PiRamid: A compact Raspberry Pi imaging box to automate small-scale time-lapse digital analysis, suitable for laboratory and field use.

Digital imaging permits the quantitation of many experiments, such as microbiological growth assays, but laboratory digital imaging systems can be expensive and too specialised. The Raspberry Pi camera platform makes automated, controlled imaging affordable with accessible customisation. When combined with open source software and open-source 3D printed hardware, the control over image quality and capture of this platform permits the rapid development of novel instrumentation. Here we present "PiRamid", a compact, portable, and inexpensive enclosure for autonomous imaging both in the laboratory and in the field. The modular three-piece 3D printed design makes it easy to incorporate different camera systems or lighting configurations (e.g., single wavelength LED for fluorescence). The enclosed design allows complete control of illumination unlike a conventional digital camera or smartphone, on a tripod or handheld, under ambient lighting. The stackable design permits rapid sample addition or camera focus adjustment, with a corresponding change in magnification and resolution. The entire unit is small enough to fit within a microbiological incubator, and cheap enough (∼£100) to scale out for larger parallel experiments. Simply, Python scripts fully automate illumination and image capture for small-scale experiments with an ∼110×85 mm area at 70-90 µm resolution. We demonstrate the versatility of PiRamid by capturing time-resolved, quantitative image data for a wide range of assays. Bacterial growth kinetics was captured for conventional microbiology (agar Petri dishes), 3D printed custom microbiology labware and microfluidic microbiology. To illustrate application beyond microbiology, we demonstrate time-lapse imaging of crystal growth and degradation of salad leaves. Minor modifications permit epi-illumination by addition of a LED ring to the camera module. We conclude that PiRamid permits inexpensive digital capture and quantitation of a wide range of experiments by time-lapse imaging to simplify both laboratory and field imaging.

Rapid Bacterial Motility Monitoring Using Inexpensive 3D-Printed OpenFlexure Microscopy Allows Microfluidic Antibiotic Susceptibility Testing.

Antibiotic susceptibility testing is vital to tackle the emergence and spread of antimicrobial resistance. Inexpensive digital CMOS cameras can be converted into portable digital microscopes using 3D printed x-y-z stages. Microscopic examination of bacterial motility can rapidly detect the response of microbes to antibiotics to determine susceptibility. Here, we present a new simple microdevice-miniature microscope cell measurement system for multiplexed antibiotic susceptibility testing. The microdevice is made using melt-extruded plastic film strips containing ten parallel 0.2 mm diameter microcapillaries. Two different antibiotics, ceftazidime and gentamicin, were prepared in Mueller-Hinton agar (0.4%) to produce an antibiotic-loaded microdevice for simple sample addition. This combination was selected to closely match current standard methods for both antibiotic susceptibility testing and motility testing. Use of low agar concentration permits observation of motile bacteria responding to antibiotic exposure as they enter capillaries. This device fits onto the OpenFlexure 3D-printed digital microscope using a Raspberry Pi computer and v2 camera, avoiding need for expensive laboratory microscopes. This inexpensive and portable digital microscope platform had sufficient magnification to detect motile bacteria, yet wide enough field of view to monitor bacteria behavior as they entered antibiotic-loaded microcapillaries. The image quality was sufficient to detect how bacterial motility was inhibited by different concentrations of antibiotic. We conclude that a 3D-printed Raspberry Pi-based microscope combined with disposable microfluidic test strips permit rapid, easy-to-use bacterial motility detection, with potential for aiding detection of antibiotic resistance.

3D-Printed Dip Slides Miniaturize Bacterial Identification and Antibiotic Susceptibility Tests Allowing Direct Mastitis Sample Analysis.

The early detection of antimicrobial resistance remains an essential step in the selection and optimization of antibiotic treatments. Phenotypic antibiotic susceptibility testing including the measurement of minimum inhibitory concentration (MIC) remains critical for surveillance and diagnostic testing. Limitations to current testing methods include bulky labware and laborious methods. Furthermore, the requirement of a single strain of bacteria to be isolated from samples prior to antibiotic susceptibility testing delays results. The mixture of bacteria present in a sample may also have an altered resistance profile to the individual strains, and so measuring the susceptibility of the mixtures of organisms found in some samples may be desirable. To enable simultaneous MIC and bacterial species detection in a simple and rapid miniaturized format, a 3D-printed frame was designed for a multi-sample millifluidic dip-slide device that combines panels of identification culture media with a range of antibiotics (Ampicillin, Amoxicillin, Amikacin, Ceftazidime, Cefotaxime, Ofloxacin, Oxytetracycline, Streptomycin, Gentamycin and Imipenem) diluted in Muëller-Hinton Agar. Our proof-of-concept evaluation confirmed that the direct detection of more than one bacterium parallel to measuring MIC in samples is possible, which is validated using reference strains E. coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC 10145, and Staphylococcus aureus ATCC 12600 and with mastitis milk samples collected from Reading University Farm. When mixtures were tested, a MIC value was obtained that reflected the most resistant organism present (i.e., highest MIC), suggesting it may be possible to estimate a minimum effective antibiotic concentration for mixtures directly from samples containing multiple pathogens. We conclude that this simple miniaturized approach to the rapid simultaneous identification and antibiotic susceptibility testing may be suitable for directly testing agricultural samples, which is achieved through shrinking conventional tests into a simple "dip-and-incubate" device that can be 3D printed anywhere.

