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Gachogo, Rachael W., Daniel N. Mwai, and Frank G. Onyambu. "Cost analysis of implementing HIV drug resistance testing in Kenya: a case study of a service delivery site at a tertiary level hospital in Kenya." F1000Research 9 (July 29, 2020): 793. http://dx.doi.org/10.12688/f1000research.23379.1.
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Background: HIV drug resistance (HIVDR) threatens progress achieved in response to the HIV epidemic. Understanding the costs of implementing HIVDR testing programs for patient management and surveillance in resource-limited settings is critical in optimizing resource allocation. Here, we estimate the unit cost of HIVDR testing and identify major cost drivers while documenting challenges and lessons learnt in implementation of HIVDR testing at a tertiary level hospital in Kenya. Methods: We employed a mixed costing approach to estimate the costs associated with performing a HIVDR test from the provider’s perspective. Data collection involved a time and motion study of laboratory procedures and interviewing laboratory personnel and the management personnel. Cost analysis was based on estimated 1000 HIVDR tests per year. Data entry and analysis were done using Microsoft Excel and costs converted to US dollars (2019). Results: The estimated unit cost for a HIVDR test was $271.78 per test. The main cost drivers included capital ($102.42, 37.68%) and reagents (101.50, 37.35%). Other costs included: personnel ($46.81, 17.22%), utilities ($14.69, 5.41%), equipment maintenance costs ($2.37, 0.87%) and quality assurance program ($4, 1.47%). Costs in relation to specific laboratory processes were as follows: sample collection ($2.41, 0.89%), RNA extraction ($22.79, 8.38%), amplification ($56.14, 20.66%), gel electrophoresis ($10.34, 3.80%), sequencing ($160.94, 59.22%), and sequence analysis ($19.16, 7.05%). A user-initiated modification of halving reagent volumes for some laboratory processes (amplification and sequencing) reduced the unit cost for a HIVDR test to $233.81 (13.97%) reduction. Conclusions: Capital expenditure and reagents remain the most expensive components of HIVDR testing. This cost is bound to change as the sequencing platform is utilized towards maximum capacity or leveraged for use with other tests. Cost saving in offering HIVDR testing services is also possible through reagent volume reduction without compromising on the quality of test results.
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Solsvik, Anne Elisabeth, Ann Helen Kristoffersen, Sverre Sandberg, Gro Gidske, Anne Vegard Stavelin, Joakim Eikeland, and Erik Amundsen. "A national surveillance program for evaluating new reagent lots in medical laboratories." Clinical Chemistry and Laboratory Medicine (CCLM) 60, no. 3 (January 19, 2022): 351–60. http://dx.doi.org/10.1515/cclm-2021-1262.
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Abstract Objectives Differences between laboratory results attributable to the use of different reagent lots can potentially affect the diagnosis and monitoring of patients. To minimize patient risks, all laboratories should verify that new reagent lots meet agreed analytical performance specifications (APS). We propose a simplified, pragmatic approach for laboratories that involves compilating results into a national surveillance program, and present the first results obtained when applying this approach to troponins, glycated hemoglobin (HbA1c), prostate-specific antigen (PSA) and D-dimer. Methods In the surveillance program we have (i) determined APS for selected analytes, (ii) implemented a simplified procedure for lot evaluation with patient samples used in laboratories across Norway and (iii) performed central processing of the results from the participating laboratories. Results Over a one-year period, 27 Norwegian laboratories returned results from 28 lot changes for troponin I, 11 for troponin T, and 29 for HbA1c, PSA and D-dimer. The mean difference between two reagent lots was 4.5% for troponin I (for a concentration interval of 20–32 ng/L), 5.1% for troponin T (10.7–17.5 ng/L), 2.2% for HbA1c (40–50 mmol/mol), 3.7% for PSA (3–5 μg/L) and 5.5% for D-dimer (0.4–1.0 mg/L FEU). Conclusions A novel procedure for reagent lot evaluation is proposed in which information about multiple lot changes from different medical laboratories can be accumulated nationally. Sharing this information allows simplification of lot evaluations in individual laboratories and provides real-world data about lot-to-lot variations.
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Prokhvatilova, E. V., N. N. Teteryatnikova, I. B. Zakharova, L. I. Belitskaya, D. V. Viktorov, and A. V. Toporkov. "PREPARATION FOR STATE REGISTRATION OF THE REAGENT KIT FOR THE DETECTION AND DIFFERENTIATION OF THE DNA OF BURKHOLDERIA «PSEUDOMALLEI» GROUP." Russian Clinical Laboratory Diagnostics 64, no. 3 (April 29, 2019): 180–85. http://dx.doi.org/10.18821/0869-2084-2019-64-3-180-185.
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The reagent kit designed to detect and simultaneously differentiate the DNA of three species of Burkholderia pseudomallei - causative agents of melioidosis (B. pseudomallei), glanders (B. mallei) and B. thailandensis by the set of genes of β-lactamases with B and D molecular classes using a multiplex polymerase chain reaction with electrophoretic detection was developed for clinical laboratory diagnosis. The functional properties of the reagent kit were evaluated, tests were carried out, the stages of examination and registration in the Federal Service for Surveillance on Consumer Rights’ Protection and Human Well-being were completed. During clinical testing the effectiveness of the reagent kits in the study of various samples of clinical material and isolated cultures of microorganisms was confirmed. It has been established that the indicator of diagnostic sensitivity of the reagent kit for the detection and differentiation of the glanders, melioidosis and B. thailandensis causative agents was less than 99 %, diagnostic specificity - not less than 99 % with a confidence probability of 90 % in the analysis of each of the indicators.
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Castilho-Pelloso, Marcela Peres, Dina Lúcia Morais Falavigna, and Ana Lúcia Falavigna-Guilherme. "Suspected acute toxoplasmosis in pregnant women." Revista de Saúde Pública 41, no. 1 (February 2007): 27–34. http://dx.doi.org/10.1590/s0034-89102007000100005.
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OBJECTIVE: To determine the prevalence of reagent serology for suspected acute toxoplasmosis in pregnant women and to describe clinical, laboratory and therapeutic profiles of mothers and their children. METHODS: A retrospective study was conducted with IgM-anti-Toxoplasma gondii-reagent pregnant women and their children who attended the public health system in the state of Paraná, Southern Brazil, from January 2001 to December 2003. Information were obtained from clinical, laboratory (ELISA IgM/IgG) and ultrasonographic data and from interviews with the mothers. To test the homogeneity of the IgM indices in relation to the treatment used, the Pearson's Chi-square test was applied. Comparisons were considered significant at a 5% level. RESULTS: Two hundred and ninety (1.0%) cases of suspected IgM-reagent infection were documented, with a prevalence of 10.7 IgM-reagent women per 1,000 births. Prenatal care started within the first 12 weeks for 214/290; 146/204 were asymptomatic. Frequent complaints included headaches, visual disturbance and myalgia. Ultrasonography revealed abnormalities in 13 of 204 pregnancies. Chemoprophylaxis was administered to 112/227; a single ELISA test supported most decisions to begin treatment. Pregnant women with IgM indices =2.000 tended to be treated more often. Among exposed children, 44/208 were serologically followed up and all were IgG-reagent, and three IgM-reagent cases showed clinical symptoms. CONCLUSIONS: The existence of pregnant women with laboratorially suspected acute toxoplasmosis who were not properly followed up, and of fetuses that were not adequately monitored, shows that basic aspects of the prenatal care are not being systematically observed. There is need of implementing a surveillance system of pregnant women and their children exposed to T. gondii.
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Sitko, John C., James Jordan Steel, Erin A. Almand, Christopher A. Cullenbine, Joseph W. Rohrer, Douglas P. Wickert, and Steven CM Hasstedt. "Efficiency of pooled surveillance testing in academic labs to detect and inhibit COVID-19 outbreaks." Bioanalysis 13, no. 15 (August 2021): 1177–82. http://dx.doi.org/10.4155/bio-2021-0054.
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Robust surveillance testing is a key strategic plan to prevent COVID-19 outbreaks and slow the spread of the SARS-CoV-2 pandemic; however, limited resources, facilities and time often impair the implementation of a widespread surveillance effort. To mitigate these resource limitations, we employed a strategy of pooling samples, reducing reagent cost and processing time. Through utilizing academic faculty and labs, successful pooled surveillance testing was conducted throughout Fall 2020 semester to detect positive SARS-CoV-2 infections in a population of 4400 students. During the semester, over 25,000 individual COVID status evaluations were made by pooling eight individual samples into one quantitative reverse transcription polymerase chain reaction. This pooled surveillance strategy was highly effective at detecting infection and significantly reduced financial burden and cost by $3.6 million.
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Hapsari, Rebriarina, Irfan Kesumayadi, Nani Maharani, Endang Mahati, Ferdy Kurniawan Cayami, and Sutopo Patria Jati. "The efficiency of selective pooling strategy in a COVID-19 diagnostic laboratory." Journal of Infection in Developing Countries 16, no. 08 (August 30, 2022): 1278–84. http://dx.doi.org/10.3855/jidc.14359.
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Introduction: Mass testing is essential in the surveillance strategy for fighting the COVID-19 pandemic. It allows early detection of suspected cases and subsequently early isolation to mitigate spread. However, the high cost and limited consumables and reagents hinder the mass testing strategy in developing countries such as Indonesia. The specimen pooling strategy is an option to perform mass screening with limited resources. This study aims to determine the positivity rate cut-off and to evaluate the efficiency of pooling strategy for the laboratory diagnosis of COVID-19.
Methodology: Between August 4th, 2020, and November 11th, 2020, a four-sample pooling strategy testing to detect SARS-CoV-2 was carried out at the Microbiology Diagnostic Laboratory of Diponegoro National Hospital, Semarang, Indonesia. Pools with positive results were subjected to individual specimen retesting. Spearman’s correlation and linear regression analysis were used to determine the best positivity rate cut-off to apply pooling strategy.
Results: A total of 15,216 individual specimens were pooled into 3,804 four-sample pools. Among these pools, 1,007 (26.47%) were positive. Five hundred and ten (50.64%) were 1/4 positive. A maximum positivity rate of 22% is needed to save at least 50% extraction and qRT-PCR reactions in a four-sample pooling strategy. CT values between individual specimens and pools showed a good interval agreement.
Conclusions: Pooling strategy could reduce personnel workload and reagent cost, and increase laboratory capacity by up to 50% when the positivity rate is less than 22%.
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Gordon, Ilyssa O., Jodi D. Sherman, Michael Leapman, Michael Overcash, and Cassandra L. Thiel. "Life Cycle Greenhouse Gas Emissions of Gastrointestinal Biopsies in a Surgical Pathology Laboratory." American Journal of Clinical Pathology 156, no. 4 (April 5, 2021): 540–49. http://dx.doi.org/10.1093/ajcp/aqab021.
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Abstract Objectives Given adverse health effects of climate change and contributions of the US health care sector to greenhouse gas (GHG) emissions, environmentally sustainable delivery of care is needed. We applied life cycle assessment to quantify GHGs associated with processing a gastrointestinal biopsy in order to identify emissions hotspots and guide mitigation strategies. Methods The biopsy process at a large academic pathology laboratory was grouped into steps. Each supply and reagent was catalogued and postuse treatment noted. Energy consumption was estimated for capital equipment. Two common scenarios were considered: 1 case with 1 specimen jar (scenario 1) and 1 case with 3 specimen jars (scenario 2). Results Scenario 1 generated 0.29 kg of carbon dioxide equivalents (kg CO2e), whereas scenario 2 resulted in 0.79 kg CO2e—equivalent to 0.7 and 2.0 miles driven, respectively. The largest proportion of GHGs (36%) in either scenario came from the tissue processor step. The second largest contributor (19%) was case accessioning, mostly attributable to production of single-use disposable jars. Conclusions Applied to more than 20 million biopsies performed in the US annually, emissions from biopsy processing is equivalent to yearly GHG emissions from 1,200 passenger cars. Mitigation strategies may include modification of surveillance guidelines to include the number of specimen jars.
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Mosa, Alexander I. "CRISPR-Based Diagnostics for Point-of-Care Viral Detection." International Journal of Translational Medicine 2, no. 2 (June 1, 2022): 198–203. http://dx.doi.org/10.3390/ijtm2020017.
