Forget DNA Sequencing, Protein Sequencing is Here!

Every so often I have the distinct pleasure of hearing of a new technology that I’d never imagined before. This last week, a study in Nature Biotechnology described using nanopore to perform protein sequencing. Thinking of the difficulty and complexity of sequencing just 4 nucleotides, it’s hard to conceive of the exponential difficulty to sequence 20 amino acids.

The technology is still in a nascent period working on the ability to differentiate 20 amino acids- some with very similar sizes and charges. However, as has been the case with many technologies, the speed of advancement can increase rapidly. The possibilities of this technology are very exciting.

Figure 1. Aerolysin structure from CyroEM. This pore comes from Aeromonas hydrophila, a Gram-negative bacterium associated with diarrheal diseases and deep wound infections.

Nanopores have been used mostly for sequencing DNA. Pacific Biosciences have created a platform that reads long sequences of DNA by synthesizing DNA from a single strand moving through a pore. Another company, Oxford Nanopore has made several devices that use nanopore technology to perform sequencing in portable devices. The key to most of these technologies involve functionalizing the pores to have properties desirable for specific purposes.

For protein sequencing, amino acids need to move through the pores slowly enough to have their charges accurately measured. To accomplish this goal, the French start-up, DreamPore, used a bacterial derived cytolytic pore called aerolysin. Aerolysin was found to slow down the passage of amino acids as it has a single molecule trap, which binds to single amino acids with carrier cations. This method detects 13 of the20 amino acids. Additional chemical modifications, instrumentation advances, and nanopore imaging allowed two additional amino acids to be detected. The last few were too similar in charge to be differentiated. However, this study provides a path forward where it is conceivable to sequence each of the 20 amino acids.

In the future, detecting post-translational modifications such as glycosylation and phosphorylation will be an important advance. It will be fun to follow the progress of this technology to see how it might be applied clinically.

References

  1. Iacovache I, De Carlo S, Diaz Nuria et al. Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process. Nature Communications 2016. 7:12062.
  2. Ouldali H, Sarthak K, Ensslen T et al. Electrical recognition of the twenty proteinogenic amino acids using an aerolysin nanopore. Nature Biotechnology 2020; 38: 176-181

-Jeff SoRelle, MD is a Chief Resident of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX. His clinical research interests include understanding how the lab intersects with transgender healthcare and improving genetic variant interpretation.

Microbiology Case study: A 42 Year Old Male with Bacteremia

Case History

A 42 year old male with past medical history of diabetes mellitus and essential hypertension presented to the emergency department with high fever and chills which developed two days prior. His examination revealed a painful ulcer on the planter aspect of his right toe with surrounding erythema. According to patient, the exact duration of the ulcer is unclear as it was on planter aspect of his foot and he does not inspect his feet regularly. However, the ulcer grew in size and symptoms over the past week. He denies any cough, diarrhea or abdominal pain. He is on oral anti-diabetics with well-controlled blood sugar. Complete blood count revealed leukocytosis. Blood was collected and sent to microbiology laboratory for gram stain and culture.

Laboratory identification

Cultures signaled positive after 32 hours of incubation and gram negative rods were identified on Gram stain (Image 1). The organism grew after 24 hours of incubation on 5% sheep blood, chocolate, and MacConkey agars (Images 2 & 3). MALDI-TOF mass spectrometry identified the isolate as Salmonella spp. The isolate was sent to the public health department for additional testing by molecular typing where it was identified as Salmonella enterica subsp. enterica serovar Brandenburg (Salmonella Brandenburg). Later, MRI revealed osteomyelitis of right third toe which was considered as the likely source of patient’s bacteremia.

Image 1. Gram stain of positive blood culture broth showing gram negative rods
Image 2. Non-lactose fermenting colonies growing on MacConkey agar
Image 3. Colonies producing hydrogen sulfide making them appear black on Hektoen enteric agar

Discussion

Salmonella is a genus of the family Enterobacteriaceae in the order Enterobacterales. They are non-spore forming gram negative facultative anaerobes. Salmonella spp. are lactose non-fermenters and usually produce H2S on triple sugar iron and Hektoen enteric agar.