MicroMI: A portable microbiological mobile incubator that uses inexpensive lithium power banks for field microbiology.

Incubation at controlled temperature is a key step in culture based microbiological tests. Access to culture-based microbiological testing requires access to conventional incubators in a laboratory. Portable incubators allow microbiological testing in the field and in resource-limited settings, and can eliminate the challenge of sample transportation, minimising the chance of sample degradation. Recent studies have reported low-cost portable incubator designs suitable for field or off-grid use, but these either need an external power supply (e.g. mains AC or 12 V DC), or rely on passive heating without thermostatic control. Here we report that small inexpensive uninterruptable power supply (UPS) products manufactured for consumer electronics and powered by lithium-ion battery packs allowing thermostatic temperature control in small portable incubators that can maintain precise temperatures with or without external power. We present an open-source design for a Microbiological Mobile Incubator (MicroMI) in two sizes for field use. The MicroMI is built from simple and widely available components and is easy to set up. The open source design can be customised for different numbers of samples. The smallest and most efficient design uses a vacuum insulated food flask that allows longer operation with smaller, lower capacity UPS. The larger flight case design has space for more samples, but depletes the battery faster. The UPS maintains a typical microbiology incubation temperature for up to 24 h without external power- ideal for typical incubation needed for culture methods. The battery capacity, incubator design, and external ambient temperature all affected duration of operation without requiring external power. We validated the MicroMI by conducting classical microbiological tests using agar petri dishes, slant cultures and dip slides, and biochemical tests. We conclude the MicroMI design allows inexpensive lithium battery products to be used to simplify field microbiology and increase access to vital analytical microbiology testing.

Sero-evaluation of Immune Responses to Vibrio cholerae in a Postelimination Setting.

Cholera remains a significant public health problem worldwide. In settings of declining incidence, serosurveillance may be used to augment clinical surveillance. We utilized dried blood spot sampling and cholera-specific antibody testing to examine the serologic profiles of vaccinated and unvaccinated children in southern Vietnam, where cholera was recently eliminated.

Exploiting open source 3D printer architecture for laboratory robotics to automate high-throughput time-lapse imaging for analytical microbiology.

Growth in open-source hardware designs combined with the low-cost of high performance optoelectronic and robotics components has supported a resurgence of in-house custom lab equipment development. We describe a low cost (below $700), open-source, fully customizable high-throughput imaging system for analytical microbiology applications. The system comprises a Raspberry Pi camera mounted on an aluminium extrusion frame with 3D-printed joints controlled by an Arduino microcontroller running open-source Repetier Host Firmware. The camera position is controlled by simple G-code scripts supplied from a Raspberry Pi singleboard computer and allow customized time-lapse imaging of microdevices over a large imaging area. Open-source OctoPrint software allows remote access and control. This simple yet effective design allows high-throughput microbiology testing in multiple formats including formats for bacterial motility, colony growth, microtitre plates and microfluidic devices termed 'lab-on-a-comb' to screen the effects of different culture media components and antibiotics on bacterial growth. The open-source robot design allows customization of the size of the imaging area; the current design has an imaging area of ~420 × 300mm, which allows 29 'lab-on-a-comb' devices to be imaged which is equivalent 3480 individual 1µl samples. The system can also be modified for fluorescence detection using LED and emission filters embedded on the PiCam for more sensitive detection of bacterial growth using fluorescent dyes.

Isolation of New Delhi metallo-ß-lactamase 1-producing Vibrio cholerae non-O1, non-O139 strain carrying ctxA, st and hly genes in southern Vietnam.

Vibrio cholerae non-O1, non-O139 (VC_NAG) organisms are universally present in the aquatic environment and regarded as non-pathogenic bacteria. However, considering that they do occasionally induce gastroenteritis, a study of their virulence and antibiotic resistance genes is important. The presence of enteropathogenic genes, including ctxA, VC_NAG-specific heat-stable toxin gene (st), hemolysin (hly), and zona occludens toxin (zot) was determined by PCR in 100 VC_NAG strains isolated in southern Vietnam in 2010-2013 from 94 environmental and six human origins. These 100 VC_NAG strains were also tested phenotypically and genotypically for the presence of the New Delhi metallo-ß-lactamase (NDM-1). Of the 100 VC_NAG strains tested, six were positive for ctxA; five from the environment and one of human origin. The st gene was detected in 17 isolates, 15 and two of which were of environmental and human origins, respectively. Gene hly was detected in 19 VC_NAG strains examined, two of which were isolated from humans and 17 from environments. The zot gene was not detected in any of the strains tested. Three VC_NAG strains of environmental origin were confirmed to produce NDM-1 and the blaNDM-1 gene was detected in those strains by PCR. Of note, one of the three NDM-1-producing VC_NAG strains was confirmed to carry ctxA, st and hly genes concurrently. This is the first report of isolation of NDM-1-producing VC_NAG strains in Vietnam.

