Microbiology Case Study: A 54 Year Old Male with Right Upper Quadrant Pain

Case History

A 54 year old male with a past medical history of Type II diabetes mellitus and obesity was admitted for a few days history of severe pain over right upper quadrant accompanied by fevers, chills, nausea, vomiting and diarrhea. Physical exam revealed a palpable gallbladder. Ultrasound imaging showed a distended gallbladder with a thickened, edematous and hyperemic wall that was interpreted as acalculus cholecystitis. The patient underwent percutaneous drainage of the gallbladder with plans to undergo a cholecystectomy once the acute phase of his illness stopped. The gallbladder fluid was sent to microbiology for analysis.

Laboratory Findings

Anaerobic plates obtained from organisms growing in thioglycollate broth grew low, convex opaque white colonies. The organisms did not produce the classic double zone of beta hemolysis (Image 1).  Gram stain of the culture showed gram positive bacilli that were “boxcar” shaped (Image 2). Aerobic plates had no growth. The organisms were catalase negative and non-motile. MALDI-TOF identified the organism as Clostridium perfringens.

Image 1. Growth on CNA plate in anaerobic environment
Image 2. Gram stain from the anaerobic culture shows boxcar-shaped gram positive bacilli.

The patient was also placed on piperacillin-tazobactam while in the hospital.  His condition improved and he was discharged home with a seven day course of cefpodoxime and metronidazole with a general surgery follow up appointment.

Discussion

Clostridium perfringens is a gram positive bacilli with blunt ends (boxcar shaped). These obligate anaerobes are spore formers, however these are rarely seen. When seen, they produce subterminal spores. These organisms cause of crepitant myonecrosis (gas gangrene), gangrenous cholecystitis, septicemia, and food poisoning.  They are present in large numbers as normal microbiota in the gastro-intestinal tract of humans and animals, the female genital tract and oral mucosa. Typically, infections are caused by endogenous strains gaining access to normal sterile sites due to a predisposing factor that compromise normal anatomy: surgery, trauma, or altered host defense mechanisms (diabetes, burns, immunosuppression, and aspiration).

Penicillin is recommended in most infections, however resistance has been reported. Optimal management of intra-abdominal infection is to achieve appropriate source control and drainage is important.   

References

  1. Tille P. Bailey & Scott’s Diagnostic Microbiology. Fourteenth Edition. Elsevier; 2017.
  2. Murray P. Medical Microbiology. Seventh Edition. Elsevier; 2013.

-Angela Theiss, MD is a 3rd 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.

The System

Outside the city of New Bern, in Craven County, North Carolina, there is a particular system for residents to dispose of their garbage. Locals must go to the nearest participating gas station and purchase stickers which cost about $2.00 each. These stickers must be placed on each bag of garbage generated in the household, otherwise they will not be picked up during the weekly trash collection. In order to save money, a group of widows has formed a club in which members scout out the open dumpsters in town (usually behind stores or gas stations). Then they call and let group members know where they can covertly dump their trash for free that week.

This story may seem funny, but for the most part, it is true. I have no doubt this also occurs in other parts of the country where the system for trash collection is similar. Why do people behave this way? Are they purposely trying to circumvent the trash collection system in place or is the system just not easy for locals to utilize? If you’re having difficulty getting people to change safety behaviors (like PPE compliance) in your laboratory, you might need to determine that for the systems you have in place and ask similar questions.

In one laboratory the manager struggles with staff who work part of the day in a clean office and another part in the lab itself. When the employees go into the lab for brief periods, they often fail to don their PPE. Upon further investigation, you would learn that staff are not allowed to keep their lab coats on their chairs and that all PPE is kept in one lab store room located on the opposite side away from the offices. The system is set up to reinforce PPE non-compliance.

In another lab the manager placed a permanently-mounted counter face shield in the chemistry department so that staff would be forced to use it when popping specimen caps. Staff loaded instrument racks behind the shield, but when they carried the racks over to the analyzers, their faces were not protected from splashing. Exposures continued to occur. Here the system is at play again. A face shield was put in place to change behaviors, but it was only a partial solution. In order to protect staff fully here, they would need goggles or a face shield that can be worn. Offer light-weight reusable or disposable face protection that staff can use easily. Be sure to give them a say in whatever option is chosen.

Sometimes the system issues are not apparent until there is a safety event, and unfortunately, that can result in bigger problems. If your training program does not include regular fire safety training, a small fire situation may get out of hand quickly. Does your staff have experience handling a fire extinguisher? Would they easily be able to put out a fire? Do they know their evacuation routes and meeting places, and could they get there with ease? What about the lab emergency management plan? Have staff participated in a table-top drill so they have a basic understanding of how to respond during a chaotic disaster? These are examples of some safety systems that need to be in place to keep staff ready and safe at all times.

