural infections and vaccination in field studies. We developed methods to measure antigen specific antibody responses using dried blood spot (DBS) in cholera, ETEC and typhoid fever patients as well as recipients of oral cholera vaccine (OCV).
METHODOLOGY/PRINCIPLE FINDINGS
We processed heparinized blood for preparing DBS and plasma specimens from patients with, cholera, ETEC and typhoid as well as OCV recipients. We optimized the conventional vibriocidal method to measure vibriocidal antibody response in DBS eluates. We measured responses in DBS samples and plasma (range of titer of 5 to 10240). Vibriocidal titer showed strong agreement between DBS eluates and plasma in cholera patients (ICC = 0.9) and in OCV recipients (ICC = 0.8) using the Bland-Altman analysis and a positive correlation was seen (r = 0.7, p = 0.02 and r = 0.6, p = 0.006, respectively). We observed a strong agreement of lipopolysaccharide (LPS) and cholera toxin B (CTB)-specific antibody responses between DBS eluates and plasma in cholera patients and OCV recipients. We also found agreement of heat labile toxin B (LTB) and membrane protein (MP)-specific antibody responses in DBS eluates and plasma specimen of ETEC and typhoid patients respectively.
CONCLUSION
Our results demonstrate that dried blood specimens can be used as an alternate method for preservation of samples to measure antibody responses in enteric diseases and vaccine trials and can be applied to assessment of responses in humanitarian crisis and other adverse field settings.
Topics: Antibody Formation; Blood Specimen Collection; Cholera; Cholera Vaccines; Dried Blood Spot Testing; Enterotoxigenic Escherichia coli; Female; Humans; Intestinal Diseases; Male; Middle Aged; Typhoid Fever
PubMed: 31206533
DOI: 10.1371/journal.pone.0218353 -
Journal of Human Genetics Aug 2017Gaucher disease (GD) is an inherited metabolic disorder that involves accumulation of glycolipid glucocerebroside in monocyte-macrophage cells, which can result in...
Gaucher disease (GD) is an inherited metabolic disorder that involves accumulation of glycolipid glucocerebroside in monocyte-macrophage cells, which can result in multiple organ damage. Enzyme replacement and substrate reduction therapies have improved the potential for early diagnosis and treatment. Determining the true incidence of this rare disease is critical for relevant policy establishment. Newborn screening allows for early diagnosis and an comparatively accurate incidence of GD. A fluorometric method to detect acid β-glucocerebrosidase (GBA) activity on a dried blood spot punch was developed. Validity and feasibility of the fluorometric method was demonstrated by examining 116 healthy controls, 19 confirmed GD patients and 19 obligate carriers. GBA activity was measured on dried blood spots of 80 855 newborns. Samples from positively screened newborns were reanalyzed by a leukocyte GBA activity test and GBA gene analysis. Plasma glucosylsphingosine level was determined as a biomarker of the pathophysiology of GD. GD patients were distinguished from healthy controls and obligate carriers using the fluorometric method. Mean GBA activity in newborn screening specimens was 145.69±44.76 μmol l h (n=80 844). Three children had low GBA activity, of which one child had low GBA activity on the second dried blood spot specimen. Leukocyte, genetic and biomarker analysis confirmed the diagnosis and indicated that this child was in the early stages of GD. In conclusion, the incidence of GD in Shanghai of China is approximately 1 in 80 855. Screening for GD by fluorometric analysis of GBA activity is an efficient and feasible technology in newborns.
Topics: Adolescent; Adult; Blood Specimen Collection; Case-Control Studies; Child; Child, Preschool; China; Dried Blood Spot Testing; Female; Fluorometry; Gaucher Disease; Glucosylceramidase; Humans; Infant, Newborn; Male; Middle Aged; Neonatal Screening; Psychosine; Young Adult
PubMed: 28356566
DOI: 10.1038/jhg.2017.36 -
AIDS (London, England) May 2017: We evaluated detection of HIV-1 RNA from dried blood spots (DBS) and oral fluid specimens. Between February 2010 and August 2014, HIV-1 was newly diagnosed in eight...
