High-Resolution Mass Spectrometric Rapid Identification of Candida auris

A recent study published in the Journal of Fungi used a novel OrbitrapTM high-resolution mass spectrometric technology coupled with liquid chromatography to identify geographically different clades of Candida auris (C. auris) isolates. This proof-of-concept methodology could accurately detect C. auris in the microbiology laboratory.

Background

Over a decade ago, C. auris was first found in East Asia, causing bloodstream infections. Although this fungal infection was initially found in India, South America, South Africa, and the Middle East, it soon prevailed globally. 

C. auris soon became a common nosocomial fungal pathogen, particularly among intensive care unit (ICU) patients. As a result, the Centers for Disease Control and Prevention (CDC) has classified C. auris as an urgent threat pathogen.

An important factor that allows C. auris outbreaks worldwide is the improper identification of yeast pathogens in hospital laboratories. Hence, there is an urgent need for accurate and rapid identification of C. auris in hospital laboratories, which can reduce their transmission in healthcare facilities.

Genomic analysis of worldwide C. auris isolates has indicated that around five clades have emerged in the last 20 years, independently and simultaneously. These five distinct geographically restricted clades are clade I: South Asia, clade II: East Asia, clade III: Africa, clade IV: South America, and clade V: Iran. Each clade differs from the other by around ten thousand single-nucleotide polymorphisms. 

Each clade has differential resistance to antifungal agents; for example, clade I is more resistant to fluconazole, while clade II exhibits susceptibility. Currently, C. auris isolates belonging to these clades have been introduced to many countries worldwide. Scientists have highlighted the importance of quickly identifying and monitoring these clades to restrict further spread. 

C. auris possesses several structurally unique sphingolipids and mannoproteins, enabling it to adhere to medical devices and hospital environments persistently. These proteins also aid in biofilm formation and prevent elimination by common disinfectants.

Several studies have indicated that molecular techniques fail to identify C. auris, whereas matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) technology can accurately identify this fungus at the species level.

The Study and its Findings

102 clinical C. auris strains were selected, representing all five clades. These clades were determined based on a short tandem repeat (STR) typing assay, which was subsequently compared to whole-genome sequencing results.

The current study applied OrbitrapTM high-resolution mass spectrometric technology to identify C. auris based on protein analysis methods. This technique was combined with liquid chromatography (LC) for initial separation. In this method, electrospray ionization (ESI) transfers proteins into the gas phase for ionization and is subsequently introduced to the mass spectrometer (LC-MS).

Mass analysis is conducted by either fragment ions or intact mass (MS) through tandem mass spectrometry (MS/MS). Some of the key features of the OrbitrapTM mass analyzer are a high resolution of up to 200,000, a high mass-to-charge ratio of 6,000, high mass accuracy between 2 and 5 ppm, and a dynamic range greater than 104.

In addition, this method is highly sensitive and can measure the exact mass of a compound. It can also identify minor structural changes due to a translated single nucleotide polymorphism into an amino acid change.

Importantly, this newly developed technology could identify all C. auris isolates with high confidence. Furthermore, it could differentiate C. auris across clades. Even though a limited number of isolates were present from each clade, this spectrometric technology identified C. auris clades with 99.6% identification accuracy.

Based on a principal component analysis (PCA) and a subsequent affinity clustering study, the South Asian, East Asian, and Iranian C. auris clades were more proteomically closely related. Long branches in the affinity clustering analysis suggested that the C. auris strains were present as outliers that required more attention, regardless of the detection technique.

Proteomic typing results indicated the capacity to track strains of the same origin isolated from diverse geographical locations. In the future, more precise matching and alignment of typing schemes (based on next-generation sequencing) is required to build on these results. This would significantly reduce false identifications and classifications of unknown strains associated with new clades or lineage.

Conclusions

Although the workflow linked to mass spectrometry and next-generation sequencing are not directly comparable, their results are similar, i.e., identifying unknown clinical microbes. The standard next-generation sequencing method is a highly time-consuming process that requires many delicate time-intensive quality-control steps, particularly during multiplexed sample runs.

In contrast, the newly developed methodology can provide results within 60 minutes. Therefore, applying the high-resolution OrbitrapTM mass spectrometer to accurately and rapidly identify C. auris clades is an attractive alternative to conventional platforms.

