Hematology Case Study: Temporal Arteritis or COVID-19?

What is your least favorite test in hematology? The first things that come to my mind are those tests that are time consuming, tedious, and manual. I’ve worked in a hematology lab that did Kleihaur betke (KB) tests, and whenever I worked, I seemed to get one, or sometimes more, in a given shift. And when I worked in blood bank, we did KBs in blood bank, and I certainly did my share there, too. KBs seem to follow me around! Those, I must admit, are probably my least favorite, but I know that many techs dread parasite smears or % parasitemia, reviewing 150 or more fields or counting thousands of cells on a smear. Manual body fluid counts, manual reticulocyte counts, and manual platelet counts are likely some others on our lists of “not favorites.” Basically, anything that requires a lot of time, manual counting, and math!

One other test that probably doesn’t make many “favorites” list is the Erythrocyte Sedimentation Rate (ESR), or sed rate. Remember old fashioned Westergren Sed Rates that took an hour to do, while the ER doctor kept calling looking for their “STAT” results? There are still labs that set up manual sed rates that take an hour, and modified manual methods that take “only” 15 or 30 minutes. Some semi-automated methods can give us results in a couple minutes, but still require techs to fill a capillary tube and load the instrument. Fortunately, real help may have arrived, in the form of fully automated ESR instruments! There are instruments now that actually make ESRs almost fun. I’ve never seen techs so excited about a new instrument as they were when we got iSeds. This thing is amazing! It’s like a little Ferris wheel for sed rates. You pop the whole tube in, they go for a little ride around the Ferris wheel, then drop out, in less than 30 seconds. And you can keep loading tubes even while it’s running. A truly Stat ESR. Now that’s amazing!

Image 1. Alcor iSED Automated ESR Instrument

While these new instruments make ESR’s easier to run, with more reproducible results, and less hands-on time, they still don’t get much love, because, well…there are newer tests available for inflammation, and we know that the ESR is not a specific test for diagnosis. Across the years, some lab tests have become antiquated and obsolete…bleeding times come to mind, along with CK-MB. In 2010 an article was published that supported discontinuing laboratory tests that no longer have clinical utility in the lab. The ESR was on this list. Yet, many labs still perform ESRs. Should the ESR be phased out, or are there still valid reasons for ordering them?

Even though the test is considered non-specific, the ESR test is considered helpful in diagnosing two specific inflammatory diseasestemporal arteritis (TA) and polymyalgia rheumatica. A high ESR is one of the main test results used to confirm these diagnoses. It is also used to monitor disease activity and response to therapy in both these conditions. Almost all cases present with an elevated ESR, though a normal ESR should not be used to rule out these conditions.

Case 1: A 70 year old White female was admitted to the ER complaining of throbbing headache and blurry vision. She stated that the headache started 2 days ago, had been at her temples at first but in the past few hours was getting worse. She stated that she was prompted to come to the ER because now her whole scalp hurt, and her vision was blurry. A CBC, Basic panel, CRP and ESR were ordered. The CBC results were unremarkable, other than and increased platelet count of 480,000/µL. ESR was 110 mm/hr. Basic panel results were normal. CRP was 2.51 md/dL.

The patient was started on prednisone immediately, and a temporal artery biopsy was scheduled, with a suspicion of temporal arteritis (TA), also known as giant cell arteritis (GCA). TA is an autoimmune disease that causes inflammation of the temporal arteries. Under the microscope, the inflamed cells of these arteries look giant, which is how the disease got its name. The inflammation causes constriction of the arteries, can affect chewing and eating, and may cause blindness if not treated promptly. Treatment of choice are corticosteroids, often prescribed for at least a year. Symptoms are monitored frequently and lab results, including the ESR, can be used to monitor the condition and response to treatment.

If you are still wondering if the ESR should be discontinued as a useful test, we are now seeing patients with COVID infection and elevated ESRs. Over the past 2 years, several articles have been written about elevated ESRs in COVID-19 patients. One study aimed to evaluate the usefulness of ESR in distinguishing severe from non-severe COVID-19 cases. The study suggests that severe COVID-19 cases are associated with higher elevations of ESR, as compared to non-severe cases. A case report of a patient recovering from COVID described an increased ESR. The high ESR persisted for a long time even after the patient recovered from COVID-19, while no other inflammatory processes or other conditions known to raise ESRs were found.

Case 2: My second case is a case of a 58 year old woman who presented with an earache and a pulsing temporal headache. Ear infection was ruled out and the patient was referred to ophthalmology for possible TA. The patient’s CRP was elevated but her ESR and platelet counts were within normal reference range. The patient was COVID tested as part of a pre-op workup before temporal artery biopsy and the COVID-19 test came back positive. There have been cases in literature in the last year of this new set of symptoms in COVID-19 patients. The conclusion from these cases is that if a patient appears with symptoms consistent with TA with an elevated CRP but with a normal ESR and platelet counts, that the patients should be tested for COVID.

The ESR is one of the oldest laboratory tests still in use. The study of the sedimentation of blood was one of the principles on which ancient Greek medicine was based. In the 1700’s, physicians noticed that the rate of red blood cell sedimentation changed during illness. This theory was first introduced as a laboratory test over 100 years ago. Depending on the historic accounts and articles you read, it was first described by a Polish physician, Edmund Biernacki, in 1897, or by a Swedish physician, Robert Fahraeus, in 1915. Biernacki proved the connection between the rate of sedimentation and the amount of fibrinogen in the blood and suggested using the ESR in diagnostics. Alf Vilhelm Albertsson Westergren (a familiar name!) also presented a similar description of the ESR. In the early 1920’s. Dr Westergren went further to develop the blood drawing technique and defined standards for the ESR. To this day, the Westergren Erythrocyte Sedimentation Rate method is recognized as the gold standard reference method for ESR measurement.

Image 2. Manual Westergren ESRs

The sed rate measures the rate at which erythrocytes sediment by gravity, in mm/hour. RBCs usually repel each other due to zeta potential and aggregation is inhibited. In conditions with increased fibrinogen or immunoglobulins, these proteins coat the RBCs, promoting aggregation. The RBCs form rouleaux which settle faster than individual RBCs. In conditions such as anemia, the ESR will be high because with a lower hematocrit, the velocity of the upward flow of plasma is altered and red blood cell aggregates fall faster. In polycythemia the increased blood viscosity can cause a lower ESR. In sickle cell anemia, and other conditions such as spherocytosis, the RBCs are abnormally shaped and will not form rouleaux easily, thus decreasing the ESR.

The ESR is an easy, inexpensive, non-specific test that has been used for many years to help diagnose conditions associated with acute and chronic inflammation. An elevated ESR is not associated with a specific diagnosis; therefore, it must be used in conjunction with other tests. Conversely, a normal ESR cannot be used to exclude the presence of significant disease. The ESR should also not be used as a screening test in asymptomatic patients. Since fibrinogen is an acute-phase reactant, the ESR is increased in many inflammatory and neoplastic conditions that increase fibrinogen, including diabetes, infection, pelvic inflammatory disease, lupus. rheumatoid arthritis, acute coronary syndrome, and neoplasms. However, noninflammatory factors such as older age, female gender, and pregnancy can also cause elevation of the ESR. 

Historically, the ESR was used to indicate inflammatory conditions and monitor disease progression or response to treatment. More specific tests have been developed for many of these conditions, but the ESR still has its advantages. Interestingly enough, for a test that 12 years ago was on the ‘antiquated’ list, in the past 2 years there have been over 50 scientific journal articles written about the ESR. The ESR can eliminate unnecessary testing and help decrease medical costs. It has its advantages in small labs and in rural areas because it can provide quick results without expensive instrumentation. For labs that do not perform more sophisticated tests such as CRP and procalcitonin, the ESR can provide answers without waiting for results from reference laboratories. Even though an ESR may take 1 hour, it is much faster than send out testing. It can therefore expedite a diagnosis, or normal results can give the physician and patient timely reassurance.