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Point-of-care detection of viral infection is required for effective contact-tracing, epidemiological surveillance, and linkage to care. Traditional diagnostic platforms relying on either antigen detection or nucleic amplification are limited by sensitivity and the need for costly laboratory infrastructure, respectively. Recently, CRISPR-based diagnostics have emerged as an alternative, combining equipment light workflows with high specificity and sensitivity. However, as a nascent technology, several outstanding challenges to widespread field deployment remain. These include the need for pre-detection amplification of target molecules, the lack of standardization in sample preparation and reagent composition, and only equivocal assessments of the unit-economics relative to traditional antigen or polymerase chain reaction-based diagnostics. This review summarizes recent advances with the potential to overcome existing translational barriers, describes the events in CRISPR-based detection of target molecules, and offers perspective on how multiple approaches can be combined to decrease the limit of detection without introducing pre-amplification.
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Nov, Vandarith, Darapheak Chau, Kimsorn Pa, Keodane Hem, and Sidonn Krang. "Proficiency Testing Performances Analysis of Microbiology Laboratories Participating in Cambodia Antimicrobial Resistance (AMR) Surveillance System." Infection Control & Hospital Epidemiology 41, S1 (October 2020): s360—s361. http://dx.doi.org/10.1017/ice.2020.984.
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Background: The WHO recommends the establishment of sustainable and evidence-based surveillance systems are recommended for the prevention of microbial resistance. For these surveillance systems, all medical microbiology laboratories are required to participate an external quality assessment (EQA) program that covers antimicrobial susceptibility testing (AST). Clinical microbiology EQA panels with 3 isolates have been provided 3 times per year to antimicrobial resistance (AMR) sentinel laboratories in Cambodia since 2012. We evaluated the performance of laboratory testing implemented between 2016 and 2019, based on 4 years EQA results to highlight the main sources of unsatisfactory analytical processes and to suggest areas for improvement. Methods: We analyzed the results of microbiology EQA in 7 AMR surveillance sentinel laboratories from 2016 to 2019, which were coordinated by the National Institute of Public Health (NIPH) under the program of Pacific Paramedical Training Centre (PPTC) from New Zealand. All participating laboratories were required to identify bacteria to the species level, to verify AST results, and to answer a case study question on parasitology. Feedback results and appropriate corrective actions were reviewed to identify the root cause of nonconformity and to suggest areas for improvement. Results: Proficiency test results of participating laboratories from 9 cycles with 27 isolates were analyzed. The overall average of EQA result was 94.0%. The laboratories failed to identify the isolated pathogens in 7.0% of the tests and failed to interpret the inhibition zone of AST (ie, resistant, intermediate or susceptible) in 6.0% of tested strains. The main causes of erroneous of PT results were either preanalytical (ie, handling of the samples, timing of analysis, equipment and reagent management), analytical (ie, quality control, unsuitable methods, confusion of samples, or errors of confirmation), or postanalytical mistakes (eg, interpretation guideline, cross-checking of results, or the information management system). Followed by the root causes, internal quality control and inventory management were the highest-priority suggestions for improvement. Conclusions: All participating laboratories showed good performance on EQA for evidence-based AMR surveillance. The national antimicrobial resistance data quality is sufficiently good and the data should be shared on national and international platforms. However, the regular monitoring of national AMR surveillance system should be conducted for continued quality improvement.Funding: NoneDisclosures: None
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Votintseva, Antonina A., Phelim Bradley, Louise Pankhurst, Carlos del Ojo Elias, Matthew Loose, Kayzad Nilgiriwala, Anirvan Chatterjee, et al. "Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples." Journal of Clinical Microbiology 55, no. 5 (March 8, 2017): 1285–98. http://dx.doi.org/10.1128/jcm.02483-16.
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ABSTRACT Routine full characterization of Mycobacterium tuberculosis is culture based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near the point of care. We demonstrate a low-cost method of DNA extraction directly from patient samples for M. tuberculosis WGS. We initially evaluated the method by using the Illumina MiSeq sequencer (40 smear-positive respiratory samples obtained after routine clinical testing and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction were obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. With Illumina MiSeq/MiniSeq, the workflow from patient sample to results can be completed in 44/16 h at a reagent cost of £96/£198 per sample. We then employed a nonspecific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis strain BCG DNA and to combined culture-negative sputum DNA and BCG DNA. For flow cell version R9.4, the estimated turnaround time from patient to identification of BCG, detection of pyrazinamide resistance, and phylogenetic placement was 7.5 h, with full susceptibility results 5 h later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis DNA in direct samples.
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Fry, Norman K., John Duncan, Karen Wagner, Oceanis Tzivra, Nita Doshi, David J. Litt, Natasha Crowcroft, Elizabeth Miller, Robert C. George, and Timothy G. Harrison. "Role of PCR in the diagnosis of pertussis infection in infants: 5 years' experience of provision of a same-day real-time PCR service in England and Wales from 2002 to 2007." Journal of Medical Microbiology 58, no. 8 (August 1, 2009): 1023–29. http://dx.doi.org/10.1099/jmm.0.009878-0.
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As part of an enhanced surveillance programme for pertussis in England and Wales, a real-time PCR service for the detection of Bordetella pertussis was introduced for infants aged ≤6 months admitted to a paediatric intensive care unit or paediatric ward with a respiratory illness compatible with pertussis. Two real-time fluorescent resonance energy transfer hybridization probe LightCycler (Roche Diagnostics) PCR assays were used. One (designed in-house) targeted the pertussis toxin S1 promoter (ptxA-pr), and included an internal process control to test for sample inhibition and reagent performance. The other (already published) targeted the insertion element IS481. The analytical sensitivities of the assays were 100 and 10 fg per reaction for the ptxA-pr and IS481 PCRs, respectively. The ptxA-pr assay was specific for B. pertussis, whilst the IS481 PCR also showed some cross-reactivity with Bordetella holmesii and the type strain of Bordetella parapertussis. From April 2002 to March 2007, 848 samples were received from 774 patients and DNA was extracted. Of 824 samples that were suitable for testing, 183 (22.2 %) had evidence of Bordetella infection (18.9 % ptxA-pr and IS481; 3.3 % IS481 only), 621 (75.4 %) were negative and 20 (2.4 %) were inhibitory for the PCR. Within the targeted age group of ≤6 months, most patients (130/138) with evidence of Bordetella spp. by PCR were ≤3 months old. The overall percentage increase in laboratory-confirmed cases due to PCR compared with culture for the 5 year period described ranged from 9 to 26 % per year (mean 19 %). Real-time PCR is an invaluable tool both for enhanced epidemiological surveillance and for the provision of a rapid diagnosis of pertussis where results can affect patient and contact management.
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Garcia, Gabriela A., Anton R. Lord, Lilha M. B. Santos, Tharanga N. Kariyawasam, Mariana R. David, Dinair Couto-Lima, Aline Tátila-Ferreira, Márcio G. Pavan, Maggy T. Sikulu-Lord, and Rafael Maciel-de-Freitas. "Rapid and Non-Invasive Detection of Aedes aegypti Co-Infected with Zika and Dengue Viruses Using Near Infrared Spectroscopy." Viruses 15, no. 1 (December 20, 2022): 11. http://dx.doi.org/10.3390/v15010011.
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The transmission of dengue (DENV) and Zika (ZIKV) has been continuously increasing worldwide. An efficient arbovirus surveillance system is critical to designing early-warning systems to increase preparedness of future outbreaks in endemic countries. The Near Infrared Spectroscopy (NIRS) is a promising high throughput technique to detect arbovirus infection in Ae. aegypti with remarkable advantages such as cost and time effectiveness, reagent-free, and non-invasive nature over existing molecular tools for similar purposes, enabling timely decision making through rapid detection of potential disease. Our aim was to determine whether NIRS can differentiate Ae. aegypti females infected with either ZIKV or DENV single infection, and those coinfected with ZIKV/DENV from uninfected ones. Using 200 Ae. aegypti females reared and infected in laboratory conditions, the training model differentiated mosquitoes into the four treatments with 100% accuracy. DENV-, ZIKV-, and ZIKV/DENV-coinfected mosquitoes that were used to validate the model could be correctly classified into their actual infection group with a predictive accuracy of 100%, 84%, and 80%, respectively. When compared with mosquitoes from the uninfected group, the three infected groups were predicted as belonging to the infected group with 100%, 97%, and 100% accuracy for DENV-infected, ZIKV-infected, and the co-infected group, respectively. Preliminary lab-based results are encouraging and indicate that NIRS should be tested in field settings to evaluate its potential role to monitor natural infection in field-caught mosquitoes.
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Shastri, Aditi, Kelty R. Baker, and Perumal Thiagarajan. "Differential Effect of An Autoantibody to Thrombin on Fibrinogen Cleavage and Protein C Activation." Blood 116, no. 21 (November 19, 2010): 3652. http://dx.doi.org/10.1182/blood.v116.21.3652.3652.
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Abstract Abstract 3652 A 76 year old male experienced an unexpected blood loss of 1300 ml during an elective left shoulder replacement for degenerative joint disease. His post-operative course was uncomplicated. Further laboratory testing revealed a prolonged thrombin time of 33 seconds (control of 17.7–20.3 seconds). The reptilase time was within normal limits. He also had a normal factor V, × and fibrinogen level. The prolonged thrombin time failed to correct upon addition of normal plasma. The patient's IgG fraction was isolated by protein G-Sepharose chromatography and tested for its effect in coagulation assays. The patient's purified IgG (2.1 mg/ml) prolonged the thrombin time in normal plasma when tested with a commercial bovine thrombin time reagent. Under similar conditions, normal IgG had no effect. These laboratory tests suggest an acquired antibody to thrombin. The antibody was further characterized by testing its effect on purified human thrombin. The antibody prolonged the clotting time of human thrombin in normal plasma in a concentration dependent manner. However, the antibody had no effect on the esterolytic activity of human or bovine thrombin as measured by the proteolysis of the chromogenic thrombin substrate, S-2238. These results suggest that the epitope for the antibody overlaps the fibrinogen binding site on thrombin but does not include thrombin's active enzymatic site. We also tested the effect of the purified antibody on thrombomodulin-dependent thrombin activation of protein C. The patient's antibody was incubated with protein C, rabbit thrombomodulin and calcium containing buffer. The activation of protein C was measured using the chromogenic protein C substrate S2366. The antibody had no effect on protein C activation by thrombin. Acquired antibodies to thrombin develop after exposure to bovine thrombin, commonly used for surgical hemostasis. These antibodies are also associated with antibodies to factor V and a low factor V level. This patient's clinical and laboratory profile is intriguing, given the lack of previous exposure to bovine or human thrombin and the presence of normal Factor V levels. This novel antibody may represent formation of a spontaneous autoantibody to thrombin due to failure of immunological surveillance mechanisms. Disclosures: No relevant conflicts of interest to declare.
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Siahaan, Selma, Rukmini Rukmini, Betty Roosihermiatie, Pramita Andarwati, Rini S. Handayani, Ingan U. Tarigan, Tita Rosita, Rustika Rustika, Yuslely Usman, and Lusi Kristiana. "The Effort to Rationalize Antibiotic Use in Indonesian Hospitals: Practice and Its Implication." Journal of Tropical Medicine 2023 (February 25, 2023): 1–12. http://dx.doi.org/10.1155/2023/7701712.
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An effective strategy for combatting AMR in Indonesia is to make the use of antibiotics in hospitals more rational with the help of an Antimicrobial Resistance Control Program (AMR-CP). This study aims to analyze the implementation of the AMR-CP in hospitals by conducting in-depth interviews with health professionals from ten hospitals and health officers of ten provincial health offices in ten different provinces and observation towards its documents. The sample location was selected by purposive sampling. Informants at the hospitals were hospital directors, chairmen of the AMR-CP team, chairmen of the medical committee, persons in charge of the microbiology laboratory, clinicians, nurses, clinical pharmacists, and those program managers at the provincial health offices who are responsible for administering antibiotics. Information is first collected and then a thematic analysis is applied along with triangulation to confirm the validity of information from multiple sources, including document observation results. The analysis is adapted to the framework of the system (i.e., input, process, and output). Results show that hospitals in Indonesia already have the resources to implement AMR-CP, including AMR-CP team and microbiology laboratories. Six hospitals examined also have clinicians trained in microbiology. Though hospital leadership and its commitment to implementing AMR-CP are favorable, there is room for improvement. AMR-CP teams organize routine activities for socialization and training, develop standard operating procedures (SOPs) for antibiotic use, antibiotic patterns surveillance, and bacterial mapping. Some obstacles to implementing AMR-CP policies are posed by the human resources, facilities, budget, antibiotics and reagent shortages, and clinician compliance with SOPs. The study concludes that there was an improvement in antibiotic sensitivity patterns, rational use of antibiotics, use of microbiological laboratories, and cost-efficiency. It recommends the government and healthcare providers continue to improve AMR-CP in hospitals and promote AMR-CP policy by making the regional health office of the hospital a representative of the regional government.