Journal reference:

Jamalian, A. et al. (2023) "Fast and Accurate Identification of Candida auris by High Resolution Mass Spectrometry", Journal of Fungi, 9(2), p. 267. doi: 10.3390/jof9020267, https://www.mdpi.com/2309-608X/9/2/267

Source: News-Medical.Net 

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Finger-Prick Test Developed for Trichomonas vaginalis

A quick, affordable diagnostic test developed by a Washington State University researcher may help curb one of the most prevalent but least discussed sexually transmitted infections.

More common than chlamydia or gonorrhea, Trichomonas vaginalis, also known as trich, causes no symptoms in about 70% of those infected. Even when asymptomatic, trich is linked to a host of bad health outcomes, including increased susceptibility to HIV, prostate cancer in men and infertility and pregnancy complications in women.

Trich is easily curable with a drug called metronidazole, if diagnosed, and WSU researcher John Alderete has been working for years to improve testing and make it more accessible. His latest development, detailed in the journal Pathogens, is a new finger-prick test that delivers results in five minutes and can be produced for under $20.

“We know a lot about the biology of this organism,” said Alderete, the study lead author and professor in WSU’s School of Molecular Biosciences. “There probably will never be a vaccine for trich simply because the organism is well equipped to evade our immune responses. But I’d argue we don’t need a vaccine. We just need to diagnose people, and once diagnosed, they can be cured.”

Currently trich is often only diagnosed when symptoms are present, which can include genital itchiness and a burning sensation during urination. The tests in use now are focused on diagnosing women and involve a vaginal swab. It takes time to get results, and the tests require trained personnel as well as specialized equipment. Other methods recently approved by the Food and Drug Administration also have similar limitations.

As detailed in this study, the new test requires only a drop of blood to detect an antibody specific to trich. Alderete previously identified this biomarker, an alpha-actinin protein unique to this organism named ACT::SOE3 in earlier research. Both men and women make the antibody when they are infected.

Alderete, working with co-author Hermes Chan from the company MedMira, used the diagnostic platform of the company for his finger-prick test to detect for antibody to the target protein. Similar to COVID‑19 and pregnancy tests, the results are displayed in a window with a dot appearing if the antibody is present, indicating infection. It is a point-of-care diagnostic test meaning positive results lead to immediate treatment and cure of a person. The test does not require specialized training and equipment to administer.

The goal was to meet World Health Organization “ASSURED” standards for disease detection, which stands for affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end users. Since trich is a world-wide problem with an estimated 156 million new cases each year, Alderete hopes the test can ultimately be used in many low-resource countries, particularly in places like Africa where trich is suspected to be a contributing factor in the spread of HIV.  

The test can also have many benefits in the U.S. as well. Alderete estimated there are more than 9.2 million cases annually based on incidence rates and census data. One study found that 50% of pregnant women had persistent undiagnosed infections — a significant concern since trich is associated with pre‑term membrane rupture, preterm birth and low infant birth weight.

The first step is to make more people aware of the problem, Alderete said.

“Trich is the most common STI you’ve probably never heard of,” he said. “This STI may be the most neglected among the other curable STIs. We just have not done a good job in medicine to educate people. One of the major problems is that most people are asymptomatic. In other words, you may have it, but you don’t know you have it until you have a really bad problem.”

Patent protection on the new development is in process.

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Wayne State Researchers Develop New Technology to Easily Detect Active TB

A team of faculty from Wayne State University has discovered new technology that will quickly and easily detect active Mycobacterium tuberculosis (TB) infection antibodies. Their work, “Discovery of Novel Transketolase Epitopes and the Development of IgG-Based Tuberculosis Serodiagnostics,” was published in a recent edition of Microbiology Spectrum, a journal published by the American Society for Microbiology. The team is led by Lobelia Samavati, M.D., professor in the Center for Molecular Medicine and Genetics in the School of Medicine. Samavati was joined by Jaya Talreja, Ph.D, and Changya Peng, research scientists in Wayne State’s Department of Internal Medicine.

TB remains a global health threat, with 10 million new cases and 1.7 million deaths annually. According to the latest World Health Organization report, TB is the 13th leading cause of death and the second leading infectious killer after COVID-19. Latent tuberculous infection (LTBI) is considered a reservoir for TB bacteria and subjects can progress to active TB. One-third of the world’s population is infected with TB and, on average, 5 to 10% of those infected with LTBI will develop active TB disease over the course of their lives, usually within the first five years after initial infection.