What is your favorite or least favorite test in hematology? Let me know and I can highlight it in a future blog!

References

  1. Au, Benjamin Wai Yin MBBS, MMed (Ophth); Ku, Dominic J. BMed, MSurg; Sheth, Shivanand J. MBBS, MS (Ophthal) Thinking Beyond Giant Cell Arteritis in COVID-19 Times, Journal of Neuro-Ophthalmology: March 2022 – Volume 42 – Issue 1 – p e137-e139
  2. Brigden ML. Clinical utility of the erythrocyte sedimentation rate. Am Fam Physician. 1999 Oct 1;60(5):1443-50. PMID: 10524488.
  3. Hale AJ, Ricotta DN, Freed JA. Evaluating the Erythrocyte Sedimentation Rate. JAMA. 2019;321(14):1404–1405. doi:10.1001/jama.2019.1178
  4. Pu, Sheng-Lan et al. “Unexplained elevation of erythrocyte sedimentation rate in a patient recovering from COVID-19: A case report.” World journal of clinical cases vol. 9,6 (2021): 1394-1401. doi:10.12998/wjcc.v9.i6.1394
  5. Tishkowski K, Gupta V. Erythrocyte Sedimentation Rate. [Updated 2021 May 9]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK557485/
  6. Alan H. B. Wu, PhDKent Lewandrowski, MD, et al. Antiquated Tests Within the Clinical Pathology Laboratory. The American Journal of Managed Care. September 2010, Volume 16, Issue 9
  7. https://emedicine.medscape.com/article/332483-workup
Socha-small

-Becky Socha, MS, MLS(ASCP)CMBBCM graduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 40 years and has taught as an adjunct faculty member at Merrimack College, UMass Lowell and Stevenson University for over 20 years.  She has worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. She currently works at Mercy Medical Center in Baltimore, Md. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.

Microbiology Case Study: A 26 Year Old Female with Diarrhea

Case Description

A 26 year old female with a past medical history of Hemoglobin SC disease (Hb SC) and iron deficiency anemia presented to the emergency department with lower abdominal pain and diarrhea for three days. She began having multiple episodes of watery diarrhea, followed by bloody diarrhea after eating at a restaurant. During this time, she also had fever, chills, body aches, and headache. The patient had been on a course of ceftriaxone and metronidazole started three weeks prior for sore throat, ear infection, and bacterial vaginosis. She completed her metronidazole course prior to the current illness. Abdominal computed tomography revealed splenomegaly and a mildly dilated, fluid-filled appendix without evidence of infectious or inflammatory abnormalities. Hemoglobin on admission was 11.1 mg/dL (Reference Range: 11.2- 15.7 mg/dL) and MCV 62.9 fL (Reference Range: 79.4- 94.8 fL), which is similar to her baseline.

Laboratory Identification

The patient underwent work up for community-acquired diarrhea. Stool cultures grew non-typhoidal Salmonella (Image 1). Blood cultures performed at the time of admission flagged positive with gram negative rods which were also identified as Salmonella species by MALDI-TOF. The organism was susceptible to ampicillin, ceftriaxone, ciprofloxacin, and trimethoprim/sulfamethoxazole. The patient continued on intravenous ceftriaxone and responded to therapy. She was discharged home on oral ciprofloxacin.

Image 1. Salmonella Microbiologic Diagnosis using Xylose Lysine Deoxycholate agar and Triple Sugar Iron slant. A) Non-typhoidal strains of Salmonella are lactose non-fermenting, hydrogen sulfide producing (black colonies) enteric Gram-negative rods on Xylose Lysine Deoxycholate agar (XLD agar). B) Non-typhoidal strains of Salmonella are Alkaline (pink) over Acid (yellow) with the production of copious amounts of hydrogen sulfide on Triple Sugar Iron agar (TSI).

Discussion

Hemoglobin SC disease (Hb SC) is the second most common hemoglobinopathy after Sickle Cell Disease (SCD, Hb SS) globally.1 Hb SC disease occurs when a patient inherits both hemoglobin S and hemoglobin C alleles. Hemoglobin S and C variants are caused by point mutations in the hemoglobin beta- chain, and both variants lead to reduced affinity to the alpha-chain. While hemoglobin C is an abnormal form of hemoglobin that does not cause sickling on its own, when co-inherited with hemoglobin S, the beta chains polymerize, causing red cell sickling when oxygen tension is lowered in the blood.2 Patients develop anemia due to reduced red cell lifespan (27-29 days for Hb SC vs. 15-17 days for Hb SS) and subsequent destruction of red blood cells.3

Complications arise from vascular occlusion and destruction of red blood cells, leading to gallstones, pulmonary infarction, priapism, and/or cerebral infarction. Other complications include avascular necrosis of the femoral head, bone marrow necrosis, renal papillary necrosis, retinopathies, splenomegaly, and recurrent pregnancy loss. Although Hb SC patients often exhibit similar symptomology to sickle cell disease, symptoms are typically milder and present later in childhood.2,3 In comparison to patients with Hb SS, Hb SC patients have milder anemia, less frequent sickle cells, and less severe hemolysis. While Hb SC patients have fewer sickling episodes compared to Hb SS patients, Hb SC patients have more severe retinopathy and splenomegaly. It is also important to note that the enlargement of the spleen is often caused by red blood cell sequestration and the optimal function of the spleen is significantly reduced (functional hyposplenia), which can lead to increased risk of infection from encapsulated bacteria.

Diagnosis of Hb SC disease is typically made by performing hemoglobin electrophoresis (Image 2). Hemoglobin electrophoresis separates the differing varieties of hemoglobin by size and electrical charge. Capillary electrophoresis separates hemoglobin variants based on the “zone” of detection where each variant hemoglobin appears based on a reference pattern. Normal hemoglobin (A, F, A2) is easily discriminated from variant hemoglobins (S, C, E, D), and quantification allows for detection of beta-thalassemia (increased A2 fraction). While useful as a screening tool, the hemoglobin variants identified in the “zones” are not specific. For example, Hb C and Hb Constant Spring share a zone, and Hb A2 shares a zone with Hb O- Arab. Variants detected by capillary electrophoresis are confirmed by a second method, and in this case Hb SC was confirmed by acid agarose gel (Sebia Hydrogel). When subjected to acid gel electrophoresis, Hb C and Hb S migrate in separate bands, while Hb A, A2, D, and E comigrate in the “A” band, and the “F” band may contain F in addition to the glycated fraction of normal adult Hb A. Patients with Hb SC disease will have variants detected in the S and C zones in capillary electrophoresis and lack signal in the A zone.4

Image 2. Laboratory Diagnosis of Hb SC disease includes hemoglobin electrophoresis and peripheral blood smear review. A) Hemoglobin capillary electrophoresis (pH 9.4) separates F, S, C, A2, A (Sebia, Capillarys 2 Flex Piercing). B) Acid agarose gel (pH 6.0-6.2) separates hemoglobins F, A, S, and C (Sebia, Hydragel Acid QC lane).  C) Peripheral blood smear morphology showing characteristic Hb SC forms including target cells, boat shaped cells (single arrow), red cell with crystals (double arrow), and hemighost cells (triple arrow).

Examination of the peripheral blood smear from a patient with Hb SC disease (Image 2C) reveals frequent target cells, boat-shaped cells (taco shaped), and only rarely contains classic sickle cells. Hemoglobin C crystals can be seen, both free floating and inside red cells, a feature of CC and SC disease but not seen in SS disease. Hemi-ghost cells and cells with irregular membrane contractions are also more frequent in Hb SC disease. In contrast, sickle cells are rarely observed in peripheral smears from Hb SC patients.