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Kay A., Bradford. "The role of the laboratory in disease surveillance." Eastern Mediterranean Health Journal 2, no. 1 (August 31, 2021): 68–72. http://dx.doi.org/10.26719/1996.2.1.68.
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Laboratory information is critical for disease surveillance and control programmes. Before an outbreak, laboratory-supported surveillance allows early detection of cases. During an outbreak a sample of cases should be laboratory confirmed to assess changes in the etiological agent and to guide decisions about the allocation of resources. Support is provided by laboratories of differing capabilities. Field laboratories are useful in areas where resources are limited or nonexistent. More complete testing is usually done in regional laboratories. International reference laboratories may identify rare or dangerous pathogens, identify newly described organisms, and provide uncommon diagnostic reagents. Laboratory information must be accurate, timely and subjected to quality assurance procedures
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Tsutsumi, Tamao, Charles Frenette, Nancy Doherty, Jacqueline Sedman, and Ashraf Ismail. "243. Transflection Fourier Transform Infrared Spectroscopy as a Real-Time Strain Typing Technique: A Vancomycin-Resistant Enterococcus faecium (VRE) Typing Prospective Study." Open Forum Infectious Diseases 6, Supplement_2 (October 2019): S138. http://dx.doi.org/10.1093/ofid/ofz360.318.
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Abstract Background Rapid bacterial strain typing for nosocomial outbreak surveillance is critical for timely outbreak detection and implementation of appropriate infection control protocols in hospitals. Pulsed-field gel electrophoresis (PFGE) remains the gold standard for strain typing, but it has the disadvantages of being time-consuming and costly. Transflection Fourier transform infrared (FTIR) spectroscopy is a nondestructive and reagent-free technique for rapid microbial identification and subspecies-level discrimination. The potential of employing transflection FTIR spectroscopy as a rapid, real-time typing technique was evaluated in the present study. Methods Transflection FTIR spectra were acquired from vancomycin-resistant Enterococcus faecium (VRE) isolates obtained from rectal swabs (n = 36) of patients in 6 units at a Montreal hospital over a 3-month period and from environmental screening samples (n = 2). Upon confirmation as VRE using a transflection FTIR spectral database previously developed in our laboratory, isolates were further typed by unsupervised hierarchical cluster analysis and principal component analysis of the FTIR spectral data with the use of a feature selection algorithm. Results Analysis of the FTIR data identified independent cases of VRE outbreak in 2 of 6 units; these outbreaks were confirmed retrospectively by PFGE. Based on the PFGE typing results for all 38 isolates included in this study, FTIR spectral analyses successfully identified 95% (n = 18) of isolates related to the outbreaks and 95% (n = 18) of non-outbreak-related isolates, resulting in a false-positive (n = 1), and a false-negative (n = 1), rate of 5%. Additionally, the two environmental isolates were identified as part of the outbreak from one of the outbreak-positive units. Conclusion The results in this study indicate that transflection FTIR spectroscopy-based typing can be considered as an alternative typing technique to PFGE, providing real-time results to track the spread of antibiotic-resistant pathogens within hospitals. Furthermore, when combined with the use of a transflection FTIR spectral database, both identification and typing of an isolate can be achieved from a single spectral measurement, thereby reducing the time and cost required for outbreak investigation. Disclosures All authors: No reported disclosures.
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Roberts, Tamalee, Nantasit Luangasanatip, Clare L. Ling, Jill Hopkins, Risara Jaksuwan, Yoel Lubell, Manivanh Vongsouvath, H. Rogier van Doorn, Elizabeth A. Ashley, and Paul Turner. "Antimicrobial resistance detection in Southeast Asian hospitals is critically important from both patient and societal perspectives, but what is its cost?" PLOS Global Public Health 1, no. 10 (October 13, 2021): e0000018. http://dx.doi.org/10.1371/journal.pgph.0000018.
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Antimicrobial resistance (AMR) is a major threat to global health. Improving laboratory capacity for AMR detection is critically important for patient health outcomes and population level surveillance. We aimed to estimate the financial cost of setting up and running a microbiology laboratory for organism identification and antimicrobial susceptibility testing as part of an AMR surveillance programme. Financial costs for setting up and running a microbiology laboratory were estimated using a top-down approach based on resource and cost data obtained from three clinical laboratories in the Mahidol Oxford Tropical Medicine Research Unit network. Costs were calculated for twelve scenarios, considering three levels of automation, with equipment sourced from either of the two leading manufacturers, and at low and high specimen throughput. To inform the costs of detection of AMR in existing labs, the unit cost per specimen and per isolate were also calculated using a micro-costing approach. Establishing a laboratory with the capacity to process 10,000 specimens per year ranged from $254,000 to $660,000 while the cost for a laboratory processing 100,000 specimens ranged from $394,000 to $887,000. Excluding capital costs to set up the laboratory, the cost per specimen ranged from $22–31 (10,000 specimens) and $11–12 (100,000 specimens). The cost per isolate ranged from $215–304 (10,000 specimens) and $105–122 (100,000 specimens). This study provides a conservative estimate of the costs for setting up and running a microbiology laboratory for AMR surveillance from a healthcare provider perspective. In the absence of donor support, these costs may be prohibitive in many low- and middle- income country (LMIC) settings. With the increased focus on AMR detection and surveillance, the high laboratory costs highlight the need for more focus on developing cheaper and cost-effective equipment and reagents so that laboratories in LMICs have the potential to improve laboratory capacity and participate in AMR surveillance.
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Aboushady, Ahmed Taha, Olivier Manigart, Abdourahmane Sow, Walter Fuller, Abdoul-Salam Ouedraogo, Chinelo Ebruke, François-Xavier Babin, Laetitia Gahimbare, Issiaka Sombié, and John Stelling. "Surveillance of Antimicrobial Resistance in the ECOWAS Region: Setting the Scene for Critical Interventions Needed." Antibiotics 13, no. 7 (July 5, 2024): 627. http://dx.doi.org/10.3390/antibiotics13070627.
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Antimicrobial resistance poses a significant challenge to public health globally, leading to increased morbidity and mortality. AMR surveillance involves the systematic collection, analysis, and interpretation of data on the occurrence and distribution of AMR in humans, animals, and the environment for action. The West African Health Organization, part of the Economic Community of West African States (ECOWAS), is committed to addressing AMR in the region. This paper examines the status of AMR surveillance in ECOWAS countries using available WHO data from the TrACSS survey and GLASS enrollments. The analysis reveals that while progress has been made, significant challenges remain. Twelve of the fifteen ECOWAS countries are enrolled in GLASS, and ten have developed national action plans (NAPs) for AMR. However, there is a need to ensure all countries fully implement their NAPs, continue reporting to GLASS, and use the data for evidence-based actions and decision making. Surveillance systems for AMR and antimicrobial consumption/use vary across countries with some demonstrating limited capacity. All countries, except Cabo Verde, reported having a reference laboratory for AMR testing. Strengthening laboratory capabilities, data management and use, and multisectoral coordination are crucial for effective AMR surveillance and response. Based on the findings and the regional context, it is essential to prioritize capacity building, data utilization, and the adoption of standardized guidelines for AMR surveillance. Collaboration among ECOWAS countries, the WAHO, and international partners is essential to address AMR comprehensively. Ensuring a consistent supply of essential antimicrobial medications and reagents is vital.
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Bingen, E. H., E. Denamur, and J. Elion. "Use of ribotyping in epidemiological surveillance of nosocomial outbreaks." Clinical Microbiology Reviews 7, no. 3 (July 1994): 311–27. http://dx.doi.org/10.1128/cmr.7.3.311.
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Over the past few years, genotypic methods based on the study of bacterial DNA polymorphism have shown high discriminatory power for strain differentiation and superiority over most phenotypic methods commonly available in the clinical microbiology laboratory. Some of the methods used, however, required either a high level of technology and sophisticated equipment (e.g., pulsed-field gel electrophoresis) or species-specific reagents of restricted availability (randomly cloned DNA probes or gene-specific probes). Because ribotyping uses a universal probe (rRNA) and is a rather simple technology, particularly since the advent of nonradioactive labelling systems, it has been widely used for strain differentiation of most bacterial species involved in nosocomial outbreaks. In vitro and in vivo stability of the markers studied has been demonstrated. Although there may be limitation to this approach, ribotyping was found to be highly discriminative, particularly for typing members of the family Enterobacteriaceae, Pseudomonas cepacia, and Xanthomonas maltophilia. In many cases, it has improved the understanding of the mechanism of nosocomial acquisition of organisms by allowing a distinction between endogenous and exogenous infections. Among exogenous infections, it has distinguished between individual and epidemic strains, thus differentiating cross-infection from independent acquisition.
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Moreno-Contreras, Joaquín, Marco A. Espinoza, Carlos Sandoval-Jaime, Marco A. Cantú-Cuevas, Daniel A. Madrid-González, Héctor Barón-Olivares, Oscar D. Ortiz-Orozco, et al. "Pooling saliva samples as an excellent option to increase the surveillance for SARS-CoV-2 when re-opening community settings." PLOS ONE 17, no. 1 (January 25, 2022): e0263114. http://dx.doi.org/10.1371/journal.pone.0263114.
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In many countries a second wave of infections caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has occurred, triggering a shortage of reagents needed for diagnosis and compromising the capacity of laboratory testing. There is an urgent need to develop methods to accelerate the diagnostic procedures. Pooling samples represents a strategy to overcome the shortage of reagents, since several samples can be tested using one reaction, significantly increasing the number and speed with which tests can be carried out. We have reported the feasibility to use a direct lysis procedure of saliva as source for RNA to SARS-CoV-2 genome detection by reverse transcription quantitative-PCR (RT-qPCR). Here, we show that the direct lysis of saliva pools, of either five or ten samples, does not compromise the detection of viral RNA. In addition, it is a sensitive, fast, and inexpensive method that can be used for massive screening, especially considering the proximity of the reincorporation of activities in universities, offices, and schools.
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Balmaseda, Ángel, Saira Saborío Galo, Karla González, Yolanda Téllez, Nadezna García, Leonel Pérez, Lionel Gresh, and Eva Harris. "Development of in-house serological methods for diagnosis and surveillance of chikungunya." Revista Panamericana de Salud Pública 41 (June 29, 2017): 1. http://dx.doi.org/10.26633/rpsp.2017.56.
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Objective.To develop and evaluate serological methods for chikungunya diagnosis and research in Nicaragua.Methods.Two IgM ELISA capture systems (MAC-ELISA) for diagnosis of acute chikungunya virus (CHIKV) infections, and two Inhibition ELISA Methods (IEM) to measure total antibodies against CHIKV were developed using monoclonal antibodies (mAbs) and hyperimmune serum at the National Virology Laboratory of Nicaragua in 2014–2015. The sensitivity, specificity, predictive values, and agreement of the MAC-ELISAs were obtained by comparing the results of 198 samples (116 positive; 82 negative) with the Centers for Disease Control and Prevention’s IgM ELISA (Atlanta, Georgia, United States; CDC-MAC-ELISA). For clinical evaluation of the four serological techniques, 260 paired acute and convalescent phase serum samples of suspected chikungunya cases were used.Results.All four assays were standardized by determining the optimal concentrations of the different reagents. Processing times were substantially reduced compared to the CDC-MAC-ELISA. For the MAC-ELISA systems, a sensitivity of 96.6% and 97.4%, and a specificity of 98.8% and 91.5% were obtained using mAb and hyperimmune serum, respectively, compared with the CDC method. Clinical evaluation of the four serological techniques versus the CDC real-time RT-PCR assay resulted in a sensitivity of 95.7% and a specificity of 88.8%–95.9%.Conclusion.Two MAC-ELISA and two IEM systems were standardized, demonstrating very good quality for chikungunya diagnosis and research demands. This will achieve more efficient epidemiological surveillance in Nicaragua, the first country in Central America to produce its own reagents for serological diagnosis of CHIKV. The methods evaluated here can be applied in other countries and will contribute to sustainable diagnostic systems to combat the disease.
