Biology subjects

Sack, D. A.

Publications and source records attributed to Sack, D. A..

3 recordsLinked to original sources

bioRxiv2025

A colorimetric method for detecting virulent bacteriophage to Vibrio cholerae in fecal and environmental samples

BackgroundV. cholerae are often infected with vibriophages and these phages may be found associated with V. cholerae bacteria or may be detected independently from the bacteria, especially in environmental water samples. When detected, vibriophages serve as a surrogate for detection of V. cholerae and they also have an important role in the ecology of V. cholerae. Vibriophages can be detected using plaque assays or PCR, but these methods are time-consuming and require specialized laboratory resources. MethodologyTo address limitations of other methods, we developed a simple, rapid, and inexpensive colorimetric assay to detect vibriophage that can be scaled up to quickly and easily to screen a large number of samples. The assay uses resazurin and a bacterium, AC6169 that is susceptible to vibriophages ICP1, 2, and 3. Resazurin is a dye that turns color from blue to pink when added to a culture broth with growing bacteria. When a bacteria-free test sample, such as a Millipore filtered wastewater sample containing vibriophages, is added to a culture broth with AC6169, the bacteria will be lysed by the phages and will not grow, and the color of the broth will remain blue. However, if there are no phages in the sample, the bacteria will grow rapidly, and the culture broth will turn pink. We developed the assay using ICP1 spiked samples of environmental water, stool and frozen bile peptone and found it to be sensitive with a limit of detection of 4 to 40 plaque forming units/ml. ConclusionThis colorimetric assay promises to provide a convenient method to detect vibriophages on a larger scale than was possible earlier to better understand their role as a surrogate for detecting V. cholerae and to better understand their role in the pathogenesis, ecology, and epidemiology of cholera. Author SummaryWe developed a simple and inexpensive colorimetric assay to detect virulent vibriophage in samples of wastewater and stool. The assay uses resazurin, a blue dye and a unique bacterium, AC6169, that is known to be susceptible to vibriophages. Bacteria-free samples (e.g. Millipore filtrates) suspected of having vibriophage are incubated for 2 or 3 hours in a broth with AC6169, following which resazurin is added. After a further 30-minute incubation, the color of the broth is observed. If the sample contains vibriophage, the broth will remain blue, but if there are no vibriophage, the broth will turn pink. The change in color from blue to pink is due to the metabolic reaction from the growing bacteria. When vibriophage are present, they lyse the bacterium AC6169, thus preventing the color change, but if vibriophage are not present, the bacteria multiply rapidly, and the metabolic reaction causes the broth to change color to pink. Because the assay procedure is inexpensive and simple, laboratories in cholera endemic areas should find it convenient for their epidemiologic and clinical surveillance activities.

microbiology↗

bioRxiv2020

Gold-standard diagnostics are tarnished by lytic bacteriophage

BackgroundA fundamental clinical and scientific concern is how lytic bacteriophage, as well as antibiotics, impact diagnostic positivity. MethodsCholera was chosen as a model disease to investigate this important question. Patients with diarrheal disease were enrolled at two remote hospitals in Bangladesh. Diagnostic performance was assessed as a function of lytic bacteriophage detection, as well as exposure to the first-line antibiotic azithromycin detected by mass spectrometry. ResultsAmong diarrheal samples positive by nanoliter quantitative PCR for Vibrio cholerae (n=78/849), the odds that a rapid diagnostic test (RDT) or qPCR was positive was reduced by 89% (OR 0.108; 95%CI 0.002-0.872) and 87% (OR 0.130; 95%CI 0.022-0.649) when lytic bacteriophage were detected, respectively. The odds that a rapid diagnostic test (RDT) or qPCR was positive was reduced by more than 99% (OR 0.00; 95% CI: 0.00-0.28) and 89% (OR 0.11; 95% CI: 0.03-0.44) when azithromycin was detected, respectively. ConclusionsEstimations of cholera burden may improve by accommodating for the negative effect of antimicrobial exposure on diagnostic positivity. Furthermore, the findings herein challenge our current approach to interpreting and developing bacterial diagnostics given variable rates of lytic bacteriophage and antibiotic exposure.

microbiology↗

bioRxiv2019

Cholera in Internally Displaced Persons Camps in Borno State--Nigeria, 2017: A qualitative study of the multi-sectorial emergency response to stop the spread of the outbreak

Introduction/BackgroundIn August 2017, a cholera outbreak started in Muna Garage IDPs camp, Borno State-Nigeria, and >5000 cases occurred in six local government areas. This qualitative study evaluated perspectives about the emergency response to this outbreak.\n\nMethods/FindingsWe conducted 39 key informant interviews and focused group discussions, and reviewed 21 documents with participants involved with surveillance, water-sanitation-hygiene, case management, oral cholera vaccine, communications, logistics, and coordination. Qualitative data analysis used thematic techniques comprising key-words-in-context, word-repetition, and key-sector-terms.\n\nAuthorities were alerted quickly, but outbreak declaration took 12 days due to a 10 day delay waiting for culture confirmation. Outbreak investigation revealed several potential transmission channels, but a leaking latrine around the index cases house was not repaired for >7 days.\n\nUse of chlorine disinfectant was initially not accepted by the community due to rumors that it would sterilize women. This could have been avoided with improved community consultation. Initially, key messages were communicated in Hausa, although Kanuri was the primary language; later this was corrected. Planning would have benefited using exercise drills to identify weaknesses, and inventory sharing to avoid stock outs.\n\nThe response by the Rural Water Supply and Sanitation Agency was perceived to be slow and an increased risk from Eid El Jabir festival with increased movement and food sharing was not recognized. Case management was provided at treatment centers, but some partners were concerned that their work was recognized asking, \"who gets the glory and the data?\"\n\nOCV was provided to nearly one million people and it distribution benefited from a robust polio vaccine structure; however, logistical problems related to payment of staff needed resolution.\n\nInitial coordination was thought to be slow, but improved by activating an Emergency Operations Centre. The Borno Ministry of Health used an Incident Management System to coordinate multi-sectoral response activities.\n\nThese were informed by daily reviews of epi curves and geo-coordinate maps. The synergy between partners and government improved when each recognized the governments leadership role.\n\nConclusions/SignificanceDespite a timely alert of the outbreak, the delayed declaration led to a slowed initial response, but this improved during the course of the outbreak. OCV distribution was efficient and benefited from the OPV infrastructure. Improvements in laboratory capacity are urgently needed.\n\nAuthor SummaryIn August 2017, a cholera outbreak started in the Muna Garage Internally Displaced Persons (IPDs) camp in Borno State, Nigeria. By October, it appeared in six local government areas with a total of 5,340 cases reported including 61 deaths. We evaluated the perspectives of the emergency response by the government of Nigeria and implementing partners to stop the outbreak. We conducted 39 interviews and group discussions and also studied 21 documents related to the outbreak response. We found that epidemiologic surveillance timely alerted the health authorities about the outbreak, but the outbreak was declared 12 days later, awaiting for culture confirmation. This led to delays in the initial response. We also observed that conditions in the IDPs camps like overflowing latrines, overcrowding, and open defecation were highly favorable to cholera transmission. Improved IDP camp conditions are needed to prevent cholera and other water born infections and strengthened laboratory capacity is needed to enable a more rapid response.

epidemiology↗