When people take shortcuts or find ways to circumvent the system, there is usually a pretty good reason, Often, it is the design of the system. In New Bern, elderly women can’t lift large heavy trash bags, so they use smaller bags. They don’t want to pay the same price for a garbage bag sticker that others are paying for big bags. There’s a problem with the system- and those ladies found a way around it. What problems do you see in your lab safety system? If you don’t know what they are, ask around. Staff will talk. It’s better to find out what the workarounds are now and to fix them before an injury or exposure occurs.

Dan Scungio, MT(ASCP), SLS, CQA (ASQ) has over 25 years experience as a certified medical technologist. Today he is the Laboratory Safety Officer for Sentara Healthcare, a system of seven hospitals and over 20 laboratories and draw sites in the Tidewater area of Virginia. He is also known as Dan the Lab Safety Man, a lab safety consultant, educator, and trainer.

A Pathologists’ Assistant Abroad

Jennison Hartong, MLS(ASCP)CM, PA(ASCP)CM, is a Pathologists’ Assistant who recently went to Ethiopia to teach grossing techniques. The editors of Lablogatory asked her a few questions about her experiences.

Lablogatory: How’d you get involved with ASCP’s Center for Global Health?

Jennison: Dr. Milner, Chief Medical Officer of ASCP, initially reached out to one of the pathologists at M.D. Anderson to inquire if any Pathologists’ Assistants (PAs) would be interested in attending a workshop in Nigeria. I reached out and expressed my interest in teaching grossing techniques rather than public speaking (not one of my strengths). Dr. Milner then told me about this opportunity in Ethiopia where pathologists were requesting advanced, gross training in lymph node dissections on breast and colon specimens. I immediately jumped at the opportunity to help in this way.

L: What were your motivations for going?

J: Whether with basic health needs or more complex areas like cancer treatments, I’ve always wanted to use my education and experience to help others and impact lives in areas around the world where certain aspects of healthcare may not be accessible. Before becoming a PA, I was a medical technologist and was always interested in working with Doctors Without Borders, however, I did not have the years of experience to apply. I decided to go to PA school and was disappointed to learn that Doctors Without Borders does not utilize PAs. I figured that dream would have to be accomplished another way, which was why I was so eager to work with the ASCP and their global health initiatives.

Another motivation for going on this trip was experiencing the work and organizational skills required for making a trip like this successful. I am currently finishing my second master’s degree in public health with a focus in health policy and management. I was very interested in learning everything I could about planning programs to help developing countries as well as being able to network with like-minded health professionals.

L: What did you hope to accomplish while you were there?

J: My main goal of this trip was to help advance Ethiopian residents and pathologists in certain grossing techniques. More specifically, I aimed to assist with lymph node dissections and, as it turned out, how to locate and sample the radial margin in colon cancer cases.  I also wanted to experience a different culture than my own, step out of my comfort zone and challenge myself as a PA by teaching others. At the end of this experience, I can say that this trip was definitely a life changing experience and one I am extremely grateful for.

Image 1. Jennison (black scrubs) training residents from St. Paul Hospital to locate radial margins on colorectal cancer cases.

L: What did you learn about lab medicine in Ethiopia?

J: During my week in Addis Ababa, I quickly realized that it was up to me to make this trip as successful as possible. Never before in my professional career were all the decisions up to me, and at first, it was slightly uncomfortable. I was worried I would come across as too bossy or even condescending. However, after meeting Eshetu Lemma, the ASCP local representative, along with the other participants and experiencing their kindness and eagerness to learn, I was newly determined to make this trip an absolutely positive experience for everyone. I made some changes to the training sessions and after the first day, the rest of the week ran smoothly. I learned a lot about how lab medicine is practiced in Ethiopia. I learned that, in the case of a power outage, you carefully set your blade down and wait it out. I learned that resources like aprons and sleeves are not thrown away unless completely used up. I learned that due to cassette shortages, tissue submission is done quite thoughtfully- more so than in the United States. I learned that the overwhelming majority of cancer cases are presented at stage 4 due to issues surrounding resources, fear, myths, and lack of cancer education. But most importantly, I learned that the labs in Addis Ababa, Ethiopia, are doing an amazing job with the resources they are given and are eager for opportunities to positively impact patient care.

L: Is what you learned there applicable to your work in the States?

J: I’ll take what I learned there and incorporate it into my work here in the States. I’ve gained confidence in my ability as a health professional and reignited my passion to help others.