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Panwalker, Anand P., and Elizabeth Fuhse. "Nosocomial Mycobacterium gordonae Pseudoinfection From Contaminated Ice Machines." Infection Control 7, no. 2 (February 1986): 67–70. http://dx.doi.org/10.1017/s0195941700063918.
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AbstractThirty-two clinical specimens submitted to the laboratory during a 12-month period from July 1980 to June 1981 were reported to be culture-positive for Mycobacterium gordonae, an organism generally considered to be a slow-growing saprophyte with natural habitats which include soil and water. Only seven similar isolates had been recovered in the preceding 4½ year period. The discordance between clinical findings and the mycobacterial cultures suggested extrinsic contamination of the specimens. Contamination in the laboratory was believed unlikely because: 1) clinical samples obtained in an aseptic manner were never contaminated; 2) various surveillance cultures of reagents and deionized water used in the laboratory were negative; and 3) substitution of deionized water with sterile water did not control the outbreak. Extensive hospital-wide cultures of water sources implicated the use of ice and ice water from contaminated ice machines as the source of this pseudoepidemic. Cleaning of the ice machines resulted in a sharp decrease in the number of M. gordonae isolates. Pseudoinfection by M. gordonae from improperly maintained ice machines has not been reported before.
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Chidzaye, Robert W. "Assessing Barriers to Medical Laboratory Diagnostic Service Delivery in Mzuzu City." International Journal of Biomedical Science 15, no. 1 (March 15, 2019): 32–56. http://dx.doi.org/10.59566/ijbs.2019.15032.
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Medical laboratories provide confirmatory diagnosis and evidence based management of diseases, essential public health information and disease surveillance. A wide variety of research studies suggest that breakdowns in the diagnostic process result in a staggering toll of harm, patient deaths and wastage of valuable medical resources already constrained in the developing world. The objective of this study was to assess barriers to delivery of optimal laboratory diagnostic services in Mzuzu city. This was a descriptive cross-sectional study using quantitative research approach. Three categories of laboratories were selected from the public, faith based and private health systems. Stratified sampling was used to select laboratory practioners while purposive sampling was used to select administrators from each health facility. The data was analysed by the measures of central tendency mean plus measures of variability, range, standard deviation and standard error, using SPSS version 20. The findings of the study were that laboratory practioners identified several barriers to affect quality diagnostic services: 79% of laboratory staff reported short supply of laboratory supplies, work overloads (69%), frequent equipment failure (22%), scarcity of modern equipment (20%) and others. Administrators (67%) reported a limited budget allocation to the public and faith based hospitals. The study also found that there were some barriers that were more frequent than others such as shortage of laboratory supplies (84%), work overloads (70%), lack of refresher training (34%), frequent equipment failure (28%) and others. The study also found that laboratory practitioners employed several countermeasures to overcome technical barriers to diagnostics. 44% reported that they would stop tests when reagents run out of stock, wait for maintenance of equipment and stop tests (38%), higher cadres would delegate work to lower level cadres (31%), improvise on faulty equipment and expired reagents (26%) and many others. No laboratory had adequate quality management systems in place. The recommendations of this study were to strengthen human resources planning for laboratory professionals, establish or strengthen national laboratory regulatory and representative bodies to improve governance and enhance quality, and promote work and competency based in-service training to ensure that staff skills are up to date and competency is demonstrated.
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Dara, Antoine, Bourema Kouriba, Amadou Daou, Abdoul Karim Sangare, Djibril Kassogue, Charles Dara, and Abdoulaye A. Djimde. "Sequencing SARS-CoV-2 in a Malaria Research Laboratory in Mali, West Africa: The Road to Sequencing the First SARS-CoV-2 Genome in Mali." Processes 9, no. 12 (December 2, 2021): 2169. http://dx.doi.org/10.3390/pr9122169.
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Next-generation sequencing (NGS) has become a necessary tool for genomic epidemiology. Even though the utility of genomics in human health has been proved, genomic surveillance has never been as important as during the COVID-19 pandemic. This has been demonstrated by the recent use of genomic surveillance to detect new variants of SARS-CoV-2 in the United Kingdom, South Africa, and Brazil. Until recently, Malian scientists did not have access to any local NGS platform, and samples had to be shipped abroad for sequencing. Here, we report on how we adapted a laboratory setup for Plasmodium research to generate the first complete SARS-CoV-2 genome locally. Total RNA underwent a library preparation using an Illumina TruSeq stranded RNA kit. A metagenomics sequencing was performed on an Illumina MiSeq platform, which was followed by bioinformatic analyses on a local server in Mali. We recovered a full genome of SARS-CoV-2 of 29 kb with an average depth coverage of 200×. We have demonstrated our capacity to generate a high-quality genome with limited resources and highlight the need to develop genomics capacity locally to solve health problems. We discuss challenges related to access to reagents during a pandemic period and propose some home-made solutions.
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Dalpke, Alexander H., Marjeta Hofko, and Stefan Zimmermann. "Development of a Real-Time PCR Protocol Requiring Minimal Handling for Detection of Vancomycin-Resistant Enterococci with the Fully Automated BD Max System." Journal of Clinical Microbiology 54, no. 9 (June 29, 2016): 2321–29. http://dx.doi.org/10.1128/jcm.00768-16.
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Vancomycin-resistant enterococci (VRE) are an important cause of health care-associated infections, resulting in significant mortality and a significant economic burden in hospitals. Active surveillance for at-risk populations contributes to the prevention of infections with VRE. The availability of a combination of automation and molecular detection procedures for rapid screening would be beneficial. Here, we report on the development of a laboratory-developed PCR for detection of VRE which runs on the fully automated Becton Dickinson (BD) Max platform, which combines DNA extraction, PCR setup, and real-time PCR amplification. We evaluated two protocols: one using a liquid master mix and the other employing commercially ordered dry-down reagents. The BD Max VRE PCR was evaluated in two rounds with 86 and 61 rectal elution swab (eSwab) samples, and the results were compared to the culture results. The sensitivities of the different PCR formats were 84 to 100% forvanAand 83.7 to 100% forvanB; specificities were 96.8 to 100% forvanAand 81.8 to 97% forvanB. The use of dry-down reagents and the ExK DNA-2 kit for extraction showed that the samples were less inhibited (3.3%) than they were by the use of the liquid master mix (14.8%). Adoption of a cutoff threshold cycle of 35 for discrimination ofvanB-positive samples allowed an increase of specificity to 87.9%. The performance of the BD Max VRE assay equaled that of the BD GeneOhm VanR assay, which was run in parallel. The use of dry-down reagents simplifies the assay and omits any need to handle liquid PCR reagents.
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Alkholidy, Ghamdan Gamal, Labiba Saeed Anam, Ali Hamoud Almahaqri, and Yousef Khader. "Performance of the Severe Acute Respiratory Illness Sentinel Surveillance System in Yemen: Mixed Methods Evaluation Study." JMIR Public Health and Surveillance 7, no. 7 (July 9, 2021): e27621. http://dx.doi.org/10.2196/27621.
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Background The national severe acute respiratory illness (SARI) surveillance system in Yemen was established in 2010 to monitor SARI occurrence in humans and provide a foundation for detecting SARI outbreaks. Objective To ensure that the objectives of national surveillance are being met, this study aimed to examine the level of usefulness and the performance of the SARI surveillance system in Yemen. Methods The updated Centers for Disease Control and Prevention guidelines were used for the purposes of our evaluation. Related documents and reports were reviewed. Data were collected from 4 central-level managers and stakeholders and from 10 focal points at 4 sentinel sites by using a semistructured questionnaire. For each attribute, percent scores were calculated and ranked as follows: very poor (≤20%), poor (20%-40%), average (40%-60%), good (60%-80%), and excellent (>80%). Results As rated by the evaluators, the SARI surveillance system achieved its objectives. The system’s flexibility (percent score: 86%) and acceptability (percent score: 82%) were rated as “excellent,” and simplicity (percent score: 74%) and stability (percent score: 75%) were rated as “good.” The percent score for timeliness was 23% in 2018, which indicated poor timeliness. The overall data quality percent score of the SARI system was 98.5%. Despite its many strengths, the SARI system has some weaknesses. For example, it depends on irregular external financial support. Conclusions The SARI surveillance system was useful in estimating morbidity and mortality, monitoring the trends of the disease, and promoting research for informing prevention and control measures. The overall performance of the SARI surveillance system was good. We recommend expanding the system by promoting private health facilities’ (eg, private hospitals and private health centers) engagement in SARI surveillance, establishing an electronic database at central and peripheral sites, and providing the National Central Public Health Laboratory with the reagents needed for disease confirmation.
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Brangel, Polina, Sina Tureli, Barbara Mühlemann, Nicole Liechti, Daniel Zysset, Olivier Engler, Isabel Hunger-Glaser, et al. "A Global Collaborative Comparison of SARS-CoV-2 Antigenicity Across 15 Laboratories." Viruses 16, no. 12 (December 18, 2024): 1936. https://doi.org/10.3390/v16121936.
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Setting up a global SARS-CoV-2 surveillance system requires an understanding of how virus isolation and propagation practices, use of animal or human sera, and different neutralisation assay platforms influence assessment of SARS-CoV-2 antigenicity. In this study, with the contribution of 15 independent laboratories across all WHO regions, we carried out a controlled analysis of neutralisation assay platforms using the first WHO International Standard for antibodies to SARS-CoV-2 variants of concern (source: NIBSC). Live virus isolates (source: WHO BioHub or individual labs) or spike plasmids (individual labs) for pseudovirus production were used to perform neutralisation assays using the same serum panels. When comparing fold drops, excellent data consistency was observed across the labs using common reagents, including between pseudovirus and live virus neutralisation assays (RMSD of data from mean fold drop was 0.59). Utilising a Bayesian model, geometric mean titres and assay titre magnitudes (offsets) can describe the data efficiently. Titre magnitudes were seen to vary largely even for labs within the same assay group. We have observed that overall, live Microneutralisation assays tend to have the lowest titres, whereas Pseudovirus Neutralisation have the highest (with a mean difference of 3.2 log2 units between the two). These findings are relevant for laboratory networks, such as the WHO Coronavirus Laboratory Network (CoViNet), that seek to support a global surveillance system for evolution and antigenic characterisation of variants to support monitoring of population immunity and vaccine composition policy.
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Bankamp, Bettina, Raydel Anderson, Lijuan Hao, Elena Lopareva, Min-hsin Chen, Gimin Kim, R. Suzanne Beard, et al. "Building Quality Control for Molecular Assays in the Global Measles and Rubella Laboratory Network." Vaccines 12, no. 8 (July 23, 2024): 824. http://dx.doi.org/10.3390/vaccines12080824.
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More than 100 laboratories in the World Health Organization Global Measles and Rubella Laboratory Network (GMRLN) perform nucleic acid-based methods for case confirmation of measles or rubella infections and/or strain surveillance (genotyping). The quality of laboratory data is critical to ensure that diagnostic results and country reports to regional verification committees are based on accurate data. A molecular External Quality Assurance (mEQA) program was initiated by the US-CDC in 2014 to evaluate the performance of laboratories in the network. The inclusion of testing for measles and rubella viruses, with a focus on detection and genotyping, plus the diversity of assays and platforms employed required a flexible and comprehensive proficiency testing program. A stepwise introduction of new evaluation criteria gradually increased the stringency of the proficiency testing program, while giving laboratories time to implement the required changes. The mEQA program plays an important role in many processes in the GMRLN, including informing plans for the training of laboratory staff, access to reagents, and the submission of sequence data to global databases. The EQA program for Local Public Health Institutes in Japan is described as an example for national mEQA programs. As more laboratories initiate molecular testing, the mEQA will need to continue to expand and to adapt to the changing landscape for molecular testing.
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Warrington, Jill S., Jessica W. Crothers, Andrew Goodwin, Linda Coulombe, Tania Hong, Lynn Bryan, Christina Wojewoda, et al. "All Hands-On Deck and All Decks on Hand: Surmounting Supply Chain Limitations During the COVID-19 Pandemic." Academic Pathology 8 (January 1, 2021): 237428952110119. http://dx.doi.org/10.1177/23742895211011928.