Salmonellaeare flagellated gram negative bacilli that are members of the Enterobacterales. Salmonellosis is typically foodborne in nature and presents as a self-limiting acute gastroenteritis.5,6 However, these organisms can invade beyond the gastrointestinal tract resulting in bacteremia.6 This case presents Salmonella as a cause of bacteremia in a patient with Hb SC disease following a bout of gastroenteritis. Although there is a well-known association between SCD and invasive infections with Salmonella, the incidence of Salmonella infection in patients with Hb SC disease has not been well studied. Patients with SCD, particularly those in Africa, are at risk for developing invasive disease caused by non-typhoidal Salmonella, including osteomyelitis, meningitis, and bacteremia. It has been hypothesized that disruptions in the gut microbiome and increased permeability of enterocytes makes SCD patients more prone to invasive Salmonella infections.6 Furthermore, the compromised function of the spleen in both patients with SCD and Hb SC disease increases the risk of disseminated infection by encapsulated bacteria and Gram negative rods. The spleen plays an important housekeeping role removing old or damaged erythrocytes, but also has an important immunological function housing memory B cells, producing antibodies and macrophages that phagocytize circulating bacteria, particulates or other debris and then present the antigens to other immunological cells in the spleen.7 Although sepsis caused by Salmonella is an occasional progression of gastroenteritis, this patient’s Hb SC disease likely increased the likelihood of bacteremia because of her functional asplenia.

References

  1. Weatherall DJ. The inherited diseases of hemoglobin are an emerging global health burden. Blood. 2010;115(22):4331–6.
  2. Tim R. Randolph,24 – Hemoglobinopathies (structural defects in hemoglobin),Editor(s): Elaine M. Keohane, Catherine N. Otto, Jeanine M. Walenga,Rodak’s Hematology (Sixth Edition), Elsevier, 2020, Pages 394-423, ISBN 9780323530453, https://doi.org/10.1016/B978-0-323-53045-3.00033-7.
  3. (https://www.sciencedirect.com/science/article/pii/B9780323530453000337)
  4. Nathan, D. G., Orkin, S. H., & Oski, F. A. (2015). Sickle Cell Disease. In Nathan and Oski’s hematology and oncology of infancy and childhood (8th ed., pp. 675-714). Philadelphia, PA: Elsevier. Retrieved from https://www.clinicalkey.com/#!/content/book/3-s2.0-B9781455754144000206y.com/#!/content/book/3-s2.0-B9781455754144000206. Accessed 2022
  5. Bain, BJ. (2020) Haemoglobinopathy Diagnosis, Third Edition. Hoboken: John Wiley and Sons, Ltd
  6. Kurtz, J. R., Goggins, J. A., & McLachlan, J. B. (2017). Salmonella infection: Interplay between the bacteria and host immune system. Immunology letters190, 42–50. https://doi.org/10.1016/j.imlet.2017.07.006
  7. Lim, S.H., Methé, B.A., Knoll, B.M. et al. Invasive non-typhoidal Salmonella in sickle cell disease in Africa: is increased gut permeability the missing link?. J Transl Med 16, 239 (2018). https://doi.org/10.1186/s12967-018-1622-4
  8. Leone G, Pizzigallo E. Bacterial Infections Following Splenectomy for Malignant and Nonmalignant Hematologic Diseases. Mediterr J Hematol

-John Stack is a first year AP/CP resident at UT Southwestern Medical Center.

-Marisa Juntilla is an Assistant Professor in the Department of Pathology at UT Southwestern Medical Center. Dr. Juntilla is a board certified Clinical Pathologist and is certified in the subspecialty of Hematopathology.

-Dominick Cavuoti is a Professor in the Department of Pathology at UT Southwestern Medical Center. Dr. Cavuoti is a board certified AP/CP who is a practicing Clinical Microbiologist, Infectious Disease pathologist and Cytopathologist.

-Andrew Clark, PhD, D(ABMM) is an Assistant Professor at UT Southwestern Medical Center in the Department of Pathology, and Associate Director of the Clements University Hospital microbiology laboratory. He completed a CPEP-accredited postdoctoral fellowship in Medical and Public Health Microbiology at National Institutes of Health, and is interested in antimicrobial susceptibility and anaerobe pathophysiology.

-Clare McCormick-Baw, MD, PhD is an Assistant Professor of Clinical Microbiology at UT Southwestern in Dallas, Texas. She has a passion for teaching about laboratory medicine in general and the best uses of the microbiology lab in particular.

Microbiology Case Study: An Adult Presents with Hand Wound Following a Dog Bite

Case Presentation

An adult presented to the emergency department with a finger infection persisting for the past 14 days after being bitten by her dog. The finger was swollen, tender and red but the patient denied fever, chills, or purulent drainage. The patient was previously given 10 days of doxycycline and amoxicillin-clavulanic acid without any improvement. The patient underwent incision and drainage and the specimen was sent for aerobic culture and Gram stain. No organisms or WBCs were seen on the Gram stain. On day 3 of incubation, a yellow colony was observed on the chocolate agar. The colony was streaked out onto another chocolate plate for subculture (Image 1). MALDI-TOF identified this organism as Neisseria animoralis.

Image 1. Subculture of Neisseria animoralis.

Discussion

Neisseria animoralis and Neisseria zoodegmatis are primarily zoonotic organisms found as normal oral flora of cats and dogs. Both can cause wound infections in humans following animal bites. However, these organisms are under recognized animal bite pathogens, often leading to their identifications being dismissed as contaminants. While there are limited published studies on this organism, it is important to recognize its role in wound infections, as in our case. Due to lack of awareness and reduced recovery in culture, case studies have shown correlations with this organism and poor healing and chronic wound infections.

On Gram stain, N. animoralis appears as a Gram negative coccoid rod. In culture, N. animoralis is a slow growing organism that produces yellow or white colonies that are shiny and smooth. N. animaloris produces acid from glucose, but not lactose, sucrose, or maltose. MALDI-TOF is most commonly used for identification.

Limited N. animoralis treatment data are available currently. Most animal bite-related infections are polymicrobial in nature and thus, antibiotic treatment is broad spectrum to cover the most common aerobic and anaerobic organisms.

Resources

  • Johannes Elias, Matthias Frosch, and Ulrich Vogel, 2019. Neisseria, In: Carroll KC, Pfaller MA Manual of Clinical Microbiology, 12th Edition. ASM Press, Washington, DC. doi: 10.1128/9781683670438.MCM.ch36
  • Heydecke A, Andersson B, Holmdahl T, Melhus A. Human wound infections caused by Neisseria animaloris and Neisseria zoodegmatis, former CDC Group EF-4a and EF-4b. Infect Ecol Epidemiol. 2013;3:10.3402/iee.v3i0.20312. Published 2013 Aug 2. doi:10.3402/iee.v3i0.20312
  • Kathryn C. Helmig, Mark S. Anderson, Thomas F. Byrd, Camille Aubin-Lemay, Moheb S. Moneim, A Rare Case of Neisseria animaloris Hand Infection and Associated Nonhealing Wound, Journal of Hand Surgery Global Online, Volume 2, Issue 2, 2020, Pages 113-115, ISSN 2589-5141 https://doi.org/10.1016/j.jhsg.2020.01.003.
  • Merlino J, Gray T, Beresford R, Baskar SR, Gottlieb T, Birdsall J. Wound infection caused by Neisseria zoodegmatis, a zoonotic pathogen: a case report. Access Microbiol. 2021;3(3):000196. Published 2021 Feb 10. doi:10.1099/acmi.0.000196

-Paige M.K. Larkin, PhD, D(ABMM), M(ASCP)CM is the Director of Molecular Microbiology and Associate Director of Clinical Microbiology at NorthShore University HealthSystem in Evanston, IL. Her interests include mycology, mycobacteriology, point-of-care testing, and molecular diagnostics, especially next generation sequencing.

Microbiology Case Study: a 53 Year Old Man with a Black Spot on His Shoulder

Case History

A 53 year old man presents to urgent care with a primary complaint of an area of erythema and tenderness around a small black spot on his left shoulder, shortly after returning from Ecuador. He does not report any fevers, chills, or drainage from the affected area. The patient reported that he occasionally felt the area moving. An occlusive Vaseline dressing was applied to the central black spot, and the organism shown below emerged from the wound.