The gold standard tests to determine whether an infection is active TB are the sputum smear and culture tests. However, these methods require collecting sputum, which is time consuming, expensive, requires trained personnel and lacks sensitivity. The current conventional tests  differentiating LTBI from uninfected controls — such as tuberculin skin tests (TST) and/or interferongamma release assay (IGRA) — do not differentiate between active TB infection and latent TB. Despite advances in rapid molecular techniques for TB diagnostics, there is an unmet need for a simple inexpensive point-of-care (POC), rapid non-sputum-based test.

Samavati’s research group has worked for more than 15 years to develop technology for detection of antibodies in various respiratory diseases. Her lab has developed a novel non-sputum based technology and has discovered several novel immune-epitopes that differentialy bind to specific immunoglobulin (IgG) in TB-infected subjects. The levels of epitope-specific IgG  in seum can differentiate active TB from LTBI subjects, healthy contols and other respiratory diseases. This  technology can be used as a simple serum assay non-sputum based serological POC- TB test, which is highly specific and sensitveto diffentiate active TB from LTBI.

“Previously, we developed a T7 phage antigen display platform and after immunoscreening of large sets of serum samples, identified 10 clones that differentially bind to serum antibody (IgG) of active TB patients differentiating TB from other respiratory diseases,” said Samavati. “One of these high-performance clones had homology to the Transketolase (TKT) enzyme of TB bacteria that is an essential enzyme required for the intracellular growth of the bacteria in a host. We hypothesized that abundance of IgG in sera against the identified novel neoantigen that we named as TKTµ may differentiate between active TB, LTBI and other non-TB granulomatous lung diseases such as sarcoidosis. We developed a novel direct Peptide ELISA test to quantify the levels of IgG in serum samples against TKTµ. We designed two additional overlapping M.tb TKT-peptide homologs with potential antigenicity corresponding to M.tb-specific transketolase (M.tb-TKT1 and M.tb-TKT3) and hence standardized three Peptide ELISA (TKTμ, M.tb TKT1 and M.tb TKT3) for the TB serodiagnosis.”

After development and standardization of a direct peptide ELISA for three peptides, the research team tested 292 subjects, and their TKT-peptide ELISA results show that TB patients have significantly higher levels of TKT-specific antibodies compared to patients who were healthy controls and with LTBI. The increased levels of TKT-specific antibodies is presumably associated with growing M.tb bacteria in active TB patients. TKT plays a key role in the switch from the dormancy to proliferative phase and TKT specific IgG may uncover the differences between active TB and LTBI. Thus, IgG-based serodiagnosis of TB with TKT-peptide ELISA is promising.

Currently, commercially available serological TB tests show poor sensitivity and specificity. The ELISA results obtained with the Wayne State team’s discovered TKT peptides yielded high specificity and sensitivity. Their results show that IgG antibodies against transketolase can discriminate active tuberculosis. 

“Our TKT peptide ELISA test requires chemically synthesized TKT peptides to coat the wells in the ELISA plate, less than 100µl blood serum sample from patient, detection reagents and an ELISA plate reader,” said Samavati. “We are extremely enthusiastic about our technology and the fact that with a simple test we can differentiate active TB from LTBI and other respiratory diseases. We believe that our method and TKT peptide ELISA can fit the requirements of the World Health Organization and the Centers for Disease Control and Prevention as a POC screening method.”

The research team has applied a patent application on its technology and is actively seeking companies interested in investing.

This research was supported by the National Heart, Lung and Blood Institute of the National Institutes of Health, grant numbers 113508 and 148089. The Foundation for Innovative New Diagnostics (FIND, Geneva, Switzerland) provided TB and LTBI samples.

The full publication is available at https://journals.asm.org/doi/10.1128/spectrum.03377-22.

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A New Rapid ‘Glow-in-the-Dark’ Test for Infectious Diseases

PCR testing - the highly sensitive gold standard for Covid-19 detection - requires expensive equipment, expert technicians, and dedicated laboratory facilities, all leading to test times of several hours or even a day. Researchers from Eindhoven University of Technology, Rijnstate Hospital, and Fontys University of Applied Sciences have developed a new test based on a ‘glow-in-the-dark’ approach that could be used to directly detect DNA/RNA of viruses and bacteria with the same sensitivity as a PCR test, and all in just 30 minutes.

“PCR testing is very accurate and was invaluable during the pandemic,” says Maarten Merkx, from the Institute for Complex Molecular Systems (ICMS), department of Biomedical Engineering, and head of the Merkx Lab at TU/e. “But it can take several hours, and often a day, to get the result of a PCR test.”

