Rapid and specific labeling of single live
            <i>Mycobacterium tuberculosis</i>
            with a dual-targeting fluorogenic probe

Cheng, Yunfeng; Xie, Jinghang; Lee, Kyung Hyun; Gaur, Rajiv L.; Song, Aiguo; Dai, Tingting; Ren, Hongjun; Wu, Juan; Sun, Zhonghe; Banaei, Niaz; Akin, Demir; Rao, Jianghong

doi:10.1126/scitranslmed.aar4470

Cited by 65 publications

(59 citation statements)

References 45 publications

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“…While we used nucleic acid stains for sensitive detection of P. falciparum parasites in thin blood smear, different probes that are specific to a set of pathogens can also be utilized. The past decades has seen much development of pathogen specific probes [75, 76, 77, 78]. Being low-cost and highly configurable, Octopi has the potential to help realize the wide spread use of these new probes in field diagnostics.…”

Section: Discussionmentioning

confidence: 99%

Octopi: Open configurable high-throughput imaging platform for infectious disease diagnosis in the field

Soto-Montoya

Voisin

et al. 2019

Preprint

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Access to quantitative, robust, yet affordable diagnostic tools is necessary to reduce global infectious disease burden. Manual microscopy has served as a bedrock for diagnostics with wide adaptability, although at a cost of tedious labor and human errors. Automated robotic microscopes are poised to enable a new era of smart field microscopy but current platforms remain cost prohibitive and largely inflexible, especially for resource poor and field settings. Here we present Octopi, a low-cost ($250-$500) and reconfigurable autonomous microscopy platform capable of automated slide scanning and correlated bright-field and fluorescence imaging. Being highly modular, it also provides a framework for new disease-specific modules to be developed.We demonstrate the power of the platform by applying it to automated detection of malaria parasites in blood smears. Specifically, we discovered a spectral shift on the order of 10 nm for DAPI-stained Plasmodium falciparum malaria parasites. This shift allowed us to detect the parasites with a low magnification (equivalent to 10x) large field of view (2.56 mm 2 ) module.Combined with automated slide scanning, real time computer vision and machine learningbased classification, Octopi is able to screen more than 1.5 million red blood cells per minute for parasitemia quantification, with estimated diagnostic sensitivity and specificity exceeding 90% at parasitemia of 50/ul and 100% for parasitemia higher than 150/l. With different modules, we further showed imaging of tissue slice and sputum sample on the platform. With roughly two orders of magnitude in cost reduction, Octopi opens up the possibility of a large robotic microscope network for improved disease diagnosis while providing an avenue for collective efforts for development of modular instruments.One sentence summary: We developed a low-cost ($250-$500) automated imaging platform that can quantify malaria parasitemia by scanning 1.5 million red blood cells per minute.Lack of cost-effective diagnostics is a major hurdle in global fight against infectious disease, 2 specially in resource poor settings [1]. This leaves our world in a highly vulnerable position 3 with therapeutic drugs being either overused, leading to drug resistant strains or not acces-4 sible to people who actually need these treatments [2]. Since health care is delivered around 5 the world in a tiered structure, local context such as high cost, lack of trained personal or 6 low throughput of many available diagnostics tests plays a large detrimental role on quality 7 of delivered health-care [3]. 8 Because of the versatility and wide adoption of manual microscopy [4] and its role in 9 direct visual identification of parasites [5], it remains a WHO gold standard for numerous 10 diseases [1]. Despite technological advancements in related fields, the practice of conventional 11 manual microscopy has remained largely unchanged over the last half century and suffers 12 from several drawbacks. With an average lab technician spending 6 to 8 hours imaging and 13 ...

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Section: Discussionmentioning

confidence: 99%

Octopi: Open configurable high-throughput imaging platform for infectious disease diagnosis in the field

Soto-Montoya

Voisin

et al. 2019

Preprint

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“…Another small molecular probe that uses a very elegant dual-targeting strategy has been developed to specifically label mycobacteria (Cheng et al . 2018). This probe discriminates between live and dead M. bovis BCG.…”

Section: The Spacementioning

confidence: 99%

Mycobacterium tuberculosisinfection of host cells in space and time

Bussi

Gutierrez

2019

FEMS Microbiology Reviews

260

156

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Tuberculosis (TB) caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb) remains one of the deadliest infectious diseases with over a billion deaths in the past 200 years (Paulson 2013). TB causes more deaths worldwide than any other single infectious agent, with 10.4 million new cases and close to 1.7 million deaths in 2017. The obstacles that make TB hard to treat and eradicate are intrinsically linked to the intracellular lifestyle of Mtb. Mtb needs to replicate within human cells to disseminate to other individuals and cause disease. However, we still do not completely understand how Mtb manages to survive within eukaryotic cells and why some cells are able to eradicate this lethal pathogen. Here, we summarise the current knowledge of the complex host cell-pathogen interactions in TB and review the cellular mechanisms operating at the interface between Mtb and the human host cell, highlighting the technical and methodological challenges to investigating the cell biology of human host cell-Mtb interactions.

