Clinical validation of loop-mediated isothermal amplification for the detection of Escherichia coli sequence type complex 131.

The dissemination of Escherichia coli multidrug-resistant (MDR) STc131 is related to its persistence in the human gastrointestinal tract as efficient gut colonizers. Infection and prevention measures are the cornerstones for preventing STc131 spread. Oral decolonization therapies that target ST131 are being developed. There are no rapid methods available to identify STc131 in human specimens. A loop-mediated isothermal amplification (LAMP) assay (named LAMP-ST131) was developed for the detection of STc131 on well-characterized E. coli isolates and then compared to culture and PCR for urines and stool swabs. With E. coli isolates (n = 720), LAMP-ST131 had a sensitivity (sens) of 100% [95% confidence interval (C.I.) = 98.1-100%)] and a specificity (spec) of 98.9% (95% C.I. = 97.5-99.5%). On urines (n = 550), LAMP-ST131 had a sens of 97.6% (95% C.I. = 89.68-94.33%) and a spec of 92.3% (95% C.I. = 87.68-99.88%), while on stool swabs (n = 278), LAMP-ST131 had a sens of 100% (95% C.I. = 88.7-100%) and a spec of 83.9% (95% C.I. = 78.8-87.9%). LAMP-ST131 detected 10 (urines) and 100 (stool swabs) gene copies/µL. LAMP-ST131 accurately identified STc131 within E. coli isolates and human specimens. The implementation of LAMP-ST131 will aid genomic surveys, enable the rapid implementation of effective infection prevention measures, and identify patients suitable for ST131 decolonization therapies. Such approaches will curb the spread of STc131 and decrease incidence rates of global MDR E. coli infections. IMPORTANCE: We developed an accurate non-culture-based loop-mediated isothermal amplification (LAMP) methodology for the detection of (sequence type) STc131 among Escherichia coli isolates and human specimens. The use of LAMP-ST131 for global genomic surveillance studies and to identify patients that are suitable for ST131 decolonization therapies will be important for decreasing multidrug-resistant E. coli infections across the globe.

Amplicon Deep Sequencing Reveals Multiple Genetic Events Lead to Treatment Failure with Atovaquone-Proguanil in Plasmodium falciparum.

Atovaquone-proguanil (AP) is used as treatment for uncomplicated malaria, and as a chemoprophylactic agent against Plasmodium falciparum. Imported malaria remains one of the top causes of fever in Canadian returning travelers. Twelve sequential whole-blood samples before and after AP treatment failure were obtained from a patient diagnosed with P. falciparum malaria upon their return from Uganda and Sudan. Ultradeep sequencing was performed on the cytb, dhfr, and dhps markers of treatment resistance before and during the episode of recrudescence. Haplotyping profiles were generated using three different approaches: msp2-3D7 agarose and capillary electrophoresis, and cpmp using amplicon deep sequencing (ADS). A complexity of infection (COI) analysis was conducted. De novo cytb Y268C mutants strains were observed during an episode of recrudescence 17 days and 16 h after the initial malaria diagnosis and AP treatment initiation. No Y268C mutant reads were observed in any of the samples prior to the recrudescence. SNPs in the dhfr and dhps genes were observed upon initial presentation. The haplotyping profiles suggest multiple clones mutating under AP selection pressure (COI > 3). Significant differences in COI were observed by capillary electrophoresis and ADS compared to the agarose gel results. ADS using cpmp revealed the lowest haplotype variation across the longitudinal analysis. Our findings highlight the value of ultra-deep sequencing methods in the understanding of P. falciparum haplotype infection dynamics. Longitudinal samples should be analyzed in genotyping studies to increase the analytical sensitivity.

Ultrasensitive Reverse Transcriptase Loop-Mediated Isothermal Amplification (US-LAMP)-Based Detection of Malaria Infection from Dried Blood Spots.

Submicroscopic malaria diagnosis requires highly sensitive tools instead of the conventional microscopy and rapid diagnostic tests (RDTs). While polymerase chain reaction (PCR) is more sensitive than RDTs and microscopy, the required capital cost and technical expertise hinder implementation of PCR in low- and middle-income countries. This chapter describes an ultrasensitive reverse transcriptase loop-mediated isothermal amplification (US-LAMP) test for malaria with a high sensitivity and specificity, while also being practical to implement in low-complexity laboratory settings. The workflow combines a silica spin column-based total nucleic extraction from dried blood spots (DBS) with US-LAMP amplifying the Plasmodium (Pan-LAMP) target and subsequent identification Plasmodium falciparum (Pf-LAMP).