Laboratory Identification

The parasite shown above is a human botfly larva, Dermatobia hominis. The clinical history is strongly suspicious for a botfly infection, and the patient himself suggested the diagnosis.

Dermatobia hominis is identified in large part by its relatively unique presentation combined with identification of the larvae in tissue. Laboratory identification of genus and species involves comparing morphological structures including the anterior and posterior spiracles, mouthparts and cephalopharyngeal skeleton, and cuticular spines. Travel history can also be helpful for genus or species-level identification.

Discussion

The lifecycle of human botflies begins when the female botfly lays her eggs on a mosquito. Once a mosquito feeds on a host, the botfly larva drop onto the host and burrow into the skin. They may remain in that location for up to 10 weeks before dropping off the host into soil to pupate and continue the life cycle.

The human botfly is found in North America, ranging from Mexico to Paraguay and northeast Argentina. Cases in the US are primarily in travelers returning from the botfly’s native range. Measuring the incidence of infection in travelers can be difficult due to the nature of the disease. Experienced travelers may be able to remove the larva at home. In other cases the botfly larva may leave the host before the patient seeks medical care.

Testing for the presence of these larva is easy as they require oxygen coming in through a hole in the skin. Cover the lesion with a thick layer of sterile Vaseline gauze and wait approximately 5-15 minutes for the organism to emerge. Surgery is usually not required as the larva is most often removed intact. Antibiotics should be given following removal of the parasite to prevent secondary infections.

-Britt Boles, MD 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.

Microbiology Case Study: A 27 Year Old with Disseminated Joint Pain

Case History

A 27 year old male presented to the Emergency Department (ED) with complaints of right knee pain and swelling for one week. Two weeks prior, he tripped while walking to work and began to feel pain in his right calf. Upon physical examination, swelling was noted in his ankles, knee, shoulders, and fingers. The knee and shoulder were tender to palpation. In the ED, he was afebrile and vitals were normal. He denied any sort of injury, chills, or rash and no history of tobacco, alcohol, or illicit substance abuse. CT scan of the lower extremity showed no acute fracture but moderate to large knee joint effusion was observed. He and his fiancé (male partner) has been in a monogamous relationship for almost a decade, however the patient did have a history of gonorrhea nine years ago but was treated. Knee arthrocentesis was performed. The fluid was yellow and cloudy and contained 27,000 WBCs. The Gram stain of the synovial fluid showed many intracellular gram negative diplococci and the joint fluid culture grew out Neisseria gonorrhoeae. PCR of the rectal swab also detected N. gonorrhoeae.

Discussion

N. gonorrhoeae is the causative agent of gonorrhea, a sexually transmitted disease. In the United States, it is the second most commonly reported communicable disease.1 While infections can be asymptomatic, in men, gonorrhea commonly causes acute urethritis with dysuria, urethral discharge, and rarely, epididymitis.2,3,4 In women, gonorrhea can cause cervicitis and lead to pelvic inflammatory disease (PID), infertility, ectopic pregnancy, and chronic pelvic pain.5,6 Those with gonococcal endocervicitis can be co-infected with Chlamydia trachomatis and/or Trichomonas vaginalis, other causative agents of sexually transmitted diseases. N. gonorrhoeae can cause extragenital infections in the pharynx and rectum, which are most commonly seen among men who have sex with men (MSM). Disseminated gonococcal infection is rare (0.5-3% of infected individuals) and can be characterized by low grade fever, hemorrhagic skin lesions, tenosynovitis, polyarthralgia and septic arthritis. Complications of disseminated infections may include permanent joint damage, endocarditis, and meningitis. Gonococcal conjunctivitis mainly affects newborns from untreated mothers.7

Gonorrhea can be diagnosed clinically by a history and physical examination and also, by microbiological methods. Home collection kits are available to increase convenience. On a Gram stain, N. gonorrhoeae, a gram negative coccus, frequently appears within or closely associated polymorphonuclear leukocytes (PMNs) typically as diplococci pairs. Direct smears can be prepared from urethral, endocervical sites, and normally sterile or minimally contaminated sites such as joint fluid. Swab specimens (e.g. urogenital, pharyngeal, vaginal or rectal) should be collected with a Dacron or Rayon swab as calcium alginate and cotton swabs may be toxic or inhibitory for the bacteria.8 Specimens must be transported to the microbiology immediately. 9 Blood and joint fluid are also acceptable specimen types for culture for detection of disseminated gonococcal infection.

Enriched selective media for culture of N. gonorrhoeae includes MTM medium, ML medium, GC-Lect and the New York City medium. Plates should be incubated in a CO2 incubator (between 3-7%) at 35C to 37C for optimal growth.9 Gram negative diplococci recovered from urogenital sites that grow on the selective media and are oxidase-positive can be presumptively identified as N. gonorrhoeae. Another quick biochemical test that can be done is superoxol; N. gonorrhoeae produce immediate bubbling whereas N. meningitidis and N. lactamica produce weak, delayed bubbling. Confirmation using other testing methods such as carbohydrate utilization tests (e.g. N. gonorrhoeae produces acid from glucose only), immunological methods, enzymatic procedures, or DNA probe are also available.10

Compared to standard culture methods, Nucleic Acid Amplification Tests (NAAT) offer more rapid results and increased sensitivity. Additionally, NAATs may also include additional targets such as C. trachomatis, a frequent co-pathogen, as part of the assay. NAATs should be used according manufacturer’s protocols and on validated specimen types. For example, the Cepheid Xpert CT/NG test (as used by our patient here) can be used to test asymptomatic and symptomatic individuals and the acceptable specimen types are urine, pharyngeal, and rectal swabs, patient-collected vaginal swabs, and clinician-collected endocervical swabs.11 Given the legal implications of a N. gonorrhoeae diagnosis in a child, the CDC recommends that NAATs can be used to test for N. gonorrhoeae from vaginal and urine specimens from females and urine for males.12 For extragenital specimens, only validated FDA-cleared NAATs assays using pediatric specimens should be used.

The CDC recommends that uncomplicated gonorrhea be treated with ceftriaxone and azithromycin. However, between 2000-2010s, elevated MICs to both ceftriaxone and cefixime were seen and emerging azithromycin resistance is still a concern. The CLSI M100 currently recommends agar dilution or disk diffusion for antimicrobial susceptibility testing for N. gonorrhoeae. Susceptible and resistant interpretative breakpoints are available for penicillin, most cephems, tetracycline, ciprofloxacin, and spectinomycin. Of note, for azithromycin, only the susceptible category has a breakpoint.13

Image 1. Gram stain of synovial fluid showing many intracellular gram negative diplococci.
Image 2. Chlamydia trachomatis and Neisseria gonorrhoeae PCR. Orange and Brown= targets for N. gonorrhoeae; light and dark green=control genes.