Part of the time delay with PCR testing, which checks for the presence of viral DNA or RNA in a patient sample, is the need for a thermal cycling process.

“Samples tested via PCR might contain miniscule amounts of viral DNA/RNA. To make this detectable, the sample is subjected to cycles of heating and cooling. This leads to the right conditions for DNA/RNA replication,” says Harm van der Veer, who is a PhD candidate in the Merkx Lab at TU/e. “But expensive equipment is needed, making it unsuitable for use by GPs or other local health facilities, let alone as a home test.”

Enter LUNAS

To address these drawbacks, van der Veer, Merkx and colleagues from TU/e, Rijnstate Hospital, and Fontys University of Applied Sciences have developed a new sensor that can be used at the GP, gives rapid results, and has similar sensitivity and specificity as the PCR test.

And just like the PCR test, their test has a catchy acronym. It’s known as LUNAS. Their work on the test has just been published in the journal ACS Central Science. Van der Veer: “LUNAS stands for luminescent nucleic acid sensor, which means that we have made a bioluminescent sensor to detect the nucleic acids (the molecules that make up DNA or RNA) found in viruses and bacteria.”

The Merkx Lab has considerable experience with bioluminescent sensors. “Many organisms such as fireflies use bioluminescence to produce light. In our sensors, we combine proteins that bind a certain biomolecule (such as an antibody or DNA or RNA) with so-called luciferase enzymes, which emit light upon detection of the target biomolecule in a sample,” says Merkx.  

What’s more, bioluminescent sensors require no external excitation and the ‘glow-in-the-dark’ response can be detected with an ordinary digital camera that you’ll find in a smartphone.

A CRISPR helping hand

When developing LUNAS, the researchers had to solve two major problems: ‘How can the test replicate the thermal cycling amplification of DNA used in PCR testing?’ and ‘How can the sensor accurately bind to the resulting DNA in a rapid manner?’

“Our aim was a test with the sensitivity to detect a single piece of DNA in a microliter sample (a sample that is one millionth of a liter) that also happens to contains lots of other molecules. It’s like trying to find one specific, tiny fish in a sea of quadrillion (10e15) liters of water filled with lots of other fish,” says van der Veer.

First, to quickly produce more viral DNA/RNA copies in a sample, the researchers used so-called recombinase polymerase amplification (RPA), which has been used in other point-of-care (close to the patient) tests in the past, as it works at low, constant temperature unlike PCR.

Second, to ensure specific detection of the DNA/RNA in the sample the researchers turned to CRISPR for help. “Most people associate CRISPR with DNA editing, but at the heart of CRISPR is a very accurate way of identifying and latching onto pieces of DNA”, says van der Veer.

So, the researchers bonded two CRISPR proteins to a split bioluminescent protein (See image). These proteins are designed to recognize and grab pieces of viral DNA copies. When they do so, the two pieces of the bioluminescent protein are joined together and produce blue light. Then, the researchers can detect the blue light using a digital camera.

If there’s no viral DNA/RNA in the sample, little or no blue light is emitted by the sensor. Instead, a reference bioluminescent protein just emits green light.

“However, when viral DNA/RNA is present, within about 10 - 30 minutes there is a marked increase in the blue-to-green light ratio. It’s like a molecular traffic light,” adds Merkx. To validate the accuracy of the LUNAS sensor to detect viral infections, the researchers tested Covid-19 patient samples provided by Rijnstate Hospital.

iGEM beginnings and future directions

LUNAS can be traced all the way back to the iGEM Eindhoven 2019 student project when van der Veer was part of the student team, which worked on making a test for the rapid detection of bacterial pathogens and help fight antimicrobial resistance.

From working on the project, van der Veer developed his interest in biosensors, which led to his masters research, and now PhD research, at the Merkx Lab. “The fruition of my time with iGEM is LUNAS,” says van der Veer.

LUNAS also looks set to be developed to detect other illnesses. “In a collaboration involving Fontys, companies, healthcare professionals, and led by TU/e PhD researcher Yosta de Stigter, we are developing LUNAS to test for sexually transmitted disease (STDs) and a portable microfluidic device to help with its use at point-of-care (near patient or beside) locations,” says Merkx.

Commercialization is also on the minds of van der Veer and Merkx, particularly for healthcare. However, there are key barriers to this adaption in healthcare.