Rapid diagnosis of diarrhea caused by Shigella sonnei using dipsticks; comparison of rectal swabs, direct stool and stool culture.

BACKGROUND: We evaluated a dipstick test for rapid detection of Shigella sonnei on bacterial colonies, directly on stools and from rectal swabs because in actual field situations, most pathologic specimens for diagnosis correspond to stool samples or rectal swabs. METHODOLOGY/PRINCIPAL FINDINGS: The test is based on the detection of S. sonnei lipopolysaccharide (LPS) O-side chains using phase I-specific monoclonal antibodies coupled to gold particles, and displayed on a one-step immunochromatographic dipstick. A concentration as low as 5 ng/ml of LPS was detected in distilled water and in reconstituted stools in 6 minutes. This is the optimal time for lecture to avoid errors of interpretation. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 4 x 10(6) CFU/ml of S. sonnei. The specificity was 100% when tested with a battery of Shigella and different unrelated strains. When tested on 342 rectal swabs in Chile, specificity (281/295) was 95.3% (95% CI: 92.9% - 97.7%) and sensitivity (47/47) was 100%. Stool cultures and the immunochromatographic test showed concordant results in 95.5 % of cases (328/342) in comparative studies. Positive and negative predictive values were 77% (95% CI: 65% - 86.5%) and 100% respectively. When tested on 219 stools in Chile, Vietnam, India and France, specificity (190/198) was 96% (95% CI 92%-98%) and sensitivity (21/21) was 100%. Stool cultures and the immunochromatographic test showed concordant results in 96.3 % of cases (211/219) in comparative studies. Positive and negative predictive values were 72.4% (95% CI 56.1%-88.6%) and 100 %, respectively. CONCLUSION: This one-step dipstick test performed well for diagnosis of S. sonnei both on stools and on rectal swabs. These data confirm a preliminary study done in Chile.

Dipstick test for rapid diagnosis of Shigella dysenteriae 1 in bacterial cultures and its potential use on stool samples.

BACKGROUND: We describe a test for rapid detection of S. dysenteriae 1 in bacterial cultures and in stools, at the bedside of patients. METHODOLOGY/PRINCIPAL FINDINGS: The test is based on the detection of S. dysenteriae 1 lipopolysaccharide (LPS) using serotype 1-specific monoclonal antibodies coupled to gold particles and displayed on a one-step immunochromatographic dipstick. A concentration as low as 15 ng/ml of LPS was detected in distilled water and in reconstituted stools in 10 minutes. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 1.6×106 CFU/ml and 4.9×106 CFU/ml of S. dysenteriae 1, respectively. Optimal conditions to read the test have been determined to limit the risk of ambiguous results due to appearance of a faint yellow test band in some negative samples. The specificity was 100% when tested with a battery of Shigella and unrelated strains in culture. When tested on 328 clinical samples in India, Vietnam, Senegal and France by laboratory technicians and in Democratic Republic of Congo by a field technician, the specificity (312/316) was 98.7% (95% CI:96.6-99.6%) and the sensitivity (11/12) was 91.7% (95% CI:59.8-99.6%). Stool cultures and the immunochromatographic test showed concordant results in 98.4 % of cases (323/328) in comparative studies. Positive and negative predictive values were 73.3% (95% CI:44.8-91.1%) and 99.7% (95% CI:98-100%). CONCLUSION: The initial findings presented here for a simple dipstick-based test to diagnose S. dysenteriae 1 demonstrates its promising potential to become a powerful tool for case management and epidemiological surveys.

Dipstick for rapid diagnosis of Shigella flexneri 2a in stool.

BACKGROUND: Shigellosis or bacillary dysentery, an acute bloody diarrhoea, is a major public health burden in developing countries. In the absence of prompt and appropriate treatment, the infection is often fatal, particularly in young malnourished children. Here, we describe a new diagnostic test for rapid detection, in stool, at the bedside of patients, of Shigella flexneri 2a, the most predominant agent of the endemic form of the disease. METHODOLOGY/PRINCIPAL FINDINGS: The test is based on the detection of S.flexneri 2a lipopolysaccharide (LPS) using serotype 2a-specific monoclonal antibodies coupled to gold particles and displayed on one-step immunochromatographic dipstick. A concentration as low as 20 ng/ml of LPS is detected in distilled water and in reconstituted stools in under 15 minutes. The threshold of detection corresponds to a concentration of 5x10(7) CFU/ml of S. flexneri 2a, which provides an unequivocal positive reaction in three minutes in distilled water and reconstituted stools. The specificity is 100% when tested with a battery of Shigella and unrelated strains, in culture. When tested in Vietnam, on clinical samples, the specificity and sensitivity were 99.2 and 91.5%, respectively. A decrease of the sensitivity during the evaluation on stool samples was observed after five weeks at room temperature and was due to moistening of the dipsticks caused by the humidity of the air during the fifth week of the evaluation. This drawback is now overcome by improving the packaging and providing dipsticks individually wrapped in waterproof bags. CONCLUSION: This simple dipstick-bases test represents a powerful tool for case management and epidemiological surveys.