References

  1. CDC. Sexually Transmitted Disease Surveillance, 2020. Atlanta, GA: Department of Health and Human Services; April 2022.
  2. John J, Donald WH. Asymptomatic urethral gonorrhoea in men. Br J Vener Dis 1978; 54:322.
  3. Handsfield HH, Lipman TO, Harnisch JP, et al. Asymptomatic gonorrhea in men. Diagnosis, natural course, prevalence and significance. N Engl J Med 1974; 290:117.
  4. Sherrard J, Barlow D. Gonorrhoea in men: clinical and diagnostic aspects. Genitourin Med 1996; 72:422.
  5. McCormack WM, Johnson K, Stumacher RJ, Donner A, Rychwalski R. Clinical spectrum of gonococcal infection in women. Lancet, 1(8023), 1182–1185 (1977).
  6. Curran J, Rendtorff R, Chandler R, Wiser W, Robinson H. Female gonorrhea: its relation to abnormal uterine bleeding, urinary tract symptoms, and cervicitis. Obstet Gynecol, 45(2), 195–198 (1975).
  7. O’Brien JP, Goldenberg DL, Rice PA. Disseminated gonococcal infection: a prospective analysis of 49 patients and a review of pathophysiology and immune mechanisms. Medicine (Baltimore) 1983; 62:395.
  8. Laurer BA, Masters HB. Toxic effect of calcium alginate swabs on Neiserria gonorrhoeae. J Clin Microbiol 1988: 26:54-56
  9. Leber, A. 3.9 Genital Cultures. Clinical Microbiology Procedures Handbook, 4th Edition. ASM Press, Washington, DC. 2016. p.3.9.3.1-3.9.3.15. 
  10. Knapp JS. Historical perspectives and identification of Neisseria and related species. Clin Microbiol Rev 1988;1:415-431.
  11. Cepheid GeneXpert. Xpert CT/NG (English). 2019. 301-0234 Rev.K
  12. CDC. Gonococcal Infections Among Infants and Children. Sexually Transmitted Infection Treatment Guidelines, Atlanta, GA: Department of Health and Human Services; 2021.
  13. CLSI. Performance Standards for Antimicrobial Susceptibility Test. CLSI supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute; 2022, Edition 32

-Maikel Benitez Barzaga, MD is a Pathology Resident (PGY-1) at The George Washington University Hospital. His academic interest include hematology, microbiology, molecular and surgical pathology.

-Rebecca Yee, PhD, D(ABMM), M(ASCP)CM is the Chief of Microbiology, Director of Clinical Microbiology and Molecular Microbiology Laboratory at the George Washington University Hospital. Her interests include bacteriology, antimicrobial resistance, and development of infectious disease diagnostics.

BOGO: Biopsy One, Get One Free

I’ve mentioned before how important it is to know clinical history before attending a biopsy, and I cannot stress this point enough. As the first line of screening, the intermediary between clinician and pathologist, the role of the cytologist is to prepare, assess, and convey. In a cancer center, we have three main populations: the patients with the unknown primary, the patients with the suspected primary, and the patients with the suspected metastasis. In the event of a suspected metastasis, we’ll review previous relevant pathology material if we have it onsite. Unless the clinician is requesting additional prognostic markers, the review process helps us eliminate the unnecessary repetition of immunostains (IHC) by confirming that the current material is morphologically consistent with the prior material. Sometimes we still perform old-school cytology without a plethora of ancillary studies. HA!

Most of the endobronchial ultrasound (EBUS) procedures performed at our institution are for lung cancer staging or differentiation between a lung cancer metastasis and an extra-pulmonary metastasis. Not that we don’t see the occasional sarcoid- or anthracosis-related process from time to time, but our most common indication is cancer. For an 88-year-old male patient with multiple lung nodules and both mediastinal and hilar lymphadenopathy, confirmation of metastasis was the main objective of the EBUS procedure. The patient’s pertinent medical history includes former tobacco use, squamous cell carcinoma of the lung (diagnosed percutaneously in 2022), clear cell renal cell carcinoma (s/p partial nephrectomy in 2020), prostate cancer (radiated in 2007), melanoma (excised in 2001), and cutaneous squamous cell and basal cell carcinoma (also previously excised in 2002 and 2008). With an extensive cancer history, the lung nodules and thoracic nodes could be any of them, although metastatic squamous cell carcinoma of the lung was clinically favored. My awesome cytologist colleague, Kelly, attended the EBUS procedure. The Rapid Onsite Evaluation (ROSE) was a clear-cut “adequate for diagnostic material,” and the attending pathologist added “tumor cells present.” The following morning, Kelly stopped by my desk to ask my opinion of the 12R (right hilar) lymph node she was screening. She said, “look at my dots. Do these look like the same cells to you? Or are they different? Because I feel like they’re different.” Before putting the slide on my scope, I asked, “so… like a combined adenosquamous? Or a small cell component?” She replied, “not small cell. Something… I don’t know, but they look different. The patient was recently diagnosed with lung cancer and has a history of renal cell.” I fixated on the H&E cell block slides (Images 1-3) before perusing the Diff-Quik and Papanicolaou-stained slides (Images 4-5). “Uhm… Why are there two different types of tumor cells here?! The cytoplasm here is so… vacuolated, but it’s not quite like lung adeno, and the other group… even the n/c (nuclear-to-cytoplasmic) ratio is different. What is this?” Kelly replied, “okay, so there are definitely two different types of tumor here.” I looked up, “It has to be. Absolutely, yes.”

Images 1-4. Lymph node, 12R, EBUS-guided FNA. 1-3: H&E cell block sections 1, 100x; 2, 400x; 3, 100x. 4: Diff-Quik stained smear.
Image 5. Lymph Node, 12R, EBUS-guided FNA. Pap-stained smear.

Kelly entered her diagnosis into our laboratory information system and brought the case over to the pathologist on cytology service for the day. She explained her thought process, and the pathologist also questioned if it was a combined process, such as a lung adenosquamous and maybe the original lung biopsy only sampled the squamous component. With the most recent clinical history of both lung squamous cell carcinoma and clear cell renal cell carcinoma, an IHC panel was appropriately selected. Later that afternoon, the pathologist exclaimed, “IT’S BOTH! IT’S SQUAMOUS AND RCC!” The clusters of squamous cell carcinoma did not stain for PAX8 (a renal cell carcinoma marker) (Image 6), and the same cluster stained positive for p40 (a squamous cell carcinoma marker) (Image 7). Within the same level of the cell block, the cluster of cells that appeared morphologically different than squamous cluster stained positive for PAX8 (Image 8) and negative for p40 (Image 9), confirming a renal cell carcinoma component. A small focus of p40-positive cells was present next to the p40-negative renal cell carcinoma (Image 9), further demonstrating mixed histology. This finding was shared with other pathologists, and the results were immediately called to the pulmonologist as this was a critical finding. Sometimes we encounter a partially involved node where the tumor cells are intermixed with lymphocytes, sometimes the lymph node yields more tumor than the primary site, and sometimes, albeit rarely, we encounter a lymph node infiltrated by two different carcinomas.

Images 6-9. Lymph Node, 12R, EBUS-guided FNA. Cell block section immunocytochemistry. Squamous cell carcinoma cluster – 6: PAX8-negative; 7: p40-positive. Renal cell carcinoma cluster – 8: PAX8-positive, 9: p40-negative (with small focus of p40-positive squamous cell carcinoma).

Due to the patient’s bulky disease and PD-L1 expression of 30%, the medical oncologists primary aim was to treat the squamous cell carcinoma first and follow up renal cell carcinoma therapy second. After the first few cycles of treatment, the lung nodules have decreased in size, but the thoracic nodes remain unchanged. Once the squamous cell carcinoma is controlled or demonstrates a more significant response, immunotherapy may be added to target both, with a tyrosine kinase inhibitor directed at renal cell carcinoma metastases in the event of progression.

-Taryn Waraksa-Deutsch, MS, SCT(ASCP)CM, CT(IAC), has worked as a cytotechnologist at Fox Chase Cancer Center, in Philadelphia, Pennsylvania, since earning her master’s degree from Thomas Jefferson University in 2014. She is an ASCP board-certified Specialist in Cytotechnology with an additional certification by the International Academy of Cytology (IAC). She is also a 2020 ASCP 40 Under Forty Honoree.

Microbiology Case Study: A 32 Year Old with Lower Extremity Swelling

Case History

A 32 year old male with alcoholic cirrhosis presented to the emergency department with progressive lower extremity swelling. On presentation he was found to have jaundice due to hemolytic anemia secondary to spur cell anemia. Admission hemoglobin was 4.3 mg/dL (4.0-11.0 mg/dL) and bilirubin, both total and direct, were 6.3 mg/dL (0.2-1.3 mg/dL) and 2.9 mg/dL (0.0-0.5 mg/dL), respectively. He also had acute kidney injury (AKI) thought to be secondary to hepatorenal syndrome leading to the development of anasarca. A urinalysis was performed as part of the evaluation for his AKI that showed 100 WBC/HPF, > 187 RBC/HPF, and moderate bacteria which triggered a urine culture.

