Microbiology Case Study: A 75 Year Old Found Unresponsive

A 75 year old female with a past medical history of coronary artery disease, hypertension, pre-diabetes mellitus, chronic obstructive pulmonary disease, prior left lobe cavitary lesion of unknown etiology, and tobacco use presented to the ED after being found nonresponsive on the couch. Family reports the patient said she had emesis the night before and felt as if she had a “stomach bug”. MRI shows T2 hyperintensities in the right MCA distribution. CSF results as follows.

White Blood Cells300
Red Blood Cells12
Protein990
Glucose79
Cryptococcal antigenNegative
Fungal cultureNo fungi isolated
HSVNegative

Laboratory findings

CSF was sent to the microbiology lab for bacterial and fungal smears and cultures. No fungi were identified. Cryptococcal antigen was negative. HSV was also negative. CSF Gram stain shows gram positive bacilli. CSF culture showed a small, white, smooth, translucent appearance on sheep blood agar. In semi-solid agar after overnight incubation at room temperature, an umbrella shaped pattern of motility was seen. The organism was identified as Listeria monocytogenes by MALDI-TOF mass spectrometry.

Image 1. Listeria monocytogenes on sheep blood agar.
Image 2. Listeria monocytogenes showing “umbrella zone” pattern of motility on semi-solid agar.

Discussion

Listeria spp. is a genus of gram positive, aerobic, facultative intracellular, catalase positive bacteria. Listeria monocytogenes is a common colonizer in the environment (animals, soil, vegetable matter) and occasionally colonizes the human gastrointestinal tract. Listeria prefers colder environments and can be found as a food contaminant, most notably in milk, raw vegetables, cheese, and meats. In addition, colonized mothers can pass Listeria monocytogenes to the fetus.1

Listeria monocytogenes has 3 notable virulence factors:2

  1. Listeriololysin O: a hemolytic toxin that allows for survival within phagocytes
  2. Act A: induces actin polymerization that facilitate cell-to-cell spread
  3. Siderophores: organisms capable of scavenging iron from human transferrin to enhance cell growth

Neonates, immunocompromised individuals, and the elderly are more likely to acquire infection. Infection can present as bacteremia and CNS infections including meningitis, encephalitis, brain abscesses, and spinal cord infections. Listeria monocytogenes is the 3rd most common cause of meningitis behind Streptococcus pneumoniae and Neisseria Meningitidis. In neonates, an in-utero infection can cause granulomatous infantisepticum leading to systemic infection and stillbirth.3 Listeria monocytogenes can also present as gastroenteritis.

References

  1. Allerberger F. Listeria: growth, phenotypic differentiation and molecular microbiology. FEMS Immunol Med Microbiol. 2003;35(3):183-189. doi:10.1016/S0928-8244(02)00447-9
  2. Bailey & Scott’s Diagnostic Microbiology – Elsevier eBook on VitalSource, 14th Edition – 9780323433792. https://evolve.elsevier.com/cs/product/9780323433792?role=student
  3. Engelen-Lee JY, Koopmans MM, Brouwer MC, Aronica E, van de Beek D. Histopathology of Listeria Meningitis. Journal of Neuropathology & Experimental Neurology. 2018;77(10):950-957. doi:10.1093/jnen/nly077

-Nicholas Taylor, DO is a 1st year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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

Microbiology Case: A 35 Year Old Male with Left Leg Cellulitis

Clinical History

A 35 year old male with chronic bilateral lower extremity lymphedema due to obesity presented with a one-week history of subjective fevers and malaise with associated left lower extremity pain, swelling and erythema. The left leg was markedly edematous with erythema present above the knee down. The leg was tender to palpation, and multiple ruptured bullae and areas of severe desquamation with excessive serous drainage were observed. Importantly, no areas of purulence were noted (Image 2). A clinical diagnosis of severe non-purulent cellulitis was made, and the patient was admitted for parenteral antibiotic therapy of vancomycin and piperacillin-tazobactam. Necrotizing fasciitis was ruled out based on imaging, and significant clinical improvement was seen after 5 days of intravenous antibiotics. The patient was transitioned to oral therapy with amoxicillin-clavulanic acid and doxycycline for a total of 14 days of antibiotics.