Clostridioides difficile Near-Patient Testing Versus Centralized Testing: A Pragmatic Cluster Randomized Crossover Trial.

BACKGROUND: Management of suspected Clostridioides difficile infection (CDI) in the hospital setting typically results in patient isolation, laboratory testing, infection control, and presumptive treatment. We investigated whether implementation of rapid near-patient testing (NPT) reduced patient isolation time, hospital length of stay (LOS), antibiotic usage, and cost. METHODS: A 2-period pragmatic cluster randomized crossover trial was conducted. Thirty-nine wards were randomized into 2 study arms. The primary outcome measure was effect of NPT on patient isolation time using a mixed-effects generalized linear regression model. Secondary outcomes examined were hospital LOS and antibiotic therapy based on a negative binomial regression model. Natural experiment (NE), intention-to-treat (ITT), and per-protocol (PP) analyses were conducted. RESULTS: During the entire study period, a total of 656 patients received NPT for CDI and 1667 received standard-of-care testing. For the primary outcome, a significant decrease of patient isolation time with NPT was observed (NE, 9.4 hours [P < .01]; ITT, 2.3 hours [P < .05]; PP, 6.7 hours [P < .1]). A significant reduction in hospital LOS was observed with NPT for short stay (NE, 47.4% [P < .01]; ITT, 18.4% [P < .01]; PP, 34.2% [P < .01]). Each additional hour delay for a negative result increased metronidazole use (24 defined daily doses per 1000 patients; P < .05) and non-CDI-treating antibiotics by 70.13 mg (P < .01). NPT was found to save 25.48 US dollars per patient when including test cost to the laboratory and patient isolation in the hospital. CONCLUSIONS: This pragmatic cluster randomized crossover trial demonstrated that implementation of CDI NPT can contribute to significant reductions in isolation time, hospital LOS, antibiotic usage, and healthcare cost. Clinical Trials Registration. NCT03857464.

Correlation of Clinical Severity With Stool Microbiome Changes in Clostridioides difficile Infection.

CONTEXT.­: Clostridioides difficile infection (CDI) is the world-leading cause of infectious nosocomial diarrhea and pseudomembranous colitis. Antibiotics are the first line of treatment against CDI despite the high likelihood of antibiotic failure and/or recurrence. More data are needed to correlate clinical variables with 16S rRNA microbiome profiles in CDI-infected patients. OBJECTIVE.­: To determine the relationship(s) between a patient's clinical factors and the stool bacteriome of CDI-positive patients and CDI-negative patients with diarrheal symptoms. DESIGN.­: This study used stool samples and clinical data from 358 patients with nosocomial diarrhea, who were divided by their CDI diagnosis (CDI-negative: n = 180; CDI-positive; n = 178). The stool bacteriome was profiled by amplicon deep sequencing of the 16S rRNA gene, followed by correlating clinical data. RESULTS.­: The stool bacteriome was significantly different by severity assessment regardless of CDI status. Phyla and species varied significantly by CDI diagnosis. Severity, defined as a serum white blood cell count greater than 15 cells/µL and/or a creatinine level greater than 1.5 mg/dL, correlated significantly with dysbiosis of the stool bacteriome profile of CDI-positive patients compared to CDI-negative patients. Serum white blood cell count was significantly higher in patients with bacterial dysbiosis, and high levels of creatinine were associated with low bacteriome diversity. CONCLUSIONS.­: Clinical severity of CDI influences the stool microbiome of infected patients. To date, this study has the largest data set comparing 16S rRNA microbiome profiles and clinical variables between CDI-infected and noninfected individuals.

Digital Droplet PCR to Track SARS-CoV-2 Outbreak in a Hospital Transitional Care Unit.

We describe an outbreak of SARS-CoV-2 on a transition unit composed of elderly patients awaiting placement. Environmental and patient sample analyses using digital droplet PCR (ddPCR) suggested possible fomite transmission and a high viral burden source from a few individual patients. This outbreak illustrates challenges inherent to this specific patient population.