The genus has only two species: Salmonella enterica, divided into 6 subspecies and containing over 2500 serovars, and Salmonella bongori. Subspecies and serotype determination is necessary for epidemiological investigations. Serotyping is used to classify Salmonella based on bacterial surface antigens; the thermostable polysaccharide cell wall or somatic (“O”) antigens and the thermo-labile flagella proteins or “H” antigens. It is also possible to identify Salmonella serotypes on the basis of phage typing, plasmid profiling, ribotyping and pulsed field gel electrophoresis (PFGE) of DNA fragments generated from restriction enzyme digestion.

Salmonella are zoonotic bacteria that can cause abortion, metritis, and systemic illness in ewes and does. Natural reservoirs of Salmonella are domestic and wild animals, including poultry, swine, cattle, birds, dogs, rodents, tortoises, turtles and cats. Humans also serve as a natural host. The most common source of transmission of Salmonella is the consumption of contaminated poultry and meat products. Person-to-person, fecal–oral transmission does occur and has been a problem in health care facilities traced to inadequate hand washing.

Salmonella brandenburg ranked 16th among the serovars responsible for human infections. It causes acute diarrhea and severe illness in a variety of animals and was first isolated in New Zealand in 1986. Since 1996 Salmonella Brandenburg has been associated with an emerging epidemic of abortions and deaths in sheep in the southern regions of the South Island. Subsequently, the same strain was reported to cause disease in horses, goats, deer, pigs and humans. The disease is known to have high morbidity and mortality within a flock or herd, rapid local spread and an occupational, health and safety risk to farm workers and their families.

There are three clinically distinguishable forms of salmonellosis in humans. These include gastroenteritis, enteric fever and septicemia. Established Salmonella bacteremia requires aggressive antimicrobial treatment with ciprofloxacin, ceftriaxone, or less frequently trimethoprim-sulfamethoxazole. A careful search for focal metastatic disease should be undertaken, especially when relapse follows cessation of treatment. Surgical drainage of metastatic abscesses may be required, with surgical intervention. Resistance to any of the drugs used to treat invasive infection may occur, so treatment should be supported by susceptibility testing.

In the case of our patient, he was treated with ceftriaxone and underwent toe amputation. The patient had an uncomplicated hospital course and made a complete recovery.

References[EMT1] 

  1. Alvseike O., Skjerve E. (2000). Probability of detection of Salmonella using different analytical procedures, with emphasis on subspecies diarizonae serovar 61:k:1,5,(7) [S. IIIb 61:k:1,5,(7)]. International Journal of Food Microbiology, 58, 49-58.
  2. Clark G, Swanney S, Nicol C, and and Fenwick S. Salmonella Brandenburg – the 1999 Season. Proceedings of the Sheep and Beef Cattle Society of the New Zealand Veterinary Association, 151-156, 2000
  3. Bailey K.M. (1997). Sheep abortion outbreak associated with Salmonella Brandenburg. Surveillance, 24(4), 10-11
  4. Baumler A.J., Tsolis R.M., Heffron F. (2000). Virulence Mechanisms of Salmonella and their Genetic Basis. In “Salmonella in Domestic Animals” (Ed. C. Wray and A. Wray). CAB International 2000, pp. 57-72

-Ansa Mehreen, MD. 1st year AP/CP resident at University of Chicago hospital program based at Evanston Hospital. Her academic interests include gastrointestinal pathology.

-Erin McElvania, PhD, D(ABMM), is the Director of Clinical Microbiology NorthShore University Health System in Evanston, Illinois. Follow Dr. McElvania on twitter @E-McElvania. 

EUA for 2019-nCoV Test

On February 4th, the FDA announced an Emergency Use Authorization for the CDC’s 2019 Novel Coronavirus real-time RT-PCR Diagnostic Panel. Here’s the press release:

Audience: Clinical Laboratory Professionals

Subject: Laboratory Update: Information about Emergency Use Authorization for  2019 Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel

Level: Laboratory Update

This message is to ensure that clinical laboratories are aware that CDC has developed a new laboratory test kit called the CDC’s 2019 Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel, for use in testing patient respiratory specimens for 2019-nCoV. On February 4, 2020, the Food and Drug Administration (FDA) issued an Emergency Use Authorization (EUA) to enable emergency use of the test kit in the United States. All EUA documents are available on the FDA website.