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Testing during the COVID-19 pandemic has been crucial to public health surveillance and clinical care. Supply chain constraints—spanning limitations in testing kits, reagents, pipet tips, and swabs availability—have challenged the ability to scale COVID-19 testing. During the early months, sample collection kits shortages constrained planned testing expansions. In response, the University of Vermont Medical Center, University of Vermont College of Medicine, Vermont Department of Health Laboratory, Aspenti Health, and providers across Vermont including 16 area hospitals partnered to surmount these barriers. The primary objectives were to increase supply availability and manage utilization. Within the first month of Vermont’s stay-at-home order, the University of Vermont Medical Center laboratory partnered with College of Medicine to create in-house collection kits, producing 5000 per week. University of Vermont Medical Center reassigned 4 phlebotomists, laboratory educators, and other laboratory staff, who had reduced workloads, to participate (requiring a total of 5.3-7.6 full-time equivalent (FTE) during the period of study). By August, automation at a local commercial laboratory produced 22,000 vials of media in one week (reducing the required personnel by 1.2 FTE). A multisite, cross-institutional approach was used to manage specimen collection kit utilization across Vermont. Hospital laboratory directors, managers, and providers agreed to order only as needed to avoid supply stockpiles and supported operational constraints through ongoing validations and kit assembly. Throughout this pandemic, Vermont has ranked highly in number of tests per million people, demonstrating the value of local collaboration to surmount obstacles during disease outbreaks and the importance of creative allocation of resources to address statewide needs.
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Buchta, Christoph, Jeremy V. Camp, Jovana Jovanovic, Peter Chiba, Elisabeth Puchhammer-Stöckl, Maximilian Mayerhofer, Helga Plicka, et al. "The versatility of external quality assessment for the surveillance of laboratory and in vitro diagnostic performance: SARS-CoV-2 viral genome detection in Austria." Clinical Chemistry and Laboratory Medicine (CCLM) 59, no. 10 (June 30, 2021): 1735–44. http://dx.doi.org/10.1515/cclm-2021-0604.
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Abstract Objectives External quality assessment (EQA) schemes provide information on individual and general analytical performance of participating laboratories and test systems. The aim of this study was to investigate the use and performance of SARS-CoV-2 virus genome detection systems in Austrian laboratories and their preparedness to face challenges associated with the pandemic. Methods Seven samples were selected to evaluate performance and estimate variability of reported results. Notably, a dilution series was included in the panel as a measure of reproducibility and sensitivity. Several performance criteria were evaluated for individual participants as well as in the cohort of all participants. Results A total of 109 laboratories participated and used 134 platforms, including 67 different combinations of extraction and PCR platforms and corresponding reagents. There were no false positives and 10 (1.2%) false negative results, including nine in the weakly positive sample (C t ∼35.9, ∼640 copies/mL). Twenty (22%) laboratories reported results of mutation detection. Twenty-five (19%) test systems included amplification of human RNA as evidence of proper sampling. The overall linearity of C t values from individual test systems for the dilution series was good, but inter-assay variability was high. Both operator-related and systematic failures appear to have caused incorrect results. Conclusions Beyond providing certification for participating laboratories, EQA provides the opportunity for participants to evaluate their performance against others so that they may improve operating procedures and test systems. Well-selected EQA samples offer additional inferences to be made about assay sensitivity and reproducibility, which have practical applications.
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Aryastami, Ketut, Harimat Hendarwan, Vivi Setiawaty, Amir Su'udi, Ully Adhie Mulyani, Made Dewi Susilawati, Syachroni Syachroni, Nelly Puspandari, and Agus Suwandono. "Laboratory preparedness to support the Covid-19 pandemic respond in Indonesia." Health Science Journal of Indonesia 11, no. 2 (December 30, 2020): 138–46. http://dx.doi.org/10.22435/hsji.v11i2.4089.
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Latar belakang: Penyakit jenis baru COVID-19 yang disebabkan oleh virus corona menjadi sebuah pandemic di akhir tahun 2019. Kota Wuhan (China) merupakan lokasi pertama terdeteksinya kasus COVID-19. Tanpa adanya kecurigaan apapun penyakit ini dengan cepatnya menyebar ke seluruh dunia mengikuti alur mobilitas manusia. Dalam kondisi tersebut sistem kesehatan di setiap negara tampak kelabakan khususnya dalam pengendalian transmisi penyakit. Studi ini ingin mengidentifikasi kesiapan jejaring laboratorium kesehatan di Indonesia.
Metode: Penilaian cepat dilakukan terhadap ketersediaan dan kesiapan laboratoriaum dalam pennanganan pandemi Covid-19. Pengumpulan data dilakukan melalui pengisian questioner yang dikirim secara elektronik. Waktu pelaksanaan adalah minggu ketiga dan keempat, Maret 2020. Terdapat 44 laboratorium jejaring laboratorium dibawah Kementerian Kesehatan yang menjadi subjek penelitian, dan sebanyak 33 yang merespon secara lengkap Variabel ketersediaan, kecukupan dan kebutuhan bahan dan alat.
Hasil: Jejaring laboratorium kesehatan dibawah Kementerian Kesehatan sudah terbentuk sejak tahun 2009. Dengan terjadinya pandemic COVID-19 Surat Keputusan Menteri Kesehatan telah direvisi hingga dua kali agar dapat meningkatkan kapasitas dan memperluas jejaring ke seluruh wilayah NKRI. Hasil studi menunjukkan, laboratorium jejaring dibawah Kementerian Kesehatan belum siap dalam menghadapi pandemic COVID-19. Dua jenis laboratorium jejaring yaitu laboratorium surveillans maupun laboratorium diagnostic memiliki kondisi yang sama. Ketersediaan bahan dan alat laboratorium standar masih tergolong rata-rata, bahkan dari sisi kecukupannyapun masih jauh dibawah kapasitas kebutuhan dalam penanganan specimen COVID-19. Kondisi yang sama juga tampak untuk bahan pendukung laboratorium termasuk alat pelindung diri untuk petugas.
Kesimpulan: Kesiapan laboratorium sebagai bagian dari system kesehatan dalam kondisi pandemic masih lemah. Keberadaan alat penunjang diagnose khususnya untuk penyakit menular harus dilengkapi sesuai dengann type laboratorium. Pandemi COVID-19 menjadi alarm dalam menghadapi era baru dan antisipasi masalah dimasa yang akan datang.
Kata kunci: Kesiapan laboratorium, COVID-19, Indonesia
Abstract
Background: A novel coronavirus disease called COVID-19 has become pandemic in late 2019. Wuhan City was the first place detected as the source of the pandemic. Without suspicion, it spreads over the world, along with human mobility. In such a condition, every country seems quite stuttering to prepare its health system to prevent its people from the possible transmission. This paper aims to describe the preparedness of the networking laboratory in Indonesia.
Methods: We conducted a rapid assessment of laboratory availability and preparedness to respond to the Covid-19 pandemic. We held the data collection on the third and fourth week of March 2020 by sending an electronic questionnaire to all 44 networking laboratories under the Ministry of Health structure. The variables assessed in this study were the availability and the requirements of the Covid-19 related laboratory's substances, including reagents and other equipment types.
Results: The Ministry of Health established the networking laboratory in 2009, but due to the COVID-19 pandemic, it has renewed twice to enhance and expand the laboratory capacities over the country. Our studies showed preparedness among networking laboratories in Indonesia regarding this new emerging COVID-19 condition was quite devastating. Both surveillance and diagnostic laboratories have a similar situation. The availability of their primary materials was mediocre, but the adequacy was far beyond the capacity in handling the COVID-19 specimen. We found a similar case in the laboratory, supporting materials, and personal protective equipment (PPE).
Conclusion: Laboratory preparedness during initial period of time of the COVID-19 pandemic as part of the health system is still weak. The availability of the necessary equipment, supporting materials, and personal protective equipment are far beyond the requirements. The COVID-19 has alarmed the laboratory and the whole health system in Indonesia into a new era with better future preparedness.
Keywords: laboratory preparedness, COVID-19, Indonesia
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Polonis, Katarzyna, Joseph H. Blommel, Andrew E. O. Hughes, David Spencer, Joseph A. Thompson, and Molly C. Schroeder. "Innovations in Short-Read Sequencing Technologies and Their Applications to Clinical Genomics." Clinical Chemistry 71, no. 1 (January 2025): 97–108. https://doi.org/10.1093/clinchem/hvae173.
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Abstract Background Massively parallel sequencing (MPS) of nucleic acids has been a transformative technology for basic and applied genomic science, increasing efficiencies and decreasing costs to enable studies of unprecedented scope and impact. In clinical settings, these technological and scientific advances have led to the development of tests that are increasingly fast, comprehensive, and more frequently employed. Practitioners of genomic medicine have applied these tools across clinical settings, including diagnosis of inherited disorders and cancers and infectious disease detection and surveillance. In recent years, the commercial marketplace for MPS sequencers and reagents has been dominated by a few companies. The growing demand for sequencing has led to the recent emergence of several new sequencing platforms with techniques that may provide alternatives or improvements to existing workflows or allow the adoption of sequencing workflows in new settings. Clinical genomics laboratories will evaluate these platforms from a unique perspective, focusing on how technological advancements can improve patient care. Content This review describes short-read sequencing platforms provided by Illumina, Element Biosciences, MGI, PacBio, Singular Genomics, Thermo Fisher Scientific, and Ultima Genomics. This review discusses their innovative approaches, principles, workflows, and applications. Summary This review aims to inform laboratory geneticists, clinicians, and researchers about emerging short-read technologies and their applications in clinical genomics. By highlighting their principles and potential contributions, we aim to assist laboratories in selecting suitable solutions for their sequencing needs considering key factors such as applications, throughput, and integration with existing laboratory workflows.
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Esman, Anna, Anna Cherkashina, Konstantin Mironov, Dmitry Dubodelov, Svetlana Salamaikina, Anna Golubeva, Gasan Gasanov, et al. "SARS-CoV-2 Variants Monitoring Using Real-Time PCR." Diagnostics 12, no. 10 (October 1, 2022): 2388. http://dx.doi.org/10.3390/diagnostics12102388.
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According to the temporary recommendations of the 2021 World Health Organization (WHO), in addition to whole-genome sequencing, laboratories in various countries can also screen for known mutations utilizing targeted RT-PCR-based mutation detection assays. The aim of this work was to generate a laboratory technique to differentiate the main circulating SARS-CoV-2 variants in 2021–2022, when a sharp increase in morbidity was observed with the appearance of the Omicron variant. Real-time PCR methodology is available for use in the majority of scientific and diagnostic institutions in Russia, which makes it possible to increase the coverage of monitoring of variants in the territories of all 85 regions in order to accumulate information for the Central Services and make epidemiological decisions. With the methodology developed by the Central Research Institute of Epidemiology of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (FSSCRP Human Wellbeing) (CRIE), more than 6000 biological samples have been typed, and 7% of samples with the Delta variant and 92% of samples with the Omicron variant have been identified as of 25 August 2022. Reagents for 140,000 definitions have been supplied to regional organizations.
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de St. Maurice, Annabelle, Amy Hallmark, Evan Hilt, Travis Price, Daniel Uslan, Anjali Bisht, Shangxin Yang, and Omai Garner. "Implementation of Hospital-Based Candida auris Surveillance Screening Among At-Risk Patients." Infection Control & Hospital Epidemiology 41, S1 (October 2020): s277—s278. http://dx.doi.org/10.1017/ice.2020.846.
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Background:Candida auris is an emerging multidrug-resistant pathogen associated with outbreaks in hospitals and skilled nursing facilities (SNFs). Patients with C. auris can have invasive disease or asymptomatic colonization. Because C. auris can be difficult to treat and eradicate in the environment, the CDC recommends using contact precautions and sporicidal agents during patient care. After C. auris was identified in a patient from an LA County SNF (SNF-X), our institution initiated surveillance screening on high-risk patients. Methods: Nurses identified patients residing at SNF-X on admission and contacted infection prevention. These patients were placed on contact or spore precautions. Bilateral axilla and inguinal folds were swabbed with an Eswab and sent for testing by a clinical laboratory-developed RT PCR assay, which can detect C. auris with high sensitivity and specificity with a rapid turnaround time (4–6 hours). This PCR assay was based on a commercial platform IntegratedCycler (Diasorin) and reagents from the same vendor. Environmental swabs from the index patient’s room were sent for PCR by HardyCHROM Candida agar (Hardy Diagnostics) before and after cleaning with OxyCideTM. PCR-positive samples were set up for culture. Results: In total, 27 patients from SNF-X were screened by PCR. Of these patients, 15 (55%) had a tracheostomy present on admission. Moreover, 26 swabs were negative; 1 was positive in the index patient (cycle threshold [Ct] value, 26). Clinical specimens from the index patient’s blood did not grow C. auris; the tracheostomy sample grew predominantly C. albicans which made identification of C. auris challenging by culture. However, investigational testing of this sample by PCR was positive (Ct value, 31). Environmental swabs collected from the patient room were obtained before and after cleaning (Table 1); all environmental cultures were negative at 5 days. Conclusions: Developing hospital-based, high-risk patient screening for C. auris is feasible and may be useful for controlling the spread of C. auris within the community. Further study is needed to determine the usefulness of PCR for environmental testing to assess the risk of nosocomial transmission of C. auris.Funding: NoneDisclosures: None
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Yadav, Pooja, Shashi Sharma, Paban Kumar Dash, Suman Dhankher, Sandhya V. K., and S. K. Kiran. "Dry- down probe free qPCR for detection of KFD in resource limited settings." PLOS ONE 18, no. 5 (May 10, 2023): e0284559. http://dx.doi.org/10.1371/journal.pone.0284559.