Active case detection and treatment of malaria in pregnancy using LAMP technology (LAMPREG): a pragmatic randomised diagnostic outcomes trial-study protocol.

INTRODUCTION: Malaria is one of the major public health problems in sub-Saharan Africa. It contributes significantly to maternal and fetal morbidity and mortality in affected countries. This study aims to evaluate the impact of enhanced case detection using molecular testing called loop-mediated isothermal amplification (LAMP) on birth outcomes in a prospective study design. METHODS AND ANALYSIS: A pragmatic randomised diagnostic outcomes trial will be conducted in several health institutes in different Ethiopian regions. Women (n=2583) in their first and second trimesters of pregnancy will be included in the study and individually randomised to the standard of care or enhanced case detection arms, and followed until delivery. Enrolment will encompass the malaria peak transmission seasons. In the standard of care arm, a venous blood sample will be collected for malaria diagnosis only in symptomatic patients. In contrast, in the intervention arm, mothers will be tested by a commercially available Conformité Européene (CE)-approved LAMP malaria test, microscopy and rapid diagnostic test for malaria regardless of their symptoms at each antenatal care visit. The primary outcome of the study is to measure birth weight. ETHICS AND DISSEMINATION: The study was approved by the following ethical research boards: Armauer Hansen Research Institute/ALERT Ethics Review Committee (FORM AF-10-015.1, Protocol number PO/05/20), the Ethiopia Ministry of Science and Higher Education National Research Ethics Review Committee (approval SRA/11.7/7115/20), the Ethiopia Food and Drug Administration (approval 02/25/33/I), UCalgary Conjoint Health Research Ethics Board (REB21-0234). The study results will be shared with the institutions and stakeholders such as the Ethiopia Ministry of Health, the Foundation for Innovative Diagnostics, WHO's Multilateral initiative on Malaria - Tropical Diseases Research (TDR-MIM), Roll Back Malaria and the Malaria in Pregnancy Consortium. The study results will also be published in peer-reviewed journals and presented at international conferences. TRIAL REGISTRATION NUMBER: NCT03754322.

SARS-CoV-2 Viral Load Quantification, Clinical Findings and Outcomes in Children Seeking Emergency Department Care: Prospective Cohort Study.

We compared the perfomance of SARS-CoV-2 reverse transcriptase real-time polymerase chain reaction (RT-PCR) to droplet digital PCR (ddPCR). 95% and 40% of positive and negative RT-PCR specimens, respectively, were positive on ddPCR yielding sensitivities of 84% (95% CI: 74, 91) and 97% (95% CI: 89, 99), for RT-PCR and ddPCR, respectively. We found that SARS-CoV-2 RT-PCR testing in children has a concerning false-negative rate at lower nucleocapsid gene copy numbers.

The Role of Subgenomic RNA in Discordant Results From Reverse Transcription-Polymerase Chain Reaction Tests for COVID-19.

CONTEXT.­: Reverse transcription-polymerase chain reaction (RT-PCR) is the standard method of diagnosing COVID-19. An inconclusive test result occurs when 1 RT-PCR target is positive for SARS-CoV-2 and 1 RT-PCR target is negative for SARS-CoV-2 within the same sample. An inconclusive result generally requires retesting. One reason why a sample may yield an inconclusive result is that one target is at a higher concentration than another target. OBJECTIVE.­: To understand the role of subgenomic RNA transcripts in discordant results from RT-PCR tests for COVID-19. DESIGN.­: A panel of 6 droplet digital PCR assays was designed to quantify the ORF1, E-gene, and N-gene of SARS-CoV-2. This panel was used to quantify viral cultures of SARS-CoV-2 that were harvested during the eclipse phase and at peak infectivity. Eleven clinical nasopharyngeal swabs were also tested with this panel. RESULTS.­: In culture, infected cells showed higher N-gene/ORF1 copy ratios than culture supernatants. The same trends in the relative abundance of copies across different targets observed in infected cells were observed in clinical samples, although trends were more pronounced in infected cells. CONCLUSIONS.­: This study showed that a greater copy number of N-gene relative to E-gene and ORF1 transcripts could potentially explain inconclusive results for some RT-PCR tests on low viral load samples. The use of N-gene RT-PCR target(s) as opposed to ORF1 targets for routine testing is supported by these data.