The test kit will be available for ordering today from the International Reagent Resource (IRR). Formerly, U.S. diagnostic testing for 2019-nCoV was only being conducted at CDC; however, the FDA EUA and distribution of the tests will allow 2019-nCoV testing to take place at laboratories designated by CDC.  This includes U.S. state and local public health laboratories and Department of Defense (DoD) laboratories.

Clinical laboratories should NOT attempt viral isolation from specimens collected from 2019-nCoV persons under investigation (PUIs). For interim guidelines for collecting, handling, and testing clinical specimens from PUIs for 2019-nCoV, please see the CDC 2019 Novel Coronavirus website

The FDA website lists current EUA assays, and also includes a link to terminated EUA assays. Each pathogen-specific EUA includes the device-specific Letter of Authorization, fact sheets, and manufacturer instructions/package inserts. These documents are updated when amendments are made (e.g., additional specimen types, extraction methods, procedural clarifications), so check the website routinely to ensure your laboratory staff members have the most up-to-date information.

Additional Resources

If you have any questions, please contact LOCS@cdc.gov.

Microbiology Case Study: A 55 Year Old with Fever, Chills, and a Cough

Clinical History

A 55 year old patient with past medical history of stage IV non-Hodgkin’s lymphoma on rituximab and Campylobacter jejuni bacteremia 1 year prior presented to the Emergency Department on the orders of their primary care provider, after outpatient blood cultures grew gram negative bacilli resembling Campylobacter species. Their symptoms included a 1-2 month history of fatigue and weakness and a 3 week history of intermittent fevers and chills with developing productive cough, sinus pressure, sore throat, progressive dyspnea on exertion, nausea, and decreased appetite.

Laboratory Findings

Initial (outpatient) blood culture was positive in the aerobic bottle at 60.1 hours, with the initial gram stain showing no organisms. The bottle was placed back in the analyzer and flagged positive again, at which point a second gram stain was performed, which again showed no organisms. An acridine orange stain was performed (Image 1), revealing multiple spiral/”gull shaped” rods. A third gram stain (Image 2) with more time allowed for safranin staining revealed faint gram negative rods. MALDI-TOF MS was attempted with no identification. The culture growth was sent to a reference laboratory and was identified via sequencing as Helicobacter species. The organism was not viable for susceptibility testing.

Image 1. Acridine orange stain of blood culture sample: this stain causes the nucleic acids to fluoresce orange, highlighting the bacteria against the background of blood.
Image 2. Gram stain after allowing extra time for safranin staining, showing few gram negative rods.
Image 3. A replating on blood agar showed difficult to discern, thin spreading colonies.

Two sets of subsequent blood cultures also grew gram negative bacilli at 65 and 67 hours. The blood culture broth from one of these cultures was also sent to the reference lab, but again did not have viable growth for susceptibility testing.

Discussion

The genus Helicobacter includes 35 species, consisting of gram negative spiral bacilli, previously considered to be part of the Campylobacter genus. Pathogenic species are classically associated with the gastrointestinal tract as they are able to survive in the harsh acidic conditions of the human stomach. The most common clinically relevant species is H. pylori, which is associated with gastric ulcers as well as other inflammatory processes in the stomach and duodenum. In prior reports, bacteremia caused by Helicobacter species is typically associated with some other underlying disease process, such as malignancy, immunocompromised state, or disruption of the GI mucosal barrier (1, 2, 3, 4, 5, 6).

Helicobacter spp. are similar in morphology to Campylobacter spp. on a gram stain; given the patient’s prior history of C. jejuni bacteremia, it was not unreasonable for the gram smear to initially be called consistent with Campylobacter spp. However, the clinical course and antibiotic susceptibility profiles of Helicobacter and Campylobacter bacteremia cases can differ in important ways. Further, susceptibilities can differ between different species of Helicobacter. There are no established guidelines for the treatment of Helicobacter spp. bacteremia and breakpoints for antibiotic susceptibility testing for some Helicobacter species have not been established. (7)

The patient in this case was discharged on a course of azithromycin with clinical improvement: at that time, the sequencing result revealing Helicobacter had not yet been received, and the clinical team was acting on the belief that the organism in the patient’s blood was a recurrence of the previous Campylobacter infection. On a follow up outpatient appointment with Infectious Disease, wherein sequencing results were available, tetracycline was prescribed due to concern about the possibility of resistance or relapsing infection.