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Kyasanur Forest Disease is a tick-borne flavivirus is endemic in the Southern India. The recent expansion and resurgence of sporadic outbreaks in southern parts of country is the most important concern. Although only formalin inactivated vaccine is available for treatment with limited efficacy the early detection and timely identification is a only way to prevent spread of cases. If the disease can be identified prior to infection in humans like in forest areas from ticks and vectors the disease spread supposed to be managed quickly. Here we have standardized a single tube ready to use dry-down probe free real time RT-PCR targeted against virus envelope gene for detection of KFDV infection. The assay was standardized in liquid format first, later it was converted into dry-down format with addition of stabilizers with a similar sensitivity and specificity (10RNA Copies/rxn). The sensitivity was comparable to the most widely used and accepted diagnostic platform i.e. TaqMan qRT-PCR. However as the reported assay here omit the need of probes makes it cost effective and dry-down reagents makes more stability to the developed assay in this study if compare to TaqMan qPCR. The assay was evaluated with KFD positive samples and healthy sample panel which revealed high concordance with TaqMan qRT-PCR. Stability was unaffected by temperature fluctuations during transportation even in cold chain free conditions, thus reduce the maintenance of strict cold storage. These findings demonstrated that the reported assay is convenient with 100% sensitivity and specificity to TaqMan qPCR. Thus this assay has the potential usefulness for diagnosis KFDV for routine surveillance in resource limited laboratory settings omitting the use costly and heat sensitive TaqMan qRT-PCR reagents without compromising the sensitivity and specificity of the diagnosis assay.
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Ambrose, Emmanuela E., Luke R. Smart, Mwesige Charles, Arielle G. Hernandez, Adolfine Hokororo, Teresa Latham, Medard Beyanga, et al. "Geospatial Mapping of Sickle Cell Disease in Northwest Tanzania: The Tanzania Sickle Surveillance Study (TS3)." Blood 132, Supplement 1 (November 29, 2018): 3662. http://dx.doi.org/10.1182/blood-2018-99-113939.
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Abstract Tanzania ranks third in Africa for the estimated number of annual births with sickle cell disease, but these estimates are based on sparse data from small studies reported over the past 50 years. A recently completed surveillance study from Uganda documented substantial variation in the prevalence of sickle cell trait and disease across the country. Tanzania lacks a national newborn screening program, and no contemporary multi-regional screening of infants has been undertaken. We designed and conducted a prospective study to determine the prevalence of sickle cell trait and disease by region and district in northwest Tanzania, where the prevalence of sickle cell is thought to be highest. The study used existing public health infrastructure while building local capacity for accurate diagnosis of sickle cell disease. Secondary objectives included characterization of hemoglobin variants and exploration of associations between sickle cell trait, sickle cell disease, malaria, and HIV. The Tanzania Sickle Surveillance Study (TS3) is a prospective cross-sectional study of HIV-exposed infants born in 9 regions across the Lake Zone of northwest Tanzania. In Tanzania, the HIV early infant diagnosis (EID) program collects dried blood spots (DBS) from all children born to HIV-infected mothers. DBS are transported to a central laboratory for prompt detection of HIV vertical transmission. In northwest Tanzania, the DBS are transported to Bugando Medical Centre, a teaching and consultancy hospital in Mwanza, where they are tested for HIV and then stored on-site, and thus available for further testing. Isoelectric focusing (IEF) equipment was donated to Bugando Medical Centre along with reagents and supplies. Two laboratory staff were trained by a board certified hematologist, and then attended a two day seminar by the IEF manufacturer. One pediatrician completed a 2-month observership at Cincinnati Children's Hospital. All DBS samples were tested by IEF using appropriate controls. Completed gels were scored independently by two Tanzanian staff members as normal, disease, trait, variant, or uninterpretable. DBS samples scored as disease or variant were repeated for confirmation and preserved for later genotyping. Regular Skype calls were convened with US-based collaborators to improve quality and interpretation. HIV test results were obtained from the local EID program. Between February 2017 and May 2018, 232 IEF gels were completed by the local staff. After children >24 months of age were excluded to obtain a more accurate newborn prevalence, the median age of children tested was 52 days (IQR 41-93 days), and a total of 17,278 unique DBS samples were scored. The quality of laboratory testing was extremely high with only 20 samples scored as uninterpretable and 54 with missing results, and the primary analysis was performed on the 17,204 remaining samples. The overall prevalence of sickle cell trait and disease in the entire cohort was 20.3% and 1.2%, respectively, with a 0.1% prevalence of hemoglobin variants. This corresponds to an allelic frequency of 0.114 for the sickle gene mutation and demonstrates perfect Hardy-Weinberg equilibrium. No HbC or other common beta-globin variants were identified. Geospatial mapping revealed some variation across regions, with sickle trait ranging from 16.6% to 22.5% and disease ranging from 0.5% to 1.5%. Analysis of individual districts with >100 samples revealed wider geographic variability, with sickle trait ranging from 15.2% to 27.8% and disease ranging from 0.0% to 4.3%. Co-morbidity between HIV and sickle cell disease was analyzed to compare it with the effect on mortality previously observed in Uganda. The prevalence of sickle cell disease was the same among HIV-infected and HIV-negative children (1.2%), suggesting no difference in mortality. The prevalence of sickle cell trait and disease among infants born in northwest Tanzania is very high, exceeding 20% trait and 1.2% disease. All regions in the Lake Zone are affected possibly due to lack of immigration to the area and similar environmental exposures. Targeted newborn screening can be started in high prevalence districts, using existing public health infrastructure with minimal start-up cost and training. Future work will evaluate the correlation between historical malaria prevalence and sickle cell prevalence, and identify hemoglobin variants. Disclosures Ware: Addmedica: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding; Agios: Other: advisory board; Global Blood Therapeutics: Other: advisory board; Biomedomics: Research Funding; Nova Laboratories: Consultancy.
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Rud, Yu, L. Buchatsky, N. Tushnytska, and I. Hrytsyniak. "MOLECULAR DIAGNOSIS OF VIRAL DISEASES IN FISHES." Scientific and Technical Bulletin оf State Scientific Research Control Institute of Veterinary Medical Products and Fodder Additives аnd Institute of Animal Biology 22, no. 2 (October 7, 2021): 323–30. http://dx.doi.org/10.36359/scivp.2021-22-2.38.
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The paper provides information about the main viral diseases of fish found in Ukraine and methods of their diagnosis. Rapid diagnosis of fish viruses using polymerase chain reaction is an affordable and relevant way to monitor and prevent outbreaks of viral diseases. The work of the Laboratory of Biotechnology in Aquaculture of the Institute of Fisheries of NAAS is aimed to develop diagnostic kits for infectious diseases of fish and conducting research in accordance with the procedure of surveillance of fish diseases. For developing diagnostic test systems, attention is paid to the trends of cultured fish species and aquafarming as well as to viruses that circulate in Ukraine or can pass into the Ukrainian aquaculture from neighboring countries. An important role is given to the study of genetic variability of fish viruses, characterized by varying types of virulence. Examples of application the techniques in carrying out diagnostics for the purpose of identification of an infectious agent and the reason of an epizootic are resulted.
A comparative analysis of techniques, components of kits and reagents for optimizing the polymerase chain reaction and its modifications, which can significantly reduce the process of identification of fish viruses, are given. Target genes for oligonucleotide primers selection for PCR diagnosis of viral diseases of fish, namely spring viremia of carp virus (SVCV), cyprinid herpesvirus type 3 (CyHV-3 or KHV), carp edema virus (CEV), infectious pancreatic necrosis virus (IPNV) viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), Piscine orthoreovirus (PRV), Acipenser iridovirus European (AcIV-E).
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Houang, E. T. S., Y. W. Chu, T. K. Ng, and A. F. B. Cheng. "Study of the Relatedness of Isolates ofShigella flexneri and Shigella sonnei Obtained in 1986 and 1987 and in 1994 and 1995 from Hong Kong." Journal of Clinical Microbiology 36, no. 9 (1998): 2404–7. http://dx.doi.org/10.1128/jcm.36.9.2404-2407.1998.
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We used pulsed-field gel electrophoresis (PFGE) to study the genetic relatedness of 235 isolates of Shigella flexneriand Shigella sonnei collected in Hong Kong (97 isolates from 1986 and 1987 and 138 isolates from 1994 and 1995). Altogether, 13 gels were run with bacteriophage lambda ladder DNA (Pharmacia) as an external reference in every sixth lane, standardized reagents and methods, and isolates randomized for species and years. For quantitative illustration of the relationships within a large body of isolates, computer-generated dendrograms were used to determine the number of isolates in pulsotypes at Dice coefficients of similarity of 75% (PT75) and 50% (PT50). For S. flexneri, there was a significant difference in the distribution of isolates collected during the two periods in both PT75and PT50, with 68% of isolates collected in 1994 and 1995 sharing a coefficient of similarity of ≥68%. For S. sonnei, a significant difference was observed in PT50only. We also used Upholt’s formula for an approximation of the fraction of nucleotide difference between isolates and Molecular Evolutionary Genetics Analysis to determine relative genetic distances. For both species, the relative genetic distances between isolates of the earlier collection period were significantly greater (P < 0.0001), i.e., they were further apart and therefore more diverse than those of the later period. We conclude that it is possible for a typical clinical laboratory to analyze a large amount of PFGE information on Shigella isolates obtained under controlled conditions. Such data analysis should enhance surveillance capabilities and give indications of further work to be done on various aspects of bacterial pathogenicity of the species.
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Rasetti-Escargueil, Christine, and Michel R. Popoff. "Recent Developments in Botulinum Neurotoxins Detection." Microorganisms 10, no. 5 (May 10, 2022): 1001. http://dx.doi.org/10.3390/microorganisms10051001.
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Botulinum neurotoxins (BoNTs) are produced as protein complexes by bacteria of the genus Clostridium that are Gram-positive, anaerobic and spore forming (Clostridium botulinum, C. butyricum, C. baratii and C. argentinense spp.). BoNTs show a high immunological and genetic diversity. Therefore, fast, precise, and more reliable detection methods are still required to monitor outbreaks and ensure surveillance of botulism. The botulinum toxin field also comprises therapeutic uses, basic research studies and biodefense issues. This review presents currently available detection methods, and new methods offering the potential of enhanced precision and reproducibility. While the immunological methods offer a range of benefits, such as rapid analysis time, reproducibility and high sensitivity, their implementation is subject to the availability of suitable tools and reagents, such as specific antibodies. Currently, the mass spectrometry approach is the most sensitive in vitro method for a rapid detection of active or inactive forms of BoNTs. However, these methods require inter-laboratory validation before they can be more widely implemented in reference laboratories. In addition, these surrogate in vitro models also require full validation before they can be used as replacement bioassays of potency. Cell-based assays using neuronal cells in culture recapitulate all functional steps of toxin activity, but are still at various stages of development; they are not yet sufficiently robust, due to high batch-to-batch cell variability. Cell-based assays have a strong potential to replace the mouse bioassay (MBA) in terms of BoNT potency determination in pharmaceutical formulations; they can also help to identify suitable inhibitors while reducing the number of animals used. However, the development of safe countermeasures still requires the use of in vivo studies to complement in vitro immunological or cell-based approaches.