“At the moment, we lack a simple and portable device for the test, which would make it easier for use at GPs or even at home,” notes van der Veer. “In addition, it would be great to have a commercial partner that could produce the tests at a large scale, while also accounting for all of the regulatory steps that need to be adhered to when making such a test.”

Additional information

This research has been supported by the Covid-19 UF fund. The research in this project is also supported by Dutch Research Council (NWO) | Nationaal Regieorgaan Praktijkgericht Onderzoek SIA (NRPO-SIA) project RAAK.PRO02.066.

Paper title: Glow-in-the-Dark Infectious Disease Diagnostics Using CRISPR-Cas9-Based Split Luciferase Complementation, ACS Central Science, (2023).

Source: Eindhoven University of Technology Newsroom

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Stanford Researchers Develop a New Way to Identify Bacteria in Fluids

An innovative adaptation of the technology in an old inkjet printer plus AI-assisted imaging leads to a faster, cheaper way to spot bacteria in blood, wastewater, and more.

Shine a laser on a drop of blood, mucus, or wastewater, and the light reflecting back can be used to positively identify bacteria in the sample.

“We can find out not just that bacteria are present, but specifically which bacteria are in the sample – E. coli, Staphylococcus, Streptococcus, Salmonella, anthrax, and more,” said Jennifer Dionne, an associate professor of materials science and engineering and, by courtesy, of radiology at Stanford University. “Every microbe has its own unique optical fingerprint. It’s like the genetic and proteomic code scribbled in light.”

Dionne is senior author of a new study in the journal Nano Letters detailing an innovative method her team has developed that could lead to faster (almost immediate), inexpensive, and more accurate microbial assays of virtually any fluid one might want to test for microbes.

A derivative of the Stanford University logo printed from droplets containing a 1:1 mixture of Staphylococcus epidermidis bacteria and mouse red blood cells (RBCs) onto a gold-coated slide. Droplets were printed using 147 MHz acoustic transducer. (Image credit: Fareeha Safir)

Traditional culturing methods still in use today can take hours if not days to complete. A tuberculosis culture takes 40 days, Dionne said. The new test can be done in minutes and holds the promise of better and faster diagnoses of infection, improved use of antibiotics, safer foods, enhanced environmental monitoring, and faster drug development, says the team.

Old dogs, new tricks

The breakthrough is not that bacteria display these spectral fingerprints, a fact that has been known for decades, but in how the team has been able to reveal those spectra amid the blinding array of light reflecting from each sample.

Details of the printed dots on a gold-coated slide (a) where false coloring in the close-up of a single dot shows red blood calls in red and Staphylococcus epidermidis bacteria in blue. The researchers also printed onto an agar-coated slide (b) to show how the dots fare under incubation. (Image credit: Fareeha Safir)

“Not only does each type of bacterium demonstrate unique patterns of light but virtually every other molecule or cell in a given sample does too,” said first author Fareeha Safir, a PhD student in Dionne’s lab. “Red blood cells, white blood cells, and other components in the sample are sending back their own signals, making it hard if not impossible to distinguish the microbial patterns from the noise of other cells.”

A milliliter of blood – about the size of a raindrop – can contain billions of cells, only a few of which might be microbes. The team had to find a way to separate and amplify the light reflecting from the bacteria alone. To do that, they ventured along several surprising scientific tangents, combining a four-decade-old technology borrowed from computing – the inkjet printer – and two cutting-edge technologies of our time – nanoparticles and artificial intelligence.

“The key to separating bacterial spectra from other signals is to isolate the cells in extremely small samples. We use the principles of inkjet printing to print thousands of tiny dots of blood instead of interrogating a single large sample,” explained co-author Butrus “Pierre” Khuri-Yakub, a professor emeritus of electrical engineering at Stanford who helped develop the original inkjet printer in the 1980s.

“But you can’t just get an off-the-shelf inkjet printer and add blood or wastewater,” Safir emphasized. To circumvent challenges in handling biological samples, the researchers modified the printer to put samples to paper using acoustic pulses. Each dot of printed blood is then just two trillionths of a liter in volume – more than a billion times smaller than a raindrop. At that scale, the droplets are so small they may hold just a few dozen cells.

In addition, the researchers infused the samples with gold nanorods that attach themselves to bacteria, if present, and act like antennas, drawing the laser light toward the bacteria and amplifying the signal some 1500 times its unenhanced strength. Appropriately isolated and amplified, the bacterial spectra stick out like scientific sore thumbs.