References

  1. Abidi, Maheen Z., et al. “Helicobacter Canis Bacteremia in a Patient with Fever of Unknown Origin.” Journal of Clinical Microbiology, vol. 51, no. 3, 2013, pp. 1046–1048.
  2. Araoka, Hideki, et al. “Clinical Characteristics of Bacteremia Caused by Helicobacter Cinaedi and Time Required for Blood Cultures To Become Positive.” Journal of Clinical Microbiology, vol. 52, no. 7, 2014, pp. 2745–2745.
  3. De Luca, et al. “Helicobacter Pylori Bacteremia: An Unusual Finding.” Infectious Disease Reports, vol. 8, no. 3, 2016, pp. 74–75.
  4. Han, Xiang Y., et al. “Helicobacter Pylori Bacteremia with Sepsis Syndrome.” Journal of Clinical Microbiology, vol. 48, no. 12, 2010, pp. 4661–4663.
  5. Imataki, Osamu, et al. “Enteral Malakoplakia Prior to Helicobacter Cinaedi Bacteremia.” American Journal of Gastroenterology, vol. 112, no. 1, 2017, pp. 187–188.
  6. Saito, Sho, et al. “Helicobacter Fennelliae Bacteremia: Three Case Reports and Literature Review.” Medicine, vol. 95, no. 18, 2016, p. e3556.
  7. Yamamoto, Kei, et al. “Comparison of the Clinical and Microbiological Characteristics of Campylobacter and Helicobacter Bacteremia: the Importance of Time to Blood Culture Positivity Using the BACTEC Blood Culture Systems.” BMC Research Notes, vol. 10, no. 1, 2017, pp. 1–6.

-Tom Koster, DO is a 1st year Anatomic and Clinical Pathology Resident at the University of Vermont Medical Center.

-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Associate Professor at the University of Vermont.

Patient Interaction

Medical school councilors have good intentions in mind when they steer medical students who realize that direct patient care isn’t their strong suit into pathology. But I am different kind of pathologist – the one who sees (or talks to) patients every day. I am a member of unique subspecialty – Transfusion Medicine – which is the most patient-centric subspecialty of all pathology subspecialties. And, contrary to the popular wisdom, I like seeing patients.

Don’t get me wrong though, my heart and soul still live in the lab, deeply rooted in understanding test performance, troubleshooting and quality control. But direct patient care helps to put all the work I have done in the lab into a perspective.

One program that became especially dear to my heart is our chronic RBC exchange program for the kids and adults with sickle cell anemia who have high risk of developing serious complications from the disease, such as stroke, acute chest syndrome, and severe iron overload. As an apheresis physician I see these patients quite frequently due to the nature of the program – chronic RBC exchanges every 4 to 6 weeks. This also means that I quickly had to learn quite a lot not only about managing the exchanges, but also about patients’ success and failures, spend time explaining to parents the benefits of the program and engaging them to maintain compliance with rigorous schedule. The work is not immediately rewarding. All the adjustments I do to the plan of care show changes in lab values in a month or two at best. But it is not entirely about numbers. Another aspect that makes this program special is when you notice that the kids you treat are doing better at school, have less ED visits and overall live a more fulfilling life.

Sometimes the patient interaction is not as direct as in the case of the sickle cell RBC exchange program. For example, being part of the obstetric team that cares for the patient with severe hemolytic disease of fetus and newborn is also extremely rewarding. And the more challenging clinical question is the more rewarding it is in the end. Just this summer we had a patient who developed an antibody to very high frequency antigen that is present in 99.7% of the population and finding the right donor for intrauterine transfusion involved quite a few people in at least 3 cities.  When all the pages, phone calls, emails, and personal conversations between me and residents, obstetricians, anesthesiologists, pediatricians, and blood suppliers result in a positive outcome for mom and baby – I feel elated. And who wouldn’t?! That is why I enjoy what I do!

-Aleh Bobr MD is currently the medical director of blood bank and tissue services at University of Nebraska Medical Center in Omaha, NE. He did his residency in Anatomic and Clinical pathology and Fellowship in Transfusion Medicine at Mayo Clinic Rochester, MN. Prior to that he did his post-doctoral research fellowship in Immunology with focus on dendritic cell biology at University of Minnesota and Yale University. He received his medical degree from Vitebsk State Medical University in Vitebsk, Belarus. Current interests include application of apheresis, platelet refractoriness.