To put it simply, this trip has been life changing. It has allowed me to experience and accomplish a lifelong dream for which I am forever grateful. I’m hopeful that my future holds more opportunities to serve other communities and help strengthen cancer programs in developing countries.

Image 2. View from St. Paul Hospital.

-Jennison Hartong, MLS(ASCP)CM, PA(ASCP)CM is a board certified Pathologists’ Assistant, specializing in surgical and gross pathology working mainly in oncology cases. Before attending graduate school, she worked as a Medical Laboratory Scientist (MLS) at Lurie Children’s Hospital of Chicago, Illinois. Upon graduating, Jennison started working at Memorial Sloan Kettering Cancer Center. In 2018, she relocated to Houston and currently works at M. D. Anderson Cancer Center in Houston, Texas. In May of 2019, Jennison will graduate with a second Master’s in public health with a focus in health policy and management from New York Medical College. She plans to use her extensive lab experience and newfound knowledge of public health to help bring basic healthcare to communities that would otherwise not have access to these necessities.

Genetic Test Results Change Faces

As a part of my Molecular Genetic Pathology fellowship, we experience a clinical component to training in addition to all of the laboratory training we receive. This last month, I rotated through Cancer Genetics, where genetic counselors discuss genetic testing with patients with a personal or family history of cancer. The counselors describe the process of genetic testing and help chose genetic tests to look at the patient’s risk for an inherited cancer syndrome.

Patients are looking forward to the certainty that will come from a genetic test, because it is the wave of the future and they think you can learn so many things from your genetics. The truth, however, can be much less clear. Up to 30% of people receive a Variant of Uncertain Significance (VUS) as their genetic test result. This rate increases as larger panels are test more genes.

Figure 1. A set of genes and associated cancer types tested by a hereditary cancer genetic test. (Taken from Myriad MyRisk Gene Table.)

A VUS represents a variation in a person’s gene that doesn’t have enough information to say that it is benign or pathogenic. This gray zone is very uncomfortable and confusing for patients and providers alike. There are several cases where someone acted on a VUS as if it were a pathogenic variant and ended up having radical interventions like a bilateral mastectomy.

We know that as scientific and medical knowledge increases, our ability to reclassify these variants improves. For laboratories, this means periodic reanalysis of previously reported variants. If this process is not properly set up, it can be very laborious and extensive. Furthermore, not only was a timeline for variant reanalysis unknown, but also the likelihood of variants becoming upgraded or downgraded had not been described.

Two recent studies helped provide some answers to these questions. The first, published in JAMA, comes from the cancer genetic group I was working with, led by Dr. Theo Ross M.D. Ph.D., worked in conjunction with Myraid (Lab that first started testing the BRCA genes, and now tests many more) to determine how often variants were reclassified. Looking at 1.1 million individuals tested at Myriad, the average time to reclassification for a VUS was 1.2-1.9 years (Mersch J et al Jama 2018). Additionally, 90% of VUS were downgraded to benign/ likely benign representing 97% of patients with a VUS. This figure from the paper shows how the time to issuing a reclassification (amended report) has decreased (Figure 2).

Figure 2. The time to sending an amended report is shown by the year the report was first issued. From Mersch et al. JAMA 2018.

I worked on the second study, which looked at variant reclassification in childhood epilepsy genetic testing (SoRelle et al JAMA Peds 2019). The results, published in JAMA Pediatrics, also found most patients had a VUS reclassified to benign/likely benign. However, several clinically significant changes (reclassified to or from pathogenic/ likely pathogenic) occurred as well (Figure 3).

Figure 3. Patients with reclassification of gene variants from each category. Arrows that cross the red line represent an instance where a change in diagnosis would result from variant reclassification. Seven patients had both a pathogenic or likely pathogenic variant and VUS reclassified and are only represented once.

Furthermore, there was a linear relationship between the time the test was reported and the rate of variant reclassification (Figure 4). We found that 25% of patients with a VUS would experience a reclassification within 2 years.

Figure 4. Reclassification rate is plotted as the fraction of reclassified variants for each year testing was performed (VUS= black line, pathogenic or likely pathogenic= red line). Solid lines represent patients with a reclassified result and dotted lines are extrapolated slopes.

Overall, the conclusions of the two studies are somewhat similar:

  1. Most patients with a VUS experience a downgrade reclassification to likely benign or benign.
  2. Variant reclassification should be performed at least every 2 years
  3. Rates of reclassification may differ by disease type. Investigation by a similar study design should be performed in other genetic diseases.