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“…In the last decade, we and others have leveraged the trehalose metabolism of mycobacteria to mark them for detection by various imaging methods (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Exogenous trehalose molecules can be directly mycolylated at the 6 position by antigen 85 (Ag85) enzymes to form trehalose monomycolates (TMM) that are inserted into the mycobacterial cell wall, termed the mycomembrane (10).…”

Section: Introductionmentioning

confidence: 99%

“…Previous studies have used quenched trehalose fluorophores that become unquenched by Ag85 activity, allowing visualization of growing mycobacterial cells in real-time (17). A dual enzyme-targeting fluorogenic probe allowed the detection of Mtb cells within an hour using a microfluidic system (18). In addition, we reported on a solvatochromic trehalose probe (DMN-Tre) that can detect live Mtb cells in TB patient sputum samples (19).…”

Section: Introductionmentioning

confidence: 99%

TowardsMycobacterium tuberculosisdetection at the point-of-care: a brighter solvatochromic probe permits the detection of mycobacteria within minutes

Kamariza

Sgl²,

et al. 2020

Preprint

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25There is an urgent need for point-of-care tuberculosis (TB) diagnostic methods that are fast, 26 inexpensive, and operationally simple. Here, we report on a bright solvatochromic dye trehalose 27 conjugate that specifically detects Mycobacterium tuberculosis (Mtb) in minutes.3-28 hydroxychromone (3HC) dyes, known to yield high fluorescence quantum yields, exhibit shifts in 29 fluorescence intensity in response to changes in environmental polarity. We synthesized two 30 analogs of 3HC-trehalose conjugates (3HC-2-Tre and 3HC-3-Tre) and determined that 3HC-3- 31 Tre has exceptionally favorable properties for Mtb detection. 3HC-3-Tre-labeled mycobacterial 32 cells displayed a 10-fold increase in fluorescence intensity compared to our previously reports on 33 the dye 4,4-N,N-dimethylaminonapthalimide (DMN-Tre). Excitingly, we detected fluorescent Mtb 34 cells within 10 minutes of probe treatment. Thus, 3HC-3-Tre permits rapid visualization of 35 mycobacteria that ultimately could empower improved Mtb detection at the point-of-care in low-36 resource settings. 37 38 With 1.2 million deaths and 10 million new cases in 2018, tuberculosis (TB) is the most 39 lethal infectious disease in the world (1). Early detection of the bacterium Mycobacterium 40 tuberculosis (Mtb), the causative agent of TB, followed by appropriate treatment could prevent 41 most deaths (2). The gold standard for TB diagnosis remains a labor-intensive culture test that 42 requires weeks of incubation time in specialized facilities. Although more rapid tests are available, 43 they present several important limitations. PCR-based tests are expensive and require skilled 44 technicians. Microscopy-based methods are attractive in low-resource settings as they are low-45 cost, have fast turnaround times, and report on people at greatest risk of transmission and death 46 (3). As a result, the sputum smear microscopy test is the most widely used technique for TB 47 diagnosis. The century-old smear test is based on the susceptibility of fluorescent auramine dye 48 or colored Ziehl-Neelson (ZN) stain to accumulate within the highly hydrophobic mycobacterial 49 cell wall (4-7). While effective for identification of Mtb cells, this process requires multiple wash 50 steps to reduce non-specific background fluorescence, or in the case of the ZN test, a rigorous 51counterstaining procedure so that stained Mtb cells can be visualized (8,9). Moreover, the smear 52 test does not distinguish live from dead cells, and this capability is vital in order to assess 53 treatment efficacy early and accurately (2). 54In the last decade, we and others have leveraged the trehalose metabolism of 55 mycobacteria to mark them for detection by various imaging methods (10-20). Exogenous 56 trehalose molecules can be directly mycolylated at the 6 position by antigen 85 (Ag85) enzymes 57 to form trehalose monomycolates (TMM) that are inserted into the mycobacterial cell wall, termed 58 the mycomembrane (10). Researchers have shown that Ag85 enzymes are promiscuous enough 59 to ...

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Rapid and specific labeling of single live Mycobacterium tuberculosis with a dual-targeting fluorogenic probe

Cited by 65 publications

References 45 publications

Octopi: Open configurable high-throughput imaging platform for infectious disease diagnosis in the field

Octopi: Open configurable high-throughput imaging platform for infectious disease diagnosis in the field

Mycobacterium tuberculosisinfection of host cells in space and time

TowardsMycobacterium tuberculosisdetection at the point-of-care: a brighter solvatochromic probe permits the detection of mycobacteria within minutes

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