Laboratory Identification

Urine received in the microbiology laboratory was plated on Blood and MacConkey/CNA agars and grew non-hemolytic, lactose-fermenting gram negative rods (Image 1). Indole testing was negative. Given this biochemical pattern, a member of the Enterobacterales was suspected as typically seen in urine cultures. However, MALDI-TOF MS provided the surprising identification of Salmonella enterica subsp. arizonae. Xylose Lysine Deoxycholate (XLD) agar was set up to confirm the unusual identification (Image 2). Hydrogen sulfide production is typical of Salmonellae, and lactose fermentation, a trait unique to some isolates of S. enterica subsp. arizonae, was confirmed. The organism was submitted to the Texas Department of Health laboratory where the isolate was definitively identified as Salmonella enterica subsp. arizonae (IIIa 14:z4,z23) by whole genome sequencing.

Image 1. Patient isolate of S. enterica subsp. arizonae exhibiting lactose fermentation on MacConkey agar after 18 hours of incubation at 35°C (A). Lactose-fermentation is a unique hallmark of S. enterica subsp. arizonae compared to other Salmonellae (B).
Image 2. Patient isolate of S. enterica subsp. arizonae exhibiting hydrogen sulfide production and lactose fermentation on XLD agar after 18 hours at 35°C (A). Note the abundant yellow color of the medium (black arrowhead) compared to S. enterica subsp. Enterica serovar Enteritidis which does not ferment lactose, but also produces hydrogen sulfide (B, white arrowhead).

Discussion

This is a rare case of an extraintestinal infection caused by Salmonella enterica subsp. arizonae. Salmonellaeare motile, gram negative, facultatively anaerobic bacilli that are members of the Enterobacterales. The genus is composed of two species, S. enterica and S. bongori. Salmonella enterica is further subdivided into six subspecies: enterica (group I), salamae (group II), arizonae (group IIIa), diarizonae (group IIIb), houtenae (group IV), and indica (group VI). Salmonella bongori used to be classified as group V but was separated as a unique species based on genomic analysis.1 S. bongori almost exclusively causes zoonotic infections, while S. enterica subsp. enterica is the most frequent cause of human clinical disease. Salmonella taxonomy is complicated further by the division of members of S. enterica subsp. enterica into >2500 unique serovars based on immunoreactivity to lipopolysaccharide (O) and two flagellar (H) surface antigens. These are then further separated into “typhoidal” and “non-typhoidal” serovars based upon the characteristics of infection (Image 3).

Image 3. Hierarchical structure of Salmonella taxonomy. S. enterica subsp. arizonae is boxed in red to highlight is taxonomic position away from other pathogenic Salmonellae. Adapted from reference number 6.

Until recently, determinative testing was almost uniformly performed by serological confirmation of agglutination with O and H antigen-specific antisera. This has been a mainstay of epidemiological analysis of foodborne Salmonella outbreaks. Only recently has whole genome sequencing been adapted as a higher throughput and more discriminatory alternative to classical serotyping schemes. Salmonella nomenclature often uses a genus-species-subspecies format followed by serovar (e.g. Salmonella enterica subsp. enterica serovar Typhi), or it can be reported as genus-serovar for short (e.g. Salmonella Typhi). Formal identification will include information concerning the two flagellar antigens and lipopolysaccharide antigens, in addition to the formalized subspecies using the formula: genus-species-subspecies [space] O antigens [colon] Phase 1 H antigen [comma] Phase 2 H antigen. In this case, the formal identification from the state laboratory for this isolate was Salmonella enterica subsp. arizonae IIIa 14:z4,z23.

About 99% of human infections are due to Salmonella enterica subspecies enterica (group I)including the serotypes Enteritidis, Typhimurium, Typhi, Paratyphi.2 Infections due to Salmonella enterica subspecies arizonae are rare; serovar IIIa 41:z4,z23 is associated with 10-20 infections per year.3 Infection typically begins as gastroenteritis from food poising or from animal sources, particularly reptiles or poultry. Disease is typically seen in the young and immunocompromised and can progress to invasive disease including sepsis, meningitis, and osteomyelitis.4 It is unclear why there are lower rates of Salmonella enterica subspecies arizonae infections in humans as compared to Salmonella enterica subspecies enterica, but there is evidence to suggest Salmonella enterica subspecies arizonae and diarizonae have altered intestinal colonization in murine models leading to failure of Salmonella to persist in the mammalian intestinal tract.5

This patient had alcoholic cirrhosis and uncomplicated cystitis secondary to Salmonella extraintestinal infection at the time of presentation. It is unclear if this patient had gastroenteritis prior to developing cystitis and the limited medical history did not reveal exposure to reptiles or poultry. In this case, the patient completed seven days of ceftriaxone without complication or recurrence of infection.

References

  1. Agbaje M, Begum RH, Oyekunle MA, Ojo OE, Adenubi OT. Evolution of Salmonella nomenclature: a critical note. Folia Microbiol (Praha) 2011; 56(6): 497-503.
  2. Brenner FW, Villar RG, Angulo FJ, Tauxe R, Swaminathan B. Salmonella nomenclature. J Clin Microbiol 2000; 38(7): 2465-7.
  3. Shariat NW, Timme RE, Walters AT. Phylogeny of Salmonella enterica subspecies arizonae by whole-genome sequencing reveals high incidence of polyphyly and low phase 1 H antigen variability. Microb Genom 2021; 7(2).
  4. Abbott SL, Ni FC, Janda JM. Increase in extraintestinal infections caused by Salmonella enterica subspecies II-IV. Emerg Infect Dis 2012; 18(4): 637-9.
  5. Katribe E, Bogomolnaya LM, Wingert H, Andrews-Polymenis H. Subspecies IIIa and IIIb Salmonellae are defective for colonization of murine models of salmonellosis compared to Salmonella enterica subsp. I serovar typhimurium. J Bacteriol 2009; 191(8): 2843-50.
  6. Achtman M, Wain J, Weill FX, Nair S, Zhou Z, et al. (2012) Multilocus Sequence Typing as a Replacement for Serotyping in Salmonella enterica. PLOS Pathogens 8(6): e1002776. https://doi.org/10.1371/journal.ppat.1002776

Denver Niles, MD is the Medical Microbiology fellow at UT Southwestern Medical Center. Prior to his Medical Microbiology fellowship, he completed pediatric infectious disease training at Baylor College of Medicine/Texas Children’s Hospital.

Muluye Mesfin, SM(ASCP)CM is the microbiology laboratory supervisor at UT Southwestern Medical Center where he has worked for 12 years.  Prior to this, Mo completed a bachelor of science degree in medical technology at the University of Maryland.

-Clare McCormick-Baw, MD, PhD is an Assistant Professor of Clinical Microbiology at UT Southwestern in Dallas, Texas. She has a passion for teaching about laboratory medicine in general and the best uses of the microbiology lab in particular.

-Andrew Clark, PhD, D(ABMM) is an Assistant Professor at UT Southwestern Medical Center in the Department of Pathology, and Associate Director of the Clements University Hospital microbiology laboratory. He completed a CPEP-accredited postdoctoral fellowship in Medical and Public Health Microbiology at National Institutes of Health, and is interested in antimicrobial susceptibility and anaerobe pathophysiology.

Microbiology Case Study: An Adult Woman with a Pelvic Abscess

Case History

An adult woman presented to the emergency department five days after undergoing gynecological surgery. The patient presented with fever and severe right lower quadrant abdominal pain. Computed tomography (CT) scan with contrast showed a ring enhanced loculated fluid collection within the cervix, which was concerning for an abscess. The patient was admitted to the hospital and empirically started on piperacillin-tazobactam, but continued to have fevers despite the antibiotics. Blood and urine samples were sent to the microbiology lab for bacterial culture but no organisms were isolated from either source. Two days later, the patient underwent a diagnostic laparoscopy, abdominal wash-out, and drainage of the abscess. The abscess fluid was sent for aerobic and anaerobic bacterial culture. Gram stain of the specimen showed 3+ white blood cells with no organism seen. The anaerobic culture grew 4+ pinpoint white colonies on blood agar after 5 days of incubation. Further identification of these colonies by MALDI-TOF MS revealed Mycoplasma hominis.