Clinical and Infection Prevention Applications of Severe Acute Respiratory Syndrome Coronavirus 2 Genotyping: an Infectious Diseases Society of America/American Society for Microbiology Consensus Review Document.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged into a world of maturing pathogen genomics, with more than 2 million genomes sequenced at the time of writing. The rise of more transmissible variants of concern that impact vaccine and therapeutic effectiveness has led to widespread interest in SARS-CoV-2 evolution. Clinicians are also eager to take advantage of the information provided by SARS-CoV-2 genotyping beyond surveillance purposes. Here, we review the potential role of SARS-CoV-2 genotyping in clinical care. The review covers clinical use cases for SARS-CoV-2 genotyping, methods of SARS-CoV-2 genotyping, assay validation and regulatory requirements, and clinical reporting for laboratories, as well as emerging issues in clinical SARS-CoV-2 sequencing. While clinical uses of SARS-CoV-2 genotyping are currently limited, rapid technological change along with a growing ability to interpret variants in real time foretells a growing role for SARS-CoV-2 genotyping in clinical care as continuing data emerge on vaccine and therapeutic efficacy.

Clinical and Infection Prevention Applications of Severe Acute Respiratory Syndrome Coronavirus 2 Genotyping: An Infectious Diseases Society of America/American Society for Microbiology Consensus Review Document.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged into a world of maturing pathogen genomics, with >2 million genomes sequenced at this writing. The rise of more transmissible variants of concern that affect vaccine and therapeutic effectiveness has led to widespread interest in SARS-CoV-2 evolution. Clinicians are also eager to take advantage of the information provided by SARS-CoV-2 genotyping beyond surveillance purposes. Here, we review the potential role of SARS-CoV-2 genotyping in clinical care. The review covers clinical use cases for SARS-CoV-2 genotyping, methods of SARS-CoV-2 genotyping, assay validation and regulatory requirements, clinical reporting for laboratories, and emerging issues in clinical SARS-CoV-2 sequencing. While clinical uses of SARS-CoV-2 genotyping are currently limited, rapid technological change along with a growing ability to interpret variants in real time foretell a growing role for SARS-CoV-2 genotyping in clinical care as continuing data emerge on vaccine and therapeutic efficacy.

Performance of four commercial real-time PCR assays for the detection of bacterial enteric pathogens in clinical samples.

OBJECTIVES: Many laboratories use culture-independent diagnostic tests for bacterial gastroenteritis (i.e. real-time polymerase chain reaction, RT-PCR) instead of culture because of better sensitivity, automation, and faster turnaround times. To address some gaps in initial evaluations and lack of intraassay comparisons for many commercial RT-PCRs, this study compared the ability of four commercially available RT-PCR tests (Ridagene, Fast Track Diagnostics, BD Max, and Prodesse Progastro) to detect five major bacterial enteric pathogens: Campylobacter, Salmonella, Shiga-toxin producing Escherichia coli (STEC), Shigella, and Yersinia. METHODS: Clinical stool specimens and contrived samples comprising commonly circulating species, serotypes, biovars, and/or toxin subtypes were used for the comparison. RESULTS: Concordance rates for RT-PCR and culture using culture-positive and culture-negative clinical stools were >90% for Campylobacter (97.5-100%), Salmonella (97.5-100%), Shigella (100%), and STEC (90-100%). However, the agreement between RT-PCR and culture for Y. enteroccolitica ranged from 70-90%. For the contrived sample set, stx2f was detected by one of four assays. Of note, no assay could detect Yersinia non-enterocolitica and Campylobacter upsaliensis. CONCLUSIONS: Depending on the prevalence of certain stx sub-types, Yersinia species, and Campylobacter species in a laboratory's jurisdiction, without further improvement in culture-independent tests, culture methods remain critical for the detection of these pathogens.

SARS-CoV-2 variant detection with ADSSpike.