Laboratory Workup

During the admission, urinalysis revealed turbid urine with elevated protein (30 mg/dL), and 2+ blood with 5 RBC/HPF on microscopic examination. Given the presence of protein with microscopic hematuria, causes of glomerulonephritis were investigated. Workup revealed a markedly elevated anti-streptolysin O (ASO) titer of 5310 (0-330) and a total complement (CH50) level of 14, which was low given his age. Urine sediment examination revealed red blood cell casts (Image 3). These clinical and laboratory findings were consistent with post-streptococcal glomerulonephritis (PSGN) due to Streptococcus pyogenes skin and soft tissue infection.

 
Image 1. Colony appearance and biochemical testing of S. pyogenes. A) Typical gram positive cocci in chains characteristic of streptococci. B) Growth on Sheep’s Blood Agar of small, translucent colonies with a wide zone of beta-hemolysis indicative of S. pyogenes. C) Catalase-negative S. pyogenes (left) compared to catalase-positive S. aureus (right). D) PYR-positive S. pyogenes (left) compared to PYR-negative S. aureus (right).
Image 2. Left lower extremity at presentation.
Image 3: Red blood cell cast seen in urine sediment.

Discussion

Streptococcus pyogenes are gram positive bacteria that appear in pairs and/or chains by microscopy (Image 1A). In culture, these organisms produce relatively small colonies which elaborate a large zone of beta hemolysis on blood agar plates; colonies are translucent with smooth edges (Image 1B). The beta-hemolytic activity of S. pyogenes is due to the activity of two hemolysins: Streptolysin-S (oxygen-stabile) and Streptolysin-O (oxygen-labile). S. pyogenes is the primary organism which expresses the Lancefield Group A carbohydrate antigen. Less frequently encountered strains of S. anginosus and S. dysgalactiae subsp. equisimilis may also express this antigen, so biochemical identification of S. pyogenes may be helpful for a definitive diagnosis. MALDI-TOF MS may also fail to discriminate between S. pyogenes and closely related β-hemolytic streptococci (including S. dysgalactiae and S. canis), necessitating adjunctive biochemical testing. Like other streptococci, S. pyogenes is catalase negative (Image 1C). Unlike other beta-hemolytic streptococci, S. pyogenes expresses pyrrolidonyl arylamidase (PYR) making this test a rapid and useful adjunctive diagnostic tool (Figure 1D). Bacitracin susceptibility was used historically but has been largely replaced by PYR testing due to concerns over specificity and prolonged turnaround time.

Globally, S. pyogenes is responsible for a large percentage of infection-related morbidity and mortality. The organism colonizes the skin and the nasopharynx of humans, but most colonized individuals do not develop active disease. Colonization however can lead to infection or dissemination to susceptible individuals. S. pyogenes infections exhibit a diverse range of clinical manifestations which can include pharyngitis, impetigo, erysipelas, cellulitis, necrotizing fasciitis, pyomyositis, streptococcal toxic shock syndrome, and bacteremia. S. pyogenes remains susceptible to penicillin, making β-lactams first-line drugs of choice for management. Conversely, rising levels of macrolide, lincomycin, tetracycline, and fluoroquinolone resistance has been observed. Susceptibility testing may be warranted if these agents are to be used, most often in the cases of severe penicillin allergy.

S. pyogenes infection can be complicated by multiple post-infectious immune-mediated sequelae including PSGN and rheumatic fever. Post-Streptococcus glomerulonephritis (PSGN) has a global incidence of > 470,000 individuals per year and occurs due to the deposition of immune complexes in the glomeruli resulting from previous S. pyogenes pharyngitis or soft tissue infection (as seen in this case). Typical clinical presentation of PSGN includes hematuria, proteinuria, edema, hypertension, elevated serum creatinine levels, hypocomplementemia, and general malaise. The elevated ASO titer (5310) was diagnostic of an S. pyogenes acute infection as the cause of this patient’s cellulitis. The development of proteinuria and hematuria following infection further supports a clinical diagnosis of PSGN. Treatment of PSGN is largely supportive with the focus on management of the underlying infection. Most individuals with kidney failure from PSGN recover to baseline renal function; however, there may be a link between PSGN and the later development of chronic kidney disease/end-stage renal disease.