The final piece of the puzzle is the use of machine learning to compare the several spectra reflecting from each printed dot of fluid to spot the telltale signatures of any bacteria in the sample.

“It’s an innovative solution with the potential for life-saving impact. We are now excited for commercialization opportunities that can help redefine the standard of bacterial detection and single-cell characterization,” said senior co-author Amr Saleh, a former postdoctoral scholar in Dionne’s lab and now a professor at Cairo University.

Catalyst for collaboration

This sort of cross-disciplinary collaboration is a hallmark of the Stanford tradition in which experts from seemingly disparate fields bring their varying expertise to bear to solve longstanding challenges with societal impact.

This particular approach was hatched during a lunchtime meeting at a café on campus and, in 2017, was among the first recipients of a series of $3 million grants distributed by Stanford’s Catalyst for Collaborative Solutions. Catalyst grants are specifically targeted at inspiring interdisciplinary risk-taking and collaboration among Stanford researchers in high-reward fields such as health care, the environment, autonomy, and security.

While this technique was created and perfected using samples of blood, Dionne is equally confident that it can be applied to other sorts of fluids and target cells beyond bacteria, like testing drinking water for purity or perhaps spotting viruses faster, more accurately, and at lower cost than present methods.

Additional Stanford co-authors include former PhD student Loza Tadesse; research staff Kamyar Firouzi; Niaz Banaei, professor of pathology and of medicine at the School of Medicine; and Stefanie Jeffrey, the John and Marva Warnock Professor, Emerita, in the School of Medicine. Nhat Vu from Pumpkinseed Technologies is also a co-author. Banaei, Dionne, Jeffrey, and Khuri-Yakub are also members of Stanford Bio-X. Dionne is also senior associate vice provost of research platforms/shared facilities, a member of the Cardiovascular Institute and the Wu Tsai Neurosciences Institute, and an affiliate of the Precourt Institute for Energy. Jeffrey is also a member of the Stanford Cancer Institute. Khuri-Yakub is also a member of the Cardiovascular Institute, the Stanford Cancer Institute and the Wu Tsai Neurosciences Institute.

This research was funded by the Stanford Catalyst for Collaborative Solutions, the Chan Zuckerberg Biohub Investigator Program, the NIH-NCATS-CTSA, the Gates Foundation, the National Science Foundation, the NIH New Innovator Award, and from seed funds from the Stanford Center for Innovation in Global Health. Part of this work was performed at the Stanford Nano Shared Facilities (SNSF) and the Soft & Hybrid Materials Facility (SMF), which are supported by the National Science Foundation and National Nanotechnology Coordinated Infrastructure.

Source: Stanford News 

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UConn Researcher Develops Simple, Affordable HIV Testing Device

HIV is one of the world’s most serious public health challenges, and molecular detection plays a significant role in early diagnosis and antiretroviral therapy for HIV patients. The current “gold standard” of HIV testing requires expensive instruments and highly-trained personnel—leaving an unmet need for a rapid, sensitive, and affordable approach for molecular detection of HIV at the point of care.

Published in the American Chemical Society’s (ACS) journal ACS Nano, a research team led by Changchun Liu in the Department of Biomedical Engineering present a low-cost, bioinspired Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-powered biosensor for point of care testing of the HIV virus using a simple personal glucose meter—similar to diabetes home testing.

“Inspired by the multicompartment structures in living cells, we propose a membrane-separated, microfluidic, CRISPR-powered cascade reaction system,” Liu says, “by combining with personal glucose biosensing technology, it is developed into a portable, disposable diagnostic platform for molecular detection of HIV virus and other pathogens.”

Dr. David Banach in the Division of Infectious Diseases in the School of Medicine, Lori Avery in the Department of Pathology of Laboratory Medicine, and Ziyue Li, Naoki Uno, and Xiong Ding in the Department of Biomedical Engineering also contributed to this study.

CRISPR technology is on the cutting edge for highly sensitive and specific nucleic acid-based molecular detection of different pathogens. When used alongside simple isothermal amplification technologies, it becomes a powerful diagnostic tool. However, the combination of isothermal amplification reaction and CRISPR detection systems have limited capabilities, requiring separate reaction tubes and multiple manual operations which increases the risk of contamination and is not ideal for simple and effective point of care applications.