The SARS-CoV-2 coronavirus pandemic has been an unprecedented challenge to global pandemic response and preparedness. With the continuous appearance of new SARS-CoV-2 variants, it is imperative to implement tools for genomic surveillance and diagnosis in order to decrease viral transmission and prevalence. The ADSSpike workflow was developed with the goal of identifying signature SNPs from the S gene associated with SARS-CoV-2 variants through amplicon deep sequencing. Seventy-two samples were sequenced, and 30 mutations were identified. Among those, signature SNPs were linked to 2 Zeta-VOI (P.2) samples and one to the Alpha-VOC (B.1.17). An average depth of 700 reads was found to properlycorrectly identify all SNPs and deletions pertinent to SARS-CoV-2 mutants. ADSSpike is the first workflow to provide a practical, cost-effective, and scalable solution to diagnose SARS-CoV-2 VOC/VOI in the clinical laboratory, adding a valuable tool to public health measures to fight the COVID-19 pandemic for approximately $41.85 USD/reaction.

Risk Assessment of Hospitalized Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-Infected Patients Using Laboratory Data and Immune Cell Morphologic Assessment.

CONTEXT.­: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly infectious agent, with the propensity to cause severe illness. While vaccine uptake has been increasing in recent months, many regions remain at risk of significant coronavirus disease 19 (COVID-19)-related health care burden. Health systems will continue to benefit from the availability of a variety of clinical and laboratory models when other triaging models are equivocal. OBJECTIVE.­: To validate previously reported clinical laboratory abnormalities seen in COVID-19 patients and identify what laboratory parameters might be outcome predictive. DESIGN.­: We undertook an observational study of hospital-admitted COVID-19 patients (n = 113), looking at a broad selection of clinical, laboratory, peripheral blood smear, and outcome data during discrete discovery and validation periods from March 2020 to November 2020. RESULTS.­: We confirmed the findings of previous studies noting derangement of a variety of laboratory parameters in COVID-19 patients, including peripheral blood morphologic changes. We also devised a simple-to-use decision tree by which patients could be risk stratified on the basis of red blood cell count, creatinine, urea, and atypical plasmacytoid lymphocyte ("covidocyte") count. This outcome classifier performed comparably to the World Health Organization clinical classifier and the neutrophil-lymphocyte ratio. CONCLUSIONS.­: Our data add to the increasing number of studies cataloguing laboratory changes in COVID-19 and support the clinical utility of incorporating blood morphologic assessment in the workup of hospitalized COVID-19 patients.

A metagenomics workflow for SARS-CoV-2 identification, co-pathogen detection, and overall diversity.

An unbiased metagenomics approach to virus identification can be essential in the initial phase of a pandemic. Better molecular surveillance strategies are needed for the detection of SARS-CoV-2 variants of concern and potential co-pathogens triggering respiratory symptoms. Here, a metagenomics workflow was developed to identify the metagenome diversity by SARS-CoV-2 diagnosis (npositive = 65; nnegative = 60), symptomatology status (nsymptomatic = 71; nasymptomatic = 54) and anatomical swabbing site (nnasopharyngeal = 96; nthroat = 29) in 125 individuals. Furthermore, the workflow was able to identify putative respiratory co-pathogens, and the SARS-CoV-2 lineage across 29 samples. The diversity analysis showed a significant shift in the DNA-metagenome by symptomatology status and anatomical swabbing site. Additionally, metagenomic diversity differed between SARS-CoV-2 infected and uninfected asymptomatic individuals. While 31 co-pathogens were identified in SARS-CoV-2 infected patients, no significant increase in pathogen or associated reads were noted when compared to SARS-CoV-2 negative patients. The Alpha SARS-CoV-2 VOC and 2 variants of interest (Zeta) were successfully identified for the first time using a clinical metagenomics approach. The metagenomics pipeline showed a sensitivity of 86% and a specificity of 72% for the detection of SARS-CoV-2. Clinical metagenomics can be employed to identify SARS-CoV-2 variants and respiratory co-pathogens potentially contributing to COVID-19 symptoms. The overall diversity analysis suggests a complex set of microorganisms with different genomic abundance profiles in SARS-CoV-2 infected patients compared to healthy controls. More studies are needed to correlate severity of COVID-19 disease in relation to potential disbyosis in the upper respiratory tract. A metagenomics approach is particularly useful when novel pandemic pathogens emerge.

Role of Hsp90 in Plasmodium falciparum Malaria.