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Chukwudi, Ijeoma Chekwube, Kenneth Ikejiofor Ogbu, Pam Dachung Luka, Refiloe Petunia Malesa, Livio Edward Heath, Emmanuel Ikenna Ugochukwu, and Kennedy Foinkfu Chah. "Comparison of colorimetric loop-mediated isothermal amplification kit and reverse transcription-polymerase chain reaction in the diagnosis of peste des petits ruminants in sheep and goats in Southeast Nigeria." November-2020 13, no. 11 (2020): 2358–63. http://dx.doi.org/10.14202/vetworld.2020.2358-2363.
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Background and Aim: Peste des petits ruminants (PPR) is an acute, extremely contagious transboundary viral disease of small ruminants with severe economic consequences, caused by PPR virus. Cost-effective and rapid diagnosis of the disease is essential for prompt management and control. This study aimed to compare the application of a commercial colorimetric loop-mediated isothermal amplification (cLAMP) kit and reverse transcriptase-polymerase chain reaction (RT-PCR) in the diagnosis of PPR in sheep and goats in Southeast Nigeria. Materials and Methods: Nasal swab samples were collected from West African Dwarf sheep and goats showing clinical signs suggestive of PPR (n=80) and those without any clinical signs (n=140) of the disease. The diagnosis was achieved through detection of PPR viral genome in the samples using a cLAMP kit and RT-PCR. cLAMP assay was done directly on nasal swab samples without ribosomal nucleic acid extraction. A set of six primers targeting the matrix gene protein was used for the cLAMP assay. Results: PPR viral genome was detected by both cLAMP and RT-PCR in 51 (63.8%) of the 80 samples from sheep and goats with signs suggestive of PPR while 14 (10%) of those without signs tested positive for PPR by both assay methods. There was a 100% agreement in the cLAMP and RT-PCR results. However, cLAMP was a faster, easier, and less expensive method compared to RT-PCR. Conclusion: The cLAMP assay demonstrates the potential for a point of care diagnosis in the field and a valuable diagnostic tool in areas with poor electricity supply as well as in a less equipped diagnostic laboratory. Since the reagents are affordable, cLAMP can be a diagnostic tool of choice in the detection and surveillance of PPR virus in countries with limited resources.
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Mwanga, Emmanuel P., Prisca A. Kweyamba, Doreen J. Siria, Issa H. Mshani, Idrisa S. Mchola, Faraja E. Makala, Godian Seleman, et al. "Reagent-free detection of Plasmodium falciparum malaria infections in field-collected mosquitoes using mid-infrared spectroscopy and machine learning." Scientific Reports 14, no. 1 (May 27, 2024). http://dx.doi.org/10.1038/s41598-024-63082-z.
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AbstractField-derived metrics are critical for effective control of malaria, particularly in sub-Saharan Africa where the disease kills over half a million people yearly. One key metric is entomological inoculation rate, a direct measure of transmission intensities, computed as a product of human biting rates and prevalence of Plasmodium sporozoites in mosquitoes. Unfortunately, current methods for identifying infectious mosquitoes are laborious, time-consuming, and may require expensive reagents that are not always readily available. Here, we demonstrate the first field-application of mid-infrared spectroscopy and machine learning (MIRS-ML) to swiftly and accurately detect Plasmodium falciparum sporozoites in wild-caught Anopheles funestus, a major Afro-tropical malaria vector, without requiring any laboratory reagents. We collected 7178 female An. funestus from rural Tanzanian households using CDC-light traps, then desiccated and scanned their heads and thoraces using an FT-IR spectrometer. The sporozoite infections were confirmed using enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), to establish references for training supervised algorithms. The XGBoost model was used to detect sporozoite-infectious specimen, accurately predicting ELISA and PCR outcomes with 92% and 93% accuracies respectively. These findings suggest that MIRS-ML can rapidly detect P. falciparum in field-collected mosquitoes, with potential for enhancing surveillance in malaria-endemic regions. The technique is both fast, scanning 60–100 mosquitoes per hour, and cost-efficient, requiring no biochemical reactions and therefore no reagents. Given its previously proven capability in monitoring key entomological indicators like mosquito age, human blood index, and identities of vector species, we conclude that MIRS-ML could constitute a low-cost multi-functional toolkit for monitoring malaria risk and evaluating interventions.
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Hugo, Leon E., Karla van Huyssteen, Olamide Oloniniyi, Laura Donnelly, Anna Conn, Katharine A. Collins, Hayley Mitchell, James S. McCarthy, and Joanne Macdonald. "Rapid low-resource detection of Plasmodium falciparum in infected Anopheles mosquitoes." Frontiers in Tropical Diseases 5 (January 29, 2024). http://dx.doi.org/10.3389/fitd.2024.1287025.
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Vector surveillance of Plasmodium falciparum is critical for monitoring and reducing one of the most severe forms of malaria, which causes high morbidity and mortality in children under five and pregnant women. Here we developed a rapid and highly sensitive test for the detection of P. falciparum (Pf)-infected mosquitoes (Rapid Pf test), with high suitability for low-resource vector surveillance implementation. The Rapid Pf test had similar analytical sensitivity to laboratory-based tests, detecting down to 4 copies/μL of a 18S rRNA DNA standard. In addition, the Rapid Pf test could be completed in less than 30 minutes, and only required a liquid sample preparation reagent, pestle, tube, and 39°C heating block for operation, indicating amenability for low-resource implementation. Diagnostic testing was performed using Anopheles stephensi mosquitoes, either uninfected, or fed with P. falciparum gametocyte cultures. These P. falciparum fed mosquitoes were determined to have 79% infection prevalence based on parallel microscopy and qPCR testing on a subset of 19 mosquitoes. However, our Rapid Pf test determined a 90% positive test rate when testing individual infected mosquitoes (n=30), and did not detect 40 uninfected mosquitoes regardless of blood-fed status (n=40), suggesting the true prevalence of infection in the mosquitoes may have been higher than calculated by qPCR and microscopy. The Rapid Pf test was demonstrated to detect infection in individual mosquitoes (both fresh and frozen/thawed), as well as pools of 1 infected mosquito mixed with 19 known uninfected mosquitoes, and individual mosquitoes left in traps for up to 8 days. After testing on infected and uninfected mosquitoes (n=148) the Rapid Pf test was conservatively estimated to achieve 100% diagnostic sensitivity (95% confidence interval, CI: 91%-100%) and 97% diagnostic specificity (CI: 92%-99%) compared to the estimated prevalence from combined microscopy and qPCR results. These results indicate the Rapid Pf test could provide a highly effective tool for weekly surveillance of infected mosquitoes, to assist with P. falciparum monitoring and intervention studies.
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Perez, Julienne Sanchez, Michele Obeid, Maria Fariduddin, and Daniel Joseph Toft. "SAT546 Papillary Thyroid Cancer Surveillance: Thyroglobulin Levels Affected by HAMA." Journal of the Endocrine Society 7, Supplement_1 (October 2023). http://dx.doi.org/10.1210/jendso/bvad114.2017.
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Abstract Disclosure: J. Sanchez Perez: None. M. Obeid: None. M. Fariduddin: None. D.J. Toft: None. Introduction: Thyroglobulin (TG) measurement is a major means of detecting recurrence of previously treated differentiated thyroid cancers. Human-Anti Mouse Antibodies (HAMA) can interfere with lab measurements of TG levels and affect clinical decisions and treatment for these cancers.Clinical Case: A 65-year-old female was diagnosed with papillary thyroid cancer (PTC). Surgical pathology following total thyroidectomy demonstrated a tumor of 3.5 cm in its greatest dimension, unifocal, without invasion of locoregional tissue classified as Stage I (pT2N0M0) per AJCC 8th edition. Radioiodine whole body scan (WBS) demonstrated iodine uptake in the neck and 126.5 mCi of I-131 was given. In surveillance 5 years later, TSH was found to be 0.17 mcIU/ml (ref range: 0.35 - 4.00) TG 64.7 ng/ml (ref range: 1.3 - 31.8 ng/mL), and anti-Tg antibodies were undetectable. Neck US consistently showed nonspecific findings of stable small nodes with normal morphology in both supraclavicular regions. Given elevated TG levels, 250 mCi of I-131 was given. Follow-up TG levels ranged from 27.8 to 36.4 ng/ml. A repeat WBS did not reveal any uptake, but as TG remained elevated, I-131 (238 mCi) was again given. Subsequent TG throughout the following 15 years ranged from 2.8 - 58.6 ng/ml. Repeat Neck US only showed benign-appearing lymph nodes. WBS did not have any focal activity to suggest recurrent disease and PET/CT did not show any abnormal FDG uptake. The persistently high TG levels without clinical evidence of tumor recurrence raised the suspicion of lab interference. The serum sample was treated with a blocking reagent that contained mouse immunoglobulin after which the TG level decreased to 2.7 ng/ml compared to untreated value of 20.5 ng/ml.Conclusion: HAMA are human antibodies against mouse antibodies which are believed to develop in humans from direct contact with rodents, including mice, and from the recent increase in the use of mouse monoclonal antibodies for diagnostic and therapeutic purposes. These antibodies implicate an unexpected source of interference in immunoassays, as seen in our patient, which can confound laboratory results and create a clinical dilemma. This patient with PTC had a complete radiological response after surgery, but an incomplete biochemical response which caused the patient to be given a total of 614 mCi of I-131. This case highlights the importance of recognizing the possible presence of HAMA when analyzing TG levels, particularly when the TG levels are used for treatment decisions. Presentation Date: Saturday, June 17, 2023
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Balea, Rickyle, Nina M. Pollak, Jody Hobson-Peters, Joanne Macdonald, and David J. McMillan. "Development and pre-clinical evaluation of a Zika virus diagnostic for low resource settings." Frontiers in Microbiology 14 (November 20, 2023). http://dx.doi.org/10.3389/fmicb.2023.1214148.
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IntroductionZika virus (ZIKV) is a re-emerging flavivirus that poses a significant public health threat. ZIKV exhibits a wide array of non-vector borne human transmission routes, such as sexual transmission, transplacental transmission and blood transfusion. Detection and surveillance of ZIKV is considered paramount in prevention of major outbreaks. With the majority of cases reported in low-resource locations, simple, low-cost detection methods are considered highly desirable.Materials and MethodsHere we have developed a sensitive and specific ZIKV diagnostic using reverse transcription recombinase-aided amplification (RT-RAA) coupled with lateral flow detection (LFD) targeting a highly conserved region of the ZIKV NS1 gene.ResultsWe show our rapid, isothermal-ZIKV-diagnostic (Iso-ZIKV-Dx) can detect 500 copies of synthetic ZIKV RNA/μL in under 30 min at a constant 39°C. Using simulated urine samples, we observed that Iso-ZIKV-Dx also detects as low as 34.28 RNA copies/reaction of ZIKV (MR766 strain). Specificity testing confirmed that our test does not detect any co-circulating flaviviruses (dengue, West Nile, Japanese encephalitis, Murray Valley encephalitis and yellow fever viruses) or chikungunya virus. Sample processing results show complete inactivation of ZIKV (MR766 strain) in 5 min at room temperature using our novel viral RNA sample preparation reagent. Furthermore, lateral flow strips testing demonstrates positive diagnoses in as little as 5 min in running buffer.DiscussionContrary to conventional RT-qPCR, our Iso-ZIKV-Dx does not require expensive machinery, specialised laboratory settings or extensively trained personnel. Pre-clinical evaluation demonstrates that our test exhibits robust, in-field capabilities without compromising sensitivity or specificity. When compared to the gold-standard RT-qPCR, our Iso-ZIKV-Dx test offers an array of applications that extend beyond diagnostics alone, including potential for surveillance and monitoring of ZIKV vector competency.
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Kuria, Joseph N., and Stephen M. Gathogo. "Concomitant fungal and Mycobacterium bovis infections in beef cattle in Kenya." Onderstepoort J Vet Res 80, no. 1 (March 4, 2013). http://dx.doi.org/10.4102/ojvr.v80i1.585.