References

  1. Mersch J, Brown NPirzadeh-Miller SMundt ECox HCBrown KAston MEsterling LManley SRoss T. Prevalence of variant reclassification following hereditary cancer genetic testing. JAMA. 2018;320:1266–1274.
  2. SoRelle JA, Thodeson DM, Arnold S, Gotway G, Park JY. Clinical Utility of Reinterpreting Previously Reported Genomic Epilepsy Test Results for Pediatric Patients. JAMA Pediatr. 2018 Nov 5:e182302.

-Jeff SoRelle, MD is a Molecular Genetic Pathology fellow 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 advancing quality in molecular diagnostics.

Blue Chips – Troubleshooting Ion Torrent Data

Ah, the blue chip – not much fun to see after spending a day preparing the libraries and running clonal amp overnight.  There are a couple possible explanations for a blue chip, and you can figure them out by looking at the metrics of the run. 

Test Fragments

The test fragments serve as a control for the sequencing run.  They are spiked into the mixture of library ISPs before they are loaded on the chip.  These will allow you to figure out where the problem occurred if you encounter a blue chip.  If the Test Fragments are detected and are of sufficient quality, then this means the sequencing run worked and the problem most likely occurred before sequencing, during library prep or clonal amplification.  If the Test Fragments are not detected, then it could mean one of two things – one – the clonal amplification did not work for either the library or the Test Fragment ISPs, or – two – the sequencing run was somehow at fault.  Let’s take a look at both examples.

Troubleshooting a Blue Chip

In the event you see a blue chip, first, check to see what kinds of ISPs showed up after the analysis.  For the chip pictured above, there were ISPs that had product on them, as you can see in the Live category (6,475,553 ISPs or 95.3% of the ISPs, shown in the screenshot below).  This means clonal amp was successful for a small number of the library ISPs.  Next, there were also Test Fragments detected, at 433,392 ISPs or 6.7% of the total ISPs.  Scroll down to the bottom of the page, and you will see how the Test Fragments sequenced.  We like to see the Percent 50AQ17 and Percent 100AQ17 at least in the 80’s, but even still, you can see that these were detected and were sequenced.  Because of this, the sequencing run looks to be fine, so most likely the problem occurred before sequencing.  In this case, we believe the library prep did not yield the expected 100pM concentration, so the library pool was over-diluted prior to clonal amplification.  The library prep was repeated, and clonal amplification was run on the new pool of libraries, and the sequencing was successful.

In this next example, we have the other possibility.  This chip was blue as well (this is a 520 chip, instead of a 530, to explain the different sized pictures). 

First, there are only 2.7% Live ISPs, so even lower than the chip above.  But the even stranger thing was that there were 0.0% Test Fragments, and at the end of the analysis, there were absolutely no ISPs left to be analyzed, library or Test Fragment.  This was the only time we had ever seen a chip like this; generally, if we had blue chips, they were like the previous example.  We looked at our library pool quant and it was in the expected range, so we did not believe it was a library prep issue.  The sequencing initialization was successful and did not have any errors, so we did not believe it was a sequencing problem.  We repeated clonal amplification with the same library pool and had successful sequencing.  In speaking with our Field Application Scientist, it was decided it must have been a failure of one of the reagents of the clonal amplification – either a Taq was not present or something, so the clonal amplification never occurred, or something similar. 

Hopefully you will not experience too many of these blue chips, but if you do, I hope you are a little more prepared to troubleshoot!  Happy sequencing!

rapp_small

-Sharleen Rapp, BS, MB (ASCP)CM is a Molecular Diagnostics Coordinator in the Molecular Diagnostics Laboratory at Nebraska Medicine. 

Gastric Cancer: A Multidisciplinary Approach

Maryam Zenali1*, Dmitriy Akselrod2, Eric Ganguly3, Eswar Tipirneni4 and Christopher J. Anker5*

1 Department of Pathology, 2 Department of Radiology, 3 Division of Gastroenterology, and 5 Division of Radiation Oncology, The University of Vermont Medical Center (UVMMC), Burlington, VT and 4 Department of Hematology Oncology, Central Vermont Medical Center (CVMC), The University Of Vermont Health Network, Adult Primary Care, Berlin, VT

*corresponding authors

A 57 year old woman with a personal and family history of breast cancer presented with early satiety and dysphagia for 5 months. Her abdominal computed tomography (CT) scan (Image 1 A) showed marked thickening of an apparently featureless gastric wall (A, blue arrows indicating the mucosal [rightward pointing] and serosal [leftward pointing] aspects of the gastric wall). Prominent gastrohepatic lymph nodes were noted as well. Her fluoroscopic upper GI study (Image1 B), following administration of barium and effervescent crystals (a double contrast effect to allow for mucosal evaluation), showed thickened rugal folds (B red arrow) and pooling of barium within an antral ulcer (B blue arrow). A subsequent CT scan (Image 1 C) after administration of intravenous and enteric contrast, confirmed marked diffuse gastric wall thickening (C blue arrows again indicating the mucosal [rightward pointing] and serosal [leftward pointing] aspects of the gastric wall) (Image 1, composite radiographs A-C).