To improve compatibility, the researchers presented a nanoporous membrane-separated cascade reaction system and integrated it into a simple, portable CRISPR-mediated cascade reaction (MCR) biosensor for HIV nucleic acid testing using a low-cost glucose meter to eliminate the need for complex instruments and well-trained personnel.

The researchers were able to detect sensitivities of 43 copies of HIV DNA and 200 copies of HIV RNA per test—exhibiting great potential for rapid detection of HIV virus and other infectious diseases at the point of care.

“Globally, HIV infection has a disproportionate impact on underserved populations with limited access to laboratory testing,” says Banach. “This technology has the potential to bring point of care HIV testing to settings where early diagnosis and monitoring during treatment are critical.”

In September 2020, Liu was awarded a $1.4 million grant from the National Institutes of Health to develop this simple, rapid, affordable HIV testing device.

Reference

Bioinspired CRISPR-Mediated Cascade Reaction Biosensor for Molecular Detection of HIV Using a Glucose Meter. Ziyue Li, Naoki Uno, Xiong Ding, Lori Avery, David Banach, and Changchun Liu. ACS Nano 2023, 17, 4, 3966–3975. https://doi.org/10.1021/acsnano.2c12754

Abstract

HIV molecular detection plays a significant role in early diagnosis and antiretroviral therapy for HIV patients. CRISPR technology has recently emerged as a powerful tool for highly sensitive and specific nucleic acid based molecular detection when used in combination with isothermal amplification. However, it remains a challenge to improve the compatibility of such a multienzyme reaction system for simple and sensitive molecular detection. Inspired by the multicompartment structures in a living cell, we present a nanoporous membrane-separated (compartmentalized), artificial, cascade reaction system to improve the compatibility of a CRISPR-mediated multienzyme reaction. We further integrated the multienzyme cascade reaction system with a microfluidic platform and glucose biosensing technology to develop a bioinspired, CRISPR-mediated cascade reaction (CRISPR-MCR) biosensor, enabling HIV molecular detection by a simple glucose meter, analogous to diabetes home testing. We applied the bioinspired CRISPR-MCR biosensor to detect HIV DNA and HIV RNA, achieving a detection sensitivity of 43 copies and 200 copies per test, respectively. Further, we successfully validated the bioinspired biosensor by testing clinical plasma samples of HIV, demonstrating its great application potential for point-of-care testing of HIV virus and other pathogens at home or in resource-limited settings.

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Korean Scientists Develop an Ultrafast PCR Technology Based on Photothermal Nanomaterials

PCR technology is a molecular diagnostics technology that detects target nucleic acids by amplifying the DNA amount. It has brought marked progress in the life sciences field since its development in 1984.

This technology has recently become familiar to the public due to the COVID-19 pandemic, since PCR can detect nucleic acids that identify the COVID-19 virus. However, due to the technical nature of the PCR test, results cannot be immediately delivered. It takes at least one to two hours for the test as it requires repeated temperature cycles (60~95℃).

Dr. Sang Kyung Kim and Dr. Seungwon Jung's research team at the Center for Augmented Safety System with Intelligence, Sensing of the Korea Institute of Science and Technology (KIST) announced that they had developed an ultrafast PCR technology. By using photothermal nanomaterials, the ultrafast PCR shortens the test time by 10-fold, compared with the time taken for the existing test. The new method is completed in five minutes, with diagnostic performance equal to that of the existing test method.

Photothermal nanomaterials generate heat immediately upon light irradiation. As such, photothermal nanomaterials rapidly increase in temperature, but it is difficult to maintain performance due to their low stability. The KIST research team has developed a polymer composite that physically holds photothermal nanomaterials and can overcome their instability.

By applying it to a PCR system, they have successfully developed a compact PCR system without a heat plate. In addition, they implemented a multiplex diagnostic technology that detects several genes at once, enabling it to distinguish several types of COVID-19 variants in a single reaction.

The work is published in the journal ACS Nano.

Director Sang Kyung Kim states, "through additional research, we plan to miniaturize the developed ultrafast PCR technology this year, to develop a device that can be utilized anywhere. While maintaining the strength of PCR as an accurate diagnostic method, we will increase its convenience, field applicability, and promptness, by which we expect that it will become a precision diagnostic device that can be used at primary local clinics, pharmacies, and even at home. In addition, PCR technology is a universal molecular diagnostic technology that can be applied to various diseases other than infectious diseases, so it will become more applicable."

Reference:

Bong Kyun Kim et al, Ultrafast Real-Time PCR in Photothermal Microparticles, ACS Nano (2022). DOI: 10.1021/acsnano.2c07017.