Image 1. Blood agar with 4+ pinpoint translucent colonies.

Discussion

Mycoplasma hominis is often a commensal of the urogenital tract, but it can be associated with urogenital infections including pelvic inflammatory disease (PID), pregnancy-related infections, and urethritis in males. There are multiple risk factors for Mycoplasma hominis genital infection including young adult age, multiple sexual partners, and pregnancy. Immunocompromised patients have a higher risk for Mycoplasma hominis extragenital infections as nearly 50% of reported extragenital infections isolated from immunocompromised patients.2 Mycoplasma hominis can cause extragenital infections including septic arthritis,4 septicemia, osteitis, retroperitoneal abscesses3, mediastinitis,1 and pneumonia.

Laboratory diagnosis of Mycoplasma hominis is challenging due to the fastidious nature of the organism and its lack of the cell wall makes it undetectable by gram staining. The more specific tests including molecular tests for Mycoplasma hominis are not routinely ordered unless there is a strong clinical suspicion, which makes diagnosis more challenging. Mycoplasma hominis can grow on 5% sheep blood and chocolate agars; however, such growth is very slow and may take from 2 to 7 days of incubation.1 The usual growth of Mycoplasma hominis reveals tiny-sized pinpoint colonies that may be overlooked (Image 1). Once growth is observed, MALDI-TOF MS can be used for identification.6

There are multiple types of selective media for the isolation of Mycoplasma hominis including SP4 agar supplemented with arginine, Hayflick agar, A7, and A8 agars.9 Both A7 and A8 agars contain arginine to enrich Mycoplasma growth but differ in the antibiotic content used to inhibit the growth of other commensals. Agar plates should be put for incubation under 5 to 10% CO2 or under anaerobic conditions at 35°C for at least 5 days.9 On these selective agars Mycoplasma hominis has a characteristic fried egg appearance and can be seen by the aid of a stereomicroscope. However, use of specific agar is not widespread.

Molecular testing of Mycoplasma hominis using nucleic acid amplification (NAAT) assays such as polymerase chain reaction (PCR) is a more sensitive and faster method of detecting Mycoplasma hominis compared with culture. However, PCR is neither widely available nor standardized. PCR assays for Mycoplasma hominis generally use 16S rRNA as a gene target, but other targets, including gap, fstY, and yidC, have been developed.7 Clinical picture should be taken into account when evaluating the significance of a positive PCR test as Mycoplasma hominis can be a commensal organism and PCR does not distinguish between live and dead organisms.

Mycoplasma spp. lack a peptidoglycan cell wall. This makes Mycoplasma spp. intrinsically resistant to β-lactams and to all antibiotics, which target the cell wall, including glycopeptide antibiotics. Mycoplasma hominis is also resistant to rifampin, sulfonamides and trimethoprim. Tetracyclines, macrolides, and fluoroquinolones are often used. Antimicrobial susceptibility testing is rarely performed, with only a few specialized laboratories offering the testing. Clinical and laboratory standards institute guidelines (CLSI M43) is followed using microbroth dilution. Agar disc diffusion testing is not used for Mycoplasma hominis as there is no correlation between inhibitory zones and minimal inhibitory concentrations.8 Mycoplasma hominis can be evaluated for susceptibility to clindamycin, tetracycline, and levofloxacin.10

After isolation of Mycoplasma hominis was reported, doxycycline was added to the patient’s antibiotic regimen. The patient responded well with subsiding of the fever and stabilization of her vital signs.

References

  1. Xiang, L., & Lu, B. 2019. Infection due to Mycoplasma hominis after left hip replacement: case report and literature review. BMC infectious diseases, 19(1), 50. https://doi.org/10.1186/s12879-019-3686-z
  2. Meyer RD, Clough W. 1993. Extragenital Mycoplasma hominis infections in adults: emphasis on immunosuppression. Clin Infect Dis. Suppl 1:S243-9. doi: 10.1093/clinids/17.supplement_1.s243. PMID: 8399923.
  • Adams M, Bouzigard R, Al-Obaidi M, Zangeneh TT. 2020. Perinephric abscess in a renal transplant recipient due to Mycoplasma hominis: Case report and review of the literature. Transpl Infect Dis.(5):e13308. doi: 10.1111/tid.13308. Epub 2020 Jul 7. PMID: 32378787.
  • Luttrell LM, Kanj SS, Corey GR, Lins RE, Spinner RJ, Mallon WJ, Sexton DJ. 1994. Mycoplasma hominis septic arthritis: two case reports and review. Clin Infect Dis.19(6):1067-70. doi: 10.1093/clinids/19.6.1067. PMID: 7888535.
  • Wylam ME, Kennedy CC, Hernandez NM, Peters SG, Maleszewski JJ, Cassivi SD, Scott JP. 2013. Fatal hyperammonemia caused by Mycoplasma hominis. Lancet 382:1956.
  • Pereyre S, Tardy F, Renaudin H, Cauvin E, Del Pra Netto Machado L, Tricot A, Benoit F, Treilles M, Bebear C. 2013. Identification and subtyping of clinically relevant human and ruminant mycoplasmas by use of matrix-assisted laser desorption ionization–time of flight mass spectrometry. J Clin Microbiol 51:3314–3323.
  • Ferandon C, Peuchant O, Janis C, Benard A, Renaudin H, Pereyre S, Bebear C. 2011. Development of a real-time PCR targeting the yidC gene for the detection of Mycoplasma hominis and comparison with quantitative culture. Clin Microbiol Infect 17:155–159.
  • Clinical and Laboratory Standards Institute. 2011. Methods for antimicrobial susceptibility testing for human mycoplasmas; approved guideline M43-A. Clinical and Laboratory Standards Institute, Wayne, PA.
  • Stabler S, Faure E, Duployez C, Wallet F, Dessein R, Le Guren R. 2021. Mycoplasma hominis extragenital abscess. J Clin Microbiol, 59(4). https://doi.org/10.1128/JCM.02343-20
  • https://sites.uab.edu/dml/tests/

Omar Abdelsadek, MD is a PGY-1 (AP/CP) Pathology Resident at University of Chicago (NorthShore) Pritzker School of Medicine.

-Paige M.K. Larkin, PhD, D(ABMM), M(ASCP)CM is the Director of Molecular Microbiology and Associate Director of Clinical Microbiology at NorthShore University HealthSystem in Evanston, IL. Her interests include mycology, mycobacteriology, point-of-care testing, and molecular diagnostics, especially next generation sequencing.

Microbiology Case Study: Pain and Discharge from the Eye of a 27 Year Old Female

Case presentation

A 27 year old female with a history of substance abuse, presented to the ED with a swollen left eye, resulting from a fall in the shower that hit her eye on the tub. She denies losing consciousness or neck pain. Her eye has swollen since then and she noticed yellow drainage from her eye. Her past medical history was unremarkable although she was diagnosed as having sexually transmitted infections (STI) with Neisseria gonorrhoeae and Chlamydia trachomatis 3 months prior to the current event. Her HIV and syphilis screening were negative. 

The drainage from her eye was collected for culture and Gram stain and sent to the Microbiology laboratory. The initial Gram stain showed moderate neutrophils and very few gram negative diplococci (GNDC). The culture grew pure growth of the same organism on Chocolate agar during overnight incubation. It was identified as Neisseria gonorrhoeae by MALDI-ToF. The patient was reached out by the emergency nurses for the Ceftriaxone injection. 