: We evaluated detection of HIV-1 RNA from dried blood spots (DBS) and oral fluid specimens. Between February 2010 and August 2014, HIV-1 was newly diagnosed in eight (2.6%) study participants who had median blood HIV-1 RNA of 61 500 copies/ml (interquartile range 7500-146 000). RNA was detected in seven (87.5%) DBS and three (37.5%) oral fluid swabs but was not detected in either specimen from one participant. DBS may be a reasonable specimen collection method to detect acute infection.
Topics: Adult; Blood; Cross-Sectional Studies; HIV Infections; HIV-1; Humans; Male; Middle Aged; Prospective Studies; RNA, Viral; Saliva; Young Adult
PubMed: 28358729
DOI: 10.1097/QAD.0000000000001477 -
Journal of Applied Oral Science :... 2018The proper selection of polymerization cycle is important to prevent overheating of the monomer that could cause degradation, porosity and, consequently, deleterious...
The proper selection of polymerization cycle is important to prevent overheating of the monomer that could cause degradation, porosity and, consequently, deleterious effects on the denture base properties. Objective This study evaluated the porosity, water sorption and solubility of acrylic resins (Vipi Cril-VC and Vipi Wave-VW) after conventional or microwave polymerization cycles. Material and Methods Specimens (n = 10) were made and cured: 1-WB = 65°C during 90 min + boiling during 90 min (VC cycle - control group); 2-M25 = 10 min at 270 W + 5 min at 0 W + 10 min at 360 W (VW cycle); 3-M3 = 3 min at 550 W; and 4-M5 = 5 min at 650 W. Afterward, they were polished and dried in a dessicator until a constant mass was reached. Specimens were then immersed in distilled water at 37°C and weighed regularly until a constant mass was achieved. For porosity, an additional weight was made with the specimen immediately immersed in distilled water. For water sorption and solubility, the specimens were dried again until equilibrium was reached. Data were submitted to 2 way-ANOVA and Tukey HSD (α=0.05). Results Porosity mean values below 1.52% with no significant difference among groups for both materials were observed. Resins showed water sorption and solubility values without a significant difference. However, there was a significant difference among groups for these both properties (P<0.013). The highest sorption (2.43%) and solubility (0.13%) values were obtained for WB and M3, respectively. Conclusions The conventional acrylic resin could be polymerized in a microwave since both the materials showed similar performance in the evaluated properties. Shorter microwave cycles could be used for both the materials without any detectable increase in volume porosity.
Topics: Acrylic Resins; Analysis of Variance; Denture Bases; Materials Testing; Microwaves; Polymerization; Porosity; Reproducibility of Results; Solubility; Time Factors; Water
PubMed: 29742260
DOI: 10.1590/1678-7757-2017-0383 -
Journal of Clinical Microbiology Apr 2009The development and validation of dried sample spots as a method of specimen collection are urgently needed in developing countries for monitoring of human... (Comparative Study)
Comparative Study
Evaluation of different RNA extraction methods and storage conditions of dried plasma or blood spots for human immunodeficiency virus type 1 RNA quantification and PCR amplification for drug resistance testing.
The development and validation of dried sample spots as a method of specimen collection are urgently needed in developing countries for monitoring of human immunodeficiency virus (HIV) infection. Our aim was to test some crucial steps in the use of dried spots, i.e., viral recovery and storage over time. Moreover, we investigated whether dried plasma and blood spots (DPS and DBS, respectively) give comparable viral load (VL) results. Four manual RNA extraction methods from commercial HIV type 1 (HIV-1) VL assays--a QIAamp minikit (Qiagen), the Abbott Molecular sample preparation system, the Nuclisens assay (bioMarieux), and High Pure viral nucleic acid kit (Roche Applied Science)--were compared for VL quantification and PCR amplification for genotypic drug resistance testing on dried spots from spiked plasma and residual samples from HIV-1 patients (n = 47; median VL, 4.13 log(10) copies/ml). RNA recovery from DPS was efficient using Nuclisens extraction (median difference, 0.03 log(10) copies/ml) and slightly underestimated using the Abbott Molecular sample preparation system (median difference, 0.35 log(10) copies/ml). PCR amplification results were in concordance. Measurements from DBS overestimated VL for plasma, with VL results showing <3.7 log(10) copies/ml. VL was stable for up to 3 months in spiked DPS stored at 20 degrees C but for only 1 month at 37 degrees C. A faster decline was observed in PCR efficiency: DPS could be stored for 1 week at 37 degrees C and for 1 month at 20 degrees C. In conclusion, the RNA extraction method is an important factor in obtaining reliable RNA quantification and PCR amplification of HIV-1 on DPS/DBS. DBS could be used as an alternative for DPS depending on HIV RNA cutoffs for virological failure. VL measurements remain stable over a longer period than do PCR amplification results.