The gastric body distended poorly with insufflation and demonstrated thickened, erythematous, edematous folds with erosions (Image 2, endoscopy image). On endoscopic ultrasound, the total thickness of the stomach was 12 mm with expanded wall layers in the proximal stomach to the antrum and a thickness of 3.5 mm in spared areas. Biopsies were obtained; the corresponding H&E and keratin stains are provided (Image 3, composite photomicrographs A-B).

Image 1. Composite radiographs.
Image 2. Endoscopy image.
Image 3. Composite photomicrographs.

Based on the original radiographic imaging that led to the biopsy, what are the differential diagnoses?

Microbiology Case Study: A 19 Year Old Woman Passes a Worm in Her Stool

Case History

A 19 year old woman with no significant past medical history presented to an outside clinic with psychological distress after passing a worm per rectum. She had no other complaints or symptoms.

Image 1. Eggs are spherical, 31 to 43 μm in diameter, with a thick yellow-brown (bile-stained) shell having radial striations and contain a hexacanth oncosphere (Image courtesy of: Fred Patterson, Parasitologist).
Image 2. Adult proglottids are longer (up to 20 mm) than they are wide (up to 7 mm) and have a genital pore (*) at the lateral margin.
Image 3. Formalin-fixed paraffin-embedded H&E stained section of a proglottid’s lateral uterine branches on each side of a central uterine stem (20x).

Discussion

Adult cestodes (tapeworms) have long, ribbon-like bodies made up of proglottids (egg-producing segments) that develop at the posterior of a scolex (specialized structure for attachment to the small intestine of a host). Taenia has 32 species, 2 of which are medically important for causing taeniasis: Taenia saginata and Taenia solium (beef and pork tapeworm infection, respectively). These parasites are distributed worldwide, with T. saginata being more common than T. solium.

The lifecycle of Taenia involves adult, egg, and larval stages. Adults release gravid proglottids and eggs that are passed in feces. The eggs reach pasture land via soil or water and are ingested by an intermediate host. For T. saginata, the intermediate host is a herbivore (cow), as eggs of T. saginata do not infect humans, and for T. solium, the intermediate host is a pig, human, or other animal. Ingested eggs hatch and release the hexacanth oncosphere (6-hooked embryo) that can penetrate tissues. Over 2 to 3 months, infective cysticerci (0.5 to 2.0 mm in diameter larvae) develop in muscles. Of note, only T. solium can cause cysticercosis (extra-intestinal larval forms within human tissues, ie. the human becomes the intermediate host) and this can be life-threatening if cysticerci invade the brain. When humans consume raw or undercooked beef/pork meat that is infected, cysticerci will attach to the small intestinal mucosa and, over 3 to 5 months, mature into the adult form. The adult T. saginata can reach 4 to 12 meters in length and the adult T. solium can reach 1.5 to 8 meters in length. Adult tapeworms can live within intestines for over 25 years while gravid proglottids and eggs are passed in stool.

Infections are usually asymptomatic or cause mild indigestion, anorexia, and abdominal discomfort. The eggs can be identified by ova and parasite examination (Figure 1) or a cellulose tape preparation of perianal skin. The oncosphere must be visualized to avoid misidentifying a pollen grain. However, eggs of Taenia species and Echinococcus species are indistinguishable. Diagnosis is also made by recovering gravid proglottids from the anal opening or passed in feces (Figures 2 and 3). Distinguishing the two species can be done by examining gravid proglottids for the number of lateral uterine branches present on one side of a central uterine stem. T. saginata have 15 to 30 lateral uterine branches while T. solium have 7 to 13 branches. Both species have a small anterior scolex (measuring 1 to 2 mm in diameter for T. saginata and 1 mm in diameter for T. solium) with 4 suckers. Definitive identification is possible since T. solium’s scolex has a rostellum (crown) with 2 rows of hooks whereas T. saginata’s scolex bears no rostellum or hooks. Treatment is a single dose of praziquantel and successful treatment is defined as passing zero proglottids over 4 consecutive months.

-Adina Bodolan, MD is a 3rd 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.