Studies in mammalian and yeast chaperone systems have significantly advanced our understanding of the biochemistry of heat shock protein 90 (Hsp90), particularly as it pertains to structural details, interaction regulation, and function. Harnessing this body of knowledge, parasitologists have made tremendous steps in understanding the role of the Hsp90 chaperone machine in protozoal parasites, especially in malaria. Heat shock proteins are a crucial part of the signaling events involved in malaria parasite acclimatization in the host. Even though studies of the role of Hsp90 in malaria have been thorough and discovered some of the most intricate details of the utility of the Hsp90 interactome for pathogen-host interactions, the full and elaborate extent of the role of Hsp90 homologs in malaria remains to be uncovered. This chapter discusses the current knowledge about the biochemistry and cellular role of Hsp90 in Plasmodium falciparum malaria. In addition, the involvement of both parasitic and host Hsp90s by the parasite in the infected cell is discussed.

A multicenter study investigating SARS-CoV-2 in tertiary-care hospital wastewater. viral burden correlates with increasing hospitalized cases as well as hospital-associated transmissions and outbreaks.

SARS-CoV-2 has been detected in wastewater and its abundance correlated with community COVID-19 cases, hospitalizations and deaths. We sought to use wastewater-based detection of SARS-CoV-2 to assess the epidemiology of SARS-CoV-2 in hospitals. Between August and December 2020, twice-weekly wastewater samples from three tertiary-care hospitals (totaling > 2100 dedicated inpatient beds) were collected. Hospital-1 and Hospital-2 could be captured with a single sampling point whereas Hospital-3 required three separate monitoring sites. Wastewater samples were concentrated and cleaned using the 4S-silica column method and assessed for SARS-CoV-2 gene-targets (N1, N2 and E) and controls using RT-qPCR. Wastewater SARS-CoV-2 as measured by quantification cycle (Cq), genome copies and genomes normalized to the fecal biomarker PMMoV were compared to the total daily number of patients hospitalized with active COVID-19, confirmed cases of hospital-acquired infection, and the occurrence of unit-specific outbreaks. Of 165 wastewater samples collected, 159 (96%) were assayable. The N1-gene from SARS-CoV-2 was detected in 64.1% of samples, N2 in 49.7% and E in 10%. N1 and N2 in wastewater increased over time both in terms of the amount of detectable virus and the proportion of samples that were positive, consistent with increasing hospitalizations at those sites with single monitoring points (Pearson's r = 0.679, P < 0.0001, Pearson's r = 0.799, P < 0.0001, respectively). Despite increasing hospitalizations through the study period, nosocomial-acquired cases of COVID-19 (Pearson's r = 0.389, P < 0.001) and unit-specific outbreaks were discernable with significant increases in detectable SARS-CoV-2 N1-RNA (median 112 copies/ml) versus outbreak-free periods (0 copies/ml; P < 0.0001). Wastewater-based monitoring of SARS-CoV-2 represents a promising tool for SARS-CoV-2 passive surveillance and case identification, containment, and mitigation in acute- care medical facilities.

A rapid near-patient detection system for SARS-CoV-2 using saliva.

The highly infectious nature of SARS-CoV-2 necessitates the use of widespread testing to control the spread of the virus. Presently, the standard molecular testing method (reverse transcriptase-polymerase chain reaction, RT-PCR) is restricted to the laboratory, time-consuming, and costly. This increases the turnaround time for getting test results. This study sought to develop a rapid, near-patient saliva-based test for COVID-19 (Saliva-Dry LAMP) with similar accuracy to that of standard RT-PCR tests. A lyophilized dual-target reverse transcription-loop-mediated isothermal amplification (RT-LAMP) test with fluorometric detection by the naked eye was developed. The assay relies on dry reagents that are room temperature stable. A device containing a centrifuge, heat block, and blue LED light system was manufactured to reduce the cost of performing the assay. This test has a limit of detection of 1 copy/µL and achieved a positive percent agreement of 100% [95% CI 88.43% to 100.0%] and a negative percent agreement of 96.7% [95% CI 82.78-99.92%] relative to a reference standard test. Saliva-Dry LAMP can be completed in 105 min. Precision, cross-reactivity, and interfering substances analysis met international regulatory standards. The combination of ease of sample collection, dry reagents, visual detection, low capital equipment cost, and excellent analytical sensitivity make Saliva-Dry LAMP particularly useful for resource-limited settings.