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Bovine tuberculosis is an important zoonosis and accurate diagnosis is important for its surveillance. Post-mortem diagnosis may, however, be compromised by lesions caused by other pathogens. In an investigation on its prevalence in slaughter cattle in Kenya, Mycobacterium bovis and dimorphic fungi were inadvertently identified separately or concurrently in tuberculous lesions. Beef carcasses were inspected for lesions in two abattoirs in Nairobi. Tissues with lesions were collected and transported to the laboratory. Smears of lesions were stained by acid-fast procedure and examined microscopically. Lesions were cultured in Löwenstein-Jensen (LJ) and in BBL TM Mycobacterium growth indicator tubes (MGIT) media. Mycobacteria isolates in LJ medium were identified by DNA typing. Smears of BBLTM MGIT cultures were acid-fast stained and examined microscopically. Tissue sections were stained with periodic acid-Schiff reagent before examination. Of the 929 carcasses examined, 176 had granulomatous lesions. Dimorphic fungi were detected as acid-fast negative cells in 58 (32.9; 33.5%) of the lesion smears, either alone (29.0; 16.4%) or concurrently with acid-fast bacilli (29.0; 16.4%). The fungi were also detected in some BBL TM MGIT-culturesmears and lesioned tissue sections. The fungi were identified, by means of cellular morphology, as Paracoccidioides brasiliensis and Blastomyces dermatitidis. A total of 64 isolates of mycobacteria were recovered in LJ medium, 19 of which were identified as M. bovis. The present report documents native P. brasiliensis infections outside the presumed endemic region and B. dermatitidis infections in a livestock animal. The findings further indicate the importance of dimorphic fungi as a differential diagnosis of bovine tuberculosis in the region.
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Hickman, Rebecca, Jason Nguyen, Tracy D. Lee, John R. Tyson, Robert Azana, Frankie Tsang, Linda Hoang, and Natalie A. Prystajecky. "Rapid, high-throughput, cost-effective whole-genome sequencing of SARS-CoV-2 using a condensed library preparation of the Illumina DNA Prep kit." Journal of Clinical Microbiology, February 5, 2024. http://dx.doi.org/10.1128/jcm.00103-22.
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ABSTRACT The ongoing COVID-19 pandemic necessitates cost-effective, high-throughput, and timely whole-genome sequencing (WGS) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses for outbreak investigations, identifying variants of concern (VoC), characterizing vaccine breakthrough infections, and public health surveillance. In addition, the enormous demand for WGS on supply chains and the resulting shortages of laboratory supplies necessitated the use of low-reagent and low-consumable methods. Here, we report an optimized library preparation method (the BCCDC cutdown method) that can be used in a high-throughput scenario, where one technologist can perform 576 library preparations (6 plates of 96 samples) over the course of one 8-hour shift. The same protocol can also be used in a rapid turnaround time scenario, from primary samples (up to 96 samples) to loading on a sequencer in an 8-hour shift. This new method uses Freed et al.’s 1,200 bp primer sets (Biol Methods Protoc 5:bpaa014, 2020, https://doi.org/10.1093/biomethods/bpaa014 ) and a modified and condensed Illumina DNA Prep workflow (Illumina, CA, USA). Compared to the original protocol, the application of this new method using hundreds of clinical specimens demonstrated equivalent results to the full-length DNA Prep workflow at 45% of the cost, 15% of consumables required (such as pipet tips), 25% of manual hands-on time, and 15% of on-instrument time if performing on a liquid handler, with no compromise in sequence quality. Results demonstrate that this new method is a rapid, simple, cost-effective, and high-quality SARS-CoV-2 WGS protocol. IMPORTANCE Sequencing has played an invaluable role in the response to the COVID-19 pandemic. Ongoing work in this area, however, demands optimization of laboratory workflow to increase sequencing capacity, improve turnaround time, and reduce cost without compromising sequence quality. This report describes an optimized DNA library preparation method for improved whole-genome sequencing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen. The workflow advantages summarized here include significant time, cost, and consumable savings, which suggest that this new method is an efficient, scalable, and pragmatic alternative for SARS-CoV-2 whole-genome sequencing.
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Maladan, Yustinus, Hana Krismawati, Tri Wahyuni, Ratna Tanjung, Kamla Awaludin, Kholis Abdurachim Audah, and Arli Aditya Parikesit. "The whole-genome sequencing in predicting Mycobacterium tuberculosis drug susceptibility and resistance in Papua, Indonesia." BMC Genomics 22, no. 1 (November 22, 2021). http://dx.doi.org/10.1186/s12864-021-08139-3.
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Abstract Background Tuberculosis is one of the deadliest disease caused by Mycobacterium tuberculosis. Its treatment still becomes a burden for many countries including Indonesia. Drug resistance is one of the problems in TB treatment. However, a development in the molecular field through Whole-genome sequencing (WGS) can be used as a solution in detecting mutations associated with TB- drugs. This investigation intended to implement this data for supporting the scientific community in deeply understanding any TB epidemiology and evolution in Papua along with detecting any mutations in genes associated with TB-Drugs. Result A whole-genome sequencing was performed on the random samples from TB Referral Laboratory in Papua utilizing MiSeq 600 cycle Reagent Kit (V3). Furthermore, TBProfiler was used for genome analysis, RAST Server was employed for annotation, while Gview server was applied for BLAST genome mapping and a Microscope server was implemented for Regions of Genomic Plasticity (RGP). The largest genome of M. tuberculosis obtained was at the size of 4,396,040 bp with subsystems number at 309 and the number of coding sequences at 4326. One sample (TB751) contained one RGP. The drug resistance analysis revealed that several mutations associated with TB-drug resistance existed. In details, mutations of rpoB gene which were identified as S450L, D435Y, H445Y, L430P, and Q432K had caused the reduced effectiveness of rifampicin; while the mutases in katG (S315T), kasA (312S), inhA (I21V), and Rv1482c-fabG1 (C-15 T) genes had contributed to the resistance in isoniazid. In streptomycin, the resistance was triggered by the mutations in rpsL (K43R) and rrs (A514C, A514T) genes, and, in Amikacin, its resistance was led by mutations in rrs (A514C) gene. Additionally, in Ethambutol and Pyrazinamide, their reduced effectiveness was provoked by embB gene mutases (M306L, M306V, D1024N) and pncA (W119R). Conclusions The results from whole-genome sequencing of TB clinical sample in Papua, Indonesia could contribute to the surveillance of TB-drug resistance. In the drug resistance profile, there were 15 Multi Drugs Resistance (MDR) samples. However, Extensively Drug-resistant (XDR) samples have not been found, but samples were resistant to only Amikacin, a second-line drug.
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Mshani, Issa H., Frank M. Jackson, Rehema Y. Mwanga, Prisca A. Kweyamba, Emmanuel P. Mwanga, Mgeni M. Tambwe, Lorenz M. Hofer, et al. "Screening of malaria infections in human blood samples with varying parasite densities and anaemic conditions using AI-Powered mid-infrared spectroscopy." Malaria Journal 23, no. 1 (June 17, 2024). http://dx.doi.org/10.1186/s12936-024-05011-z.
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Abstract Background Effective testing for malaria, including the detection of infections at very low densities, is vital for the successful elimination of the disease. Unfortunately, existing methods are either inexpensive but poorly sensitive or sensitive but costly. Recent studies have shown that mid-infrared spectroscopy coupled with machine learning (MIRs-ML) has potential for rapidly detecting malaria infections but requires further evaluation on diverse samples representative of natural infections in endemic areas. The aim of this study was, therefore, to demonstrate a simple AI-powered, reagent-free, and user-friendly approach that uses mid-infrared spectra from dried blood spots to accurately detect malaria infections across varying parasite densities and anaemic conditions. Methods Plasmodium falciparum strains NF54 and FCR3 were cultured and mixed with blood from 70 malaria-free individuals to create various malaria parasitaemia and anaemic conditions. Blood dilutions produced three haematocrit ratios (50%, 25%, 12.5%) and five parasitaemia levels (6%, 0.1%, 0.002%, 0.00003%, 0%). Dried blood spots were prepared on Whatman™ filter papers and scanned using attenuated total reflection-Fourier Transform Infrared (ATR-FTIR) for machine-learning analysis. Three classifiers were trained on an 80%/20% split of 4655 spectra: (I) high contrast (6% parasitaemia vs. negative), (II) low contrast (0.00003% vs. negative) and (III) all concentrations (all positive levels vs. negative). The classifiers were validated with unseen datasets to detect malaria at various parasitaemia levels and anaemic conditions. Additionally, these classifiers were tested on samples from a population survey in malaria-endemic villages of southeastern Tanzania. Results The AI classifiers attained over 90% accuracy in detecting malaria infections as low as one parasite per microlitre of blood, a sensitivity unattainable by conventional RDTs and microscopy. These laboratory-developed classifiers seamlessly transitioned to field applicability, achieving over 80% accuracy in predicting natural P. falciparum infections in blood samples collected during the field survey. Crucially, the performance remained unaffected by various levels of anaemia, a common complication in malaria patients. Conclusion These findings suggest that the AI-driven mid-infrared spectroscopy approach holds promise as a simplified, sensitive and cost-effective method for malaria screening, consistently performing well despite variations in parasite densities and anaemic conditions. The technique simply involves scanning dried blood spots with a desktop mid-infrared scanner and analysing the spectra using pre-trained AI classifiers, making it readily adaptable to field conditions in low-resource settings. In this study, the approach was successfully adapted to field use, effectively predicting natural malaria infections in blood samples from a population-level survey in Tanzania. With additional field trials and validation, this technique could significantly enhance malaria surveillance and contribute to accelerating malaria elimination efforts.
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Ramos-Mandujano, Gerardo, Raik Grünberg, Yingzi Zhang, Chongwei Bi, Francisco J. Guzmán-Vega, Muhammad Shuaib, Rodion V. Gorchakov, et al. "An open-source, automated, and cost-effective platform for COVID-19 diagnosis and rapid portable genomic surveillance using nanopore sequencing." Scientific Reports 13, no. 1 (November 21, 2023). http://dx.doi.org/10.1038/s41598-023-47190-w.
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AbstractThe COVID-19 pandemic, caused by SARS-CoV-2, has emphasized the necessity for scalable diagnostic workflows using locally produced reagents and basic laboratory equipment with minimal dependence on global supply chains. We introduce an open-source automated platform for high-throughput RNA extraction and pathogen diagnosis, which uses reagents almost entirely produced in-house. This platform integrates our methods for self-manufacturing magnetic nanoparticles and qRT-PCR reagents-both of which have received regulatory approval for clinical use–with an in-house, open-source robotic extraction protocol. It also incorporates our "Nanopore Sequencing of Isothermal Rapid Viral Amplification for Near Real-time Analysis" (NIRVANA) technology, designed for tracking SARS-CoV-2 mutations and variants. The platform exhibits high reproducibility and consistency without cross-contamination, and its limit of detection, sensitivity, and specificity are comparable to commercial assays. Automated NIRVANA effectively identifies circulating SARS-CoV-2 variants. Our in-house, cost-effective reagents, automated diagnostic workflows, and portable genomic surveillance strategies provide a scalable and rapid solution for COVID-19 diagnosis and variant tracking, essential for current and future pandemic responses.
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Figueroa, Dania M., Eeva Kuisma, M. Jeremiah Matson, Alain U. Ondzie, Trent Bushmaker, Stephanie N. Seifert, Francine Ntoumi, et al. "Development and validation of portable, field-deployable Ebola virus point-of-encounter diagnostic assay for wildlife surveillance." One Health Outlook 3, no. 1 (May 24, 2021). http://dx.doi.org/10.1186/s42522-021-00041-y.
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Abstract Early detection of Ebola virus spillover into wildlife is crucial for rapid response. We developed and validated a portable, cold-chain independent Ebola virus RT-qPCR assay. Methods The field syringe-based RNA extraction method was compared with a conventional laboratory-based spin-column RNA extraction method. Next, the qPCR efficiency and limit of detection of the assay was compared to standard laboratory-based reagents and equipment. The specificity of the assay was confirmed by testing against multiple Zaire Ebolavirus (EBOV) variants and other ebolavirus species. Lastly, swabs from an EBOV-infected non-human primate carcass, stored at environmental conditions mimicking central and west Africa, were analyzed to mimic in field conditions. Results The syringe-based RNA extraction method performed comparably to a standard laboratory spin-column-based method. The developed assay was comparable in sensitivity and specificity to standard laboratory-based diagnostic assays. The assay specifically detected EBOV and not any of the other tested ebolavirus species, including Reston ebolavirus, Sudan ebolavirus, Bundibugyo ebolavirus, and Tai Forrest ebolavirus. Notably, the assays limit of detection for EBOV isolates were all below 4 genome copies/μL. The assay was able to detect EBOV in oral, nasal, thoracic cavity, and conjunctiva swabs obtained from an infected non-human primate. Conclusion We developed a field-based Ebolavirus assay which is comparable in sensitivity and specificity to laboratory-based assays. Currently, the assay is being incorporated into wildlife carcass surveillance in the Republic of the Congo and is being adapted for other infectious disease agents.