Abstract:

As the turnaround time of diagnosis becomes important, there is an increasing demand for rapid, point-of-care testing (POCT) based on polymerase chain reaction (PCR), the most reliable diagnostic tool. Although optical components in real-time PCR (qPCR) have quickly become compact and economical, conventional PCR instruments still require bulky thermal systems, making it difficult to meet emerging needs. Photonic PCR, which utilizes photothermal nanomaterials as heating elements, is a promising platform for POCT as it reduces power consumption and process time. Here, we develop a photonic qPCR platform using hydrogel microparticles. Microparticles consisting of hydrogel matrixes containing photothermal nanomaterials and primers are dubbed photothermal primer-immobilized networks (pPINs). Reduced graphene oxide is selected as the most suitable photothermal nanomaterial to generate heat in pPIN due to its superior light-to-heat conversion efficiency. The photothermal reaction volume of 100 nL (predefined by the pPIN dimensions) provides fast heating and cooling rates of 22.0 ± 3.0 and 23.5 ± 2.6 °C s–1, respectively, enabling ultrafast qPCR within 5 min only with optical components. The microparticle-based photonic qPCR facilitates multiplex assays by loading multiple encoded pPIN microparticles in a single reaction. As a proof of concept, four-plex pPIN qPCR for bacterial discrimination are successfully demonstrated.

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MALDI-TOF Mass Spec Deployed by Zimbabwe to Enhance Diagnostic Tests Aimed at Combating Antibiotic Resistance

Over reliance on antibiotics has also led to the growing silent pandemic of antimicrobial resistance (AMR) over the years. AMR happens when bacteria, viruses, fungi, and parasites evolve and stop responding to antimicrobials over time. The Extended Spectrum Beta-Lactamase Escherichia coli (ESBL Ec) Tricycle study's preliminary findings indicated that multidrug-resistant bacteria were widely distributed in Zimbabwe. Additionally, the 2015 AMR situation analysis in Zimbabwe revealed that only 25% of public health laboratories have the manpower, resources, and supplies required to perform culture and antibiotic susceptibility testing (AST) on human samples. Thus, there is a need for further investment in Zimbabwe's upgraded laboratory diagnostic capabilities.

Directed antimicrobial selection for better patient treatment and outcomes depends on the quick identification (ID) of disease-causing bacteria. In October 2022, Zimbabwe made the Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) technology available for the clinical microbiology laboratory's quick and precise identification of bacteria, mycobacteria, and fungal infections.

A large amount of time can be saved by reliably identifying bacteria, moulds, and yeast on solid media using MALDI-TOF MS. This method of phenotypic identification is quick and less expensive than the norm (after the initial purchase of the instrument). Installation of the equipment was the first step, and then 10 scientists received on-the-job training in MALDI-TOF MS technology. The training was carried out at the National Microbiology Reference Laboratory in Harare, Zimbabwe, with technical support provided by the World Health Organization (WHO).

“Reducing the time for microbial identification (ID) and antimicrobial susceptibility testing (AST) could reduce the average time to appropriate antimicrobial therapy, resulting in a decrease in mortality, a shorter hospital stay, and lower hospitalization costs,” noted Dr Raiva Simbi, Acting Director of Laboratory Services, Ministry of Health and Child Care.

To increase laboratory capacity for diagnosis, gather information on drug resistance and promote the use of MALDI-TOF MS to support the prudent use of antibiotics, the UK Fleming Fund was established. Zimbabwe benefited from the £4 million provided by the Fund.

The UK’s Development Director for Zimbabwe, Mrs Geraldine O’Callaghan is proud of the MALDI-TOF MS machine installed with the support of the UK’s Fleming Fund will make such a difference in microbial identification. As COVID-19 demonstrated, strong partnerships, better surveillance and early warning systems are key to identifying and isolating future pandemics before they take off and a One Health approach can prepare us for the next pandemic.

“Globally, the UK plays a leading role in building global resilience against threats to human health for a safer and more prosperous world. We take a ‘One Health’ and ‘all hazard's approach that focuses on strong partnerships, multilateral cooperation and leveraging the UK’s science, technology, diplomatic and development expertise,” she said.  

Optimized diagnostics and data-driven solutions play a vital role in preventing the misuse of antibiotics. They enable healthcare providers to select the most effective treatment for a given condition, thereby reducing the risk of antibiotic resistance.

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