Discussion

Neisseria gonorrhoeae belong to the Neisseriaceae family, including Kingella, Eikenella, and many other genera. Neisseria gonorrhoeae is gram negative cocci in pairs and have a distinct kidney bean shape. They thrive in the mucous membranes of the respiratory and urogenital tracts.1 While the pathogenicity tends to vary among Neisseria spp; N. gonorrhea is a primary pathogen that does not belong in the usual flora of humans in any amount, unlike other Neisseria that are opportunistic and can be part of usual flora.1 N. gonorrhoeae only has been reported in human cases. N. gonorrhoeae and N. meningitidis are considered fastidious organisms that require CO2 and iron. These organisms are aerobic bacteria that only grow on chocolate media that has RBC hemolyzed. When N. gonorrhoeae infects humans; it has a surface receptor that binds transferrin directly competing with the human host for iron supplies.1 Transferrin is a glycoprotein that delivers iron throughout the body.2

N. gonorrhoeae is typically acquired through unprotected sexual activity. Once transmitted, it can be found in vaginal, oral and anal secretions.3 The recorded cases of N. gonorrhoeae in 2018-2019 have gone up 5.9%; since 2009 there has been a 92.0% increase in cases, with a lot more young men contracting the disease since 2009.3 Improvement in screening and tracking techniques can also be a reason for the drastic increase in U.S cases.3 While most of the time N. gonorrhoeae stays in the mucosal membranes, it can also thrive in other parts of the body.1 The most common genital infection causes painful urination, a pus-like substance that discharges from the penis, and pain and tenderness in the testicular region.4 For women, it can cause increased discharge, pelvic pain, and bleeding between periods.4 Untreated genital N. gonorrhoeae can cause pelvic inflammatory disease (PID) which can lead to abortion and sterility in both men and women.1 N. gonorrhoeae also has the potential to affect but is not limited to the rectum, eyes, throat, and joints where it can cause pain, swelling, and rashes.4 Pregnant women that have an N. gonorrhoeae infection can pass it onto their offspring through vaginal delivery, and it is very important to screen for N. gonorrhoeae during pregnancy.4 In the US providers must put antimicrobial (erythromycin) eye drops in the babies born regardless of the STD status of the mother.1 A neonate exposed to N. gonorrhoeae can develop blindness and rashes.1 Untreated N. gonorrhoeaecan commonly causes recurrent rectal infection in women. While ocular gonococcal eye infection can be encountered more frequently in neonates born to infected mothers through vaginal delivery, ocular gonococcal infections in adults are extremely rare and can potentially be caused by incidental inoculation of infected genital secretions of their own (auto-infection).1 

In most cases, N. gonorrheae can be cultured from a swab of the male urethral or female endocervix, or vaginal samples. Non-genital samples, such as rectal and oral sources can also be used to diagnose extra-genital gonococcal infections. Nucleic acid amplification tests (NAAT) are commonly used for rapid diagnosis of gonococcal infection from genital, anal, or oral sources. For samples collected from other sources, culture is the primary method of the diagnostic approach. N. gonorrhoeae grows well on chocolate agar. MTM (modified Thayer martin) agar supports the growth of N. gonorrhoeae as it is a selective media for N. gonorrhoeae, containing nystatin, colistin, and vancomycin to suppress the growth of other bacteria.5 N. gonorrhoeae was susceptible to penicillin in 1976, and, by 1980, penicillinase-producing N. gonorrhoeae was discovered in Southwest Asia.1 The most common treatment is ceftriaxone intramuscularly with oral azithromycin for those who are allergic to cephalosporins like ceftriaxone gemifloxacin or injectable gentamicin.4

References

  1. Mahon, C. R., & Lehman, D. C. (2019). Textbook of diagnostic microbiology. Elsevier Saunders.
  2. Ogun, A. S. (2021, July 31). Biochemistry, transferrin. StatPearls [Internet].
  3. Centers for Disease Control and Prevention. (2014, January 29). Std facts – gonorrhea.
  4. Mayo Foundation for Medical Education and Research. (2021, October 5). Gonorrhea.
  5. Cheng, A., & Kirby , J. (2014, March). Evaluation of the hologic gen-probe panther, APTIMA Combo 2 assay in a tertiary care teaching hospital. American journal of clinical pathology. 

-Alejandro Soto, MLS(ASCP)CM

-Phyu M. Thwe, Ph.D., D(ABMM), MLS(ASCP)CM is Microbiology Technical Director at Allina Health Laboratory in Minneapolis, MN. She completed her CPEP microbiology fellowship at the University of Texas Medical Branch in Galveston, TX. Her interest includes appropriate test utilization and extra-pulmonary tuberculosis.

Microbiology Case Study: Worsening Liver Function and Bacteremia in a 35 Year Old Male

Case History

A 35 year old male with a history of alcohol use disorder in early remission, acute alcoholic hepatitis with multiple admissions for worsening liver function was admitted for acute kidney injury and worsening encephalopathy. Blood cultures were collected due to leukocytosis and the anaerobic bottle flagged positive for gram negative bacilli at 4.6 days. The organism, shown in Image 1, was sent to a reference laboratory and was identified as a Campylobacter species, unable to further identify. The patient will receive a liver transplant at another institution.

Image 1. Campylobacter species morphology in a blood smear.

Discussion

Campylobacter species are gram-negative, oxidase-positive, non-fermenting, microaerophilic, non-spore forming, motile rods typically with one or more helical turn.1,2 When two bacteria form short chains, these appear as “S” shaped and/or “gull-wing” shaped. These bacteria are generally 0.2 µm by 0.5-5.0 µm in size and can be as long as 8.0 µm.1 Campylobacter species are widely distributed in most warm-blooded animals (e.g., poultry, cattle, pigs, sheep, cats, and dogs) and they grow optimally at 37-42 °C. There are more than 20 Campylobacter species, not all of which cause illness but are potentially pathogenic. Campylobacter jejuni accounts for approximately 90% of human Campylobacter infections, while less common species such as Campylobacter coli, Campylobacter upsaliensis, Campylobacter fetus, and Campylobacter lari can also cause infection.3

Transmission of Campylobacter is believed to be foodborne via undercooked meat (particularly poultry), unpasteurized milk, or improperly treated water. Person-to-person transmission is rare, but may occur via the fecal-oral route. The infection load for Campylobacter species is relatively low, with fewer than 500 organisms causing infection.4 In human infection, these bacteria usually colonize the intestinal tract leading to diarrhea (often bloody), stomach cramps, fever, nausea, and vomiting.5 Clinical manifestation usually occurs 2 to 5 days after the individual is infected and lasts approximately a week. Diagnosis is established definitively by stool culture and sometimes by blood culture.2 In some cases, long-term effects of Campylobacter infection include an array of clinical syndromes including enteritis, bacteremia, arthritis, septic abortion, meningitis, irritable bowel disease, and Guillain-Barre syndrome [4]. Individuals with a greater risk for infection include those 65-years or older, pregnant women, and those with weakened immune systems.5

Campylobacteriosis is the most common form of acute infectious diarrhea in developed countries with a higher incidence than both Salmonella and Shigella.1 The Center for Disease Control and Prevention estimates that 1.5 million people in the United States are affected by Campylobacter infection each year—making it the most common bacterial cause of diarrheal illness in the United States.3 Unfortunately, the incidence of hepatitis associated with Campylobacter species infection is unknown, as few case-reports related to Campylobacter colitis6and Campylobacter jejuni 7,8,9,10 have been published. Although the liver is often involved in systemic infections resulting in various types of abnormal liver function tests, mild to severe hepatocellular dysfunction is an uncommon observation in those with Campylobacter infection.

Most individuals infected with any Campylobacter species recover with only fluid replenishment while the diarrhea lasts and no antibiotic treatment. However, those with or at risk for severe illness should be considered for antibiotic treatment. The antibiotics that are used to treat infection are azithromycin and fluoroquinolones (usually resistant). Antimicrobial susceptibility testing can help guide appropriate therapy.3

References

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-Amelia M. Lamberty is a MS in Pathology student at the Larner College of Medicine at the University of Vermont.

-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.