Topics: Blood; Desiccation; Drug Resistance, Viral; HIV Infections; HIV-1; Humans; Molecular Diagnostic Techniques; Plasma; Polymerase Chain Reaction; RNA, Viral; Reagent Kits, Diagnostic; Specimen Handling; Temperature; Time Factors; Viral Load
PubMed: 19193835
DOI: 10.1128/JCM.02255-08 -
Scientific Reports Feb 2022At-home sampling is key to large scale seroprevalence studies. Dried blood spot (DBS) self-sampling removes the need for medical personnel for specimen collection but...
At-home sampling is key to large scale seroprevalence studies. Dried blood spot (DBS) self-sampling removes the need for medical personnel for specimen collection but facilitates specimen referral to an appropriately accredited laboratory for accurate sample analysis. To establish a highly sensitive and specific antibody assay that would facilitate self-sampling for prevalence and vaccine-response studies. Paired sera and DBS eluates collected from 439 sero-positive, 382 sero-negative individuals and DBS from 34 vaccine recipients were assayed by capture ELISAs for IgG and IgM antibody to SARS-CoV-2. IgG and IgM combined on DBS eluates achieved a diagnostic sensitivity of 97.9% (95%CI 96.6 to 99.3) and a specificity of 99.2% (95% CI 98.4 to 100) compared to serum, displaying limits of detection equivalent to 23 and 10 WHO IU/ml, respectively. A strong correlation (r = 0.81) was observed between serum and DBS reactivities. Reactivity remained stable with samples deliberately rendered inadequate, (p = 0.234) and when samples were accidentally damaged or 'invalid'. All vaccine recipients were sero-positive. This assay provides a secure method for self-sampling by DBS with a sensitivity comparable to serum. The feasibility of DBS testing in sero-prevalence studies and in monitoring post-vaccine responses was confirmed, offering a robust and reliable tool for serological monitoring at a population level.
Topics: Antibodies, Viral; Biomarkers; COVID-19; COVID-19 Testing; COVID-19 Vaccines; Dried Blood Spot Testing; Feasibility Studies; Female; Humans; Immunoglobulin G; Immunoglobulin M; Male; SARS-CoV-2; Sensitivity and Specificity; Seroepidemiologic Studies; Specimen Handling
PubMed: 35115570
DOI: 10.1038/s41598-022-05640-x -
Journal of Clinical Microbiology May 2009A novel method for the collection and transportation of dried-blood-plasma samples, SampleTanker (ST), was developed and compared to standard shipping protocols for... (Comparative Study)
Comparative Study
A novel method for the collection and transportation of dried-blood-plasma samples, SampleTanker (ST), was developed and compared to standard shipping protocols for frozen-plasma specimens containing human immunodeficiency virus type 1 (HIV-1) and/or hepatitis C virus (HCV). Matched frozen and dried 1-ml EDTA-containing plasma samples were collected and analyzed by several molecular-based virologic assays. After addition of 1.175 ml of reconstitution buffer, 1.035 ml of dried plasma was recovered. Mean intra-assay variances were 0.05, 0.05, and 0.06 log(10) copies/ml for the Versant, Amplicor, and NucliSens QT HIV-1 load assays, respectively (P, not significant). However, mean HIV-1 viral load was consistently reduced in dried samples by 0.32 to 0.51 log(10) copies/ml, depending on assay type (P < 0.05). Infectious HIV-1 was not recovered from dried ST plasma. There was no significant difference in HIV-1 viral load results obtained using ST after 8 weeks of storage at ambient temperature. Compared to frozen plasma, HIV-1 genotypic results were >99% concordant at the nucleotide and amino acid levels, as well as for resistance-associated mutations. We further demonstrated successful detection of multiple analytes, including HIV-1 viral load, HIV-1 antiretroviral resistance genotype, and HCV genotype, from a single ST unit. Dried plasma collected with ST yielded comparable results to frozen samples for multiple-analyte clinical testing. As such, ST could be a useful alternative for virologic tests and clinical trials worldwide by significantly diminishing transportation cost and the sample volume restrictions associated with dried-blood-spot technology.
Topics: Desiccation; Genotype; HIV; HIV Infections; Hepacivirus; Hepatitis C; Humans; Microbial Sensitivity Tests; Plasma; Reproducibility of Results; Specimen Handling; Viral Load
PubMed: 19321732
DOI: 10.1128/JCM.02354-08 -
PloS One 2023Proficiency testing (PT) has been hard to set up due to cost limitations and technical capacity. Conventional Xpert MTB/RIF PT programs use liquid and culture spots...
BACKGROUND
Proficiency testing (PT) has been hard to set up due to cost limitations and technical capacity. Conventional Xpert MTB/RIF PT programs use liquid and culture spots which require stringent storage and transportation conditions with cross-contamination chances prevalent. These setbacks prompted the use of dried tube specimens (DTS) for Ultra assay PT. For continuity of PT provision, stability of DTS and compatibility with testing protocols when kept for a long period needs to be established.
METHODS
DTS were prepared from known isolates inactivated using a hot air oven at 85°C. 100μl of bacterial suspensions were aliquoted and dried inside a Biosafety cabinet. Panel validation was done to establish the baseline Deoxyribonucleic acid (DNA) concentration in terms of cycle threshold (Ct) value. DTS aliquots were shipped to participants to test and report within six weeks. The remaining DTS were kept at 2-8°C and room temperature for one year with testing at six months. Twenty (20) DTS samples per set remaining at one year were heated at 55°C for two weeks before testing. The means of the different samples were compared to validation data using paired t-tests. Boxplots were designed to visualize the differences in the medians of the DTS.
RESULTS
Overall mean Ct value increased by 4.4 from the validation to testing after one year at the different storage conditions. Samples heated at 55°C showed a 6.4 Ct difference from validation data. Testing done at six months on 2-8°C stored items showed no statistical difference. At all the remaining testing times and conditions, P-values were less than 0.008 although the absolute mean Ct when compared showed slight increments and accommodated differences for the detection of MTB and rifampicin resistance. Median values for samples stored at 2-8°C were lower compared to those at room temperature.
CONCLUSION
DTS stored at 2-8°C remain more stable for one year compared to higher temperatures and can be consistently used as PT materials in more than one PT round for biannual PT providers.
Topics: Humans; Resource-Limited Settings; Uganda; Laboratory Proficiency Testing; Rifampin; Mycobacterium tuberculosis; Sensitivity and Specificity
PubMed: 36897841
DOI: 10.1371/journal.pone.0282650 -
Journal of Virological Methods Dec 2022This study is the first proof of concept of the DBS technology for Bovine alphaherpesvirus 1 (BoHV-1) antibody detection by ELISA after fully automated DBS extraction....
This study is the first proof of concept of the DBS technology for Bovine alphaherpesvirus 1 (BoHV-1) antibody detection by ELISA after fully automated DBS extraction. DBS were prepared from nine BoHV-1 seropositive plasma samples spiked with erythrocytes. Spots were extracted automatically on a DBS-MS 500 HCT autosampler, as well as manually using a 3.2 mm puncher. DBS were equally prepared from 20 bovine seronegative EDTA-blood samples and extracted automatically. Extracts were tested in a commercial BoHV-1 antibody ELISA and results were compared with those from liquid plasma. Eight seropositive DBS samples were additionally tested in the ELISA after storage for four weeks at different conditions. After automated extraction all DBS samples yielded qualitatively correct results and were in full accordance with those obtained from liquid plasma. Automated extraction using a 6 mm extraction head was more sensitive than a 4 mm head. Stability of DBS was highest at - 20 °C and decreased with increasing temperature. Even after four weeks at 37 °C, most seropositive samples yielded a positive result in the ELISA. The minimal invasiveness, biosafety, and simplicity of DBS collection together with automated extraction represents an interesting, high-throughput compatible alternative to liquid blood samples for BoHV-1 monitoring or eradication programs.
Topics: Tandem Mass Spectrometry; Dried Blood Spot Testing; Edetic Acid; Enzyme-Linked Immunosorbent Assay; Specimen Handling
PubMed: 36182002
DOI: 10.1016/j.jviromet.2022.114626