References

  1. De la Maza LM, Pezzlo MT, Bittencourt CE, Peterson EM. 2020. Color Atlas of Medical Bacteriology, 3rd edition. ASM Press. Pg. 11-23
  2. Madaio MP, Harrington JT. 2001. The diagnosis of glomerular diseases: acute glomerulonephritis and the nephrotic syndrome. Arch Intern Med. 161(1):Pg. 25-34. doi: 10.1001/archinte.161.1.25.
  3. Stevens DL, Bisno AL, Chambers HF, Dellinger EP, Goldstein EJC, Gorbach SL, Hirschmann JV, Kaplan SL, Montoya JG, Wade JC. 2014. Practice Guidelines for the Diagnosis and Management of Skin and Soft Tissue Infections: 2014 Update by the Infectious Diseases Society of America. Clin Infect Dis. 59(2): Pg. e10-e52, https://doi.org/10.1093/cid/ciu296.
  4. Walker MJ, Barnett TC, McArthur JD, Cole JN, Gillen CM, Henningham A, Sriprakash KS, Sanderson-Smith ML, Nizet V. 2014. Disease manifestations and pathogenic mechanisms of Group A Streptococcus. Clin Microbiol Rev. (2): Pg. 264-301. doi: 10.1128/CMR.00101-13.
  5. Wong CH, Khin LW, Heng KS, Tan KC, Low CO. 2014. The LRINEC (Laboratory Risk Indicator for Necrotizing Fasciitis) score: a tool for distinguishing necrotizing fasciitis from other soft tissue infections. Crit Care Med. 32(7): Pg. 1535-41. doi: 10.1097/01.ccm.0000129486.35458.7d.

-John Markantonis, DO is the former Medical Microbiology fellow at UT Southwestern and has recently completed his clinical pathology residency. He is also interested in Transfusion Medicine and parasitic diseases.

-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: Genotypic-to-phenotypic Discordant Results

Case History

Scenario 1: A 51 year old male with a history of diabetes, hypertension, coronary artery disease, gastric ulcer, chronic kidney disease and bilateral below knee amputation presented with epigastric pain, nausea, and vomiting. He was febrile and tachycardic. Computerized scan showed ascending/ transverse colitis and cholelithiasis. Blood cultures grew gram negative rods; the Biofire BCIDv2 panel reported Enterobacter cloacae with no genotypic, resistance markers detected. Phenotypic antimicrobial susceptibility testing (AST) from the Microscan Walkaway revealed resistance to ertapenem (>1mg/ml) but susceptibility to meropenem (£ 1mg/ml). Additionally, the isolate was resistant to 3rd-generation cephalosporins, fluoroquinolones, and intermediate-resistant to tetracyclines. Identification was confirmed by the MALDI-TOF MS upon growth on agar plates. The isolate was subbed with a meropenem disk to select for carbapenem resistance for further confirmatory testing. A Cepheid Carba-R test was ran on a sweep of the isolate growing near the carbapenem disk, which resulted in no carbapenemases detected. Results from E-tests with meropenem and ertapenem were consistent with original phenotypic result. Here, we reported the discrepant phenotypic result and genotypic results as is.

Image 1. Phenotypic testing results (E-test) for meropenem (MP,left) and ertapenem (ETP, right) of Enterobacter cloacae isolate described in scenario 1. E-test results were consistent with original phenotypic results which also identified the isolate as meropenem susceptible and ertapenem resistant. (Photo credit: Gizachew Demessie, Lead Tech, George Washington Hospital.)

Scenario 2: An 80 year old female underwent a Whipple procedure for a pancreatic mass. A wound culture was submitted from the operating room which grew both Streptococcus anginosus and Enterobacter cloacae complex. Phenotypic AST for the E. cloacae revealed susceptibility to ertapenem (≤0.5 mg/ml) but resistance to meropenem (4 mg/ml). The isolate was pan-susceptible to other drug classes (aside from intrinsic resistance). Similar to Case 1 above, identification was confirmed by the MALDI-TOF MS and the isolate was subcultured with selective pressure. A Cepheid Carba-R test did not detect any carbapenemases. However, upon repeating a phenotypic test, both ertapenem and meropenem were susceptible. Our investigation here led to the avoidance of reporting an incorrect phenotypic AST result.

Discussion

Genotype-to-phenotype discrepancies may occur in antimicrobial susceptibility testing. For example, an antimicrobial resistance (AMR) gene may be detected in a phenotypically susceptible isolate or an AMR gene may not be detected in a phenotypically resistant isolate. Such discordant results should be investigated so appropriate antimicrobial therapy is used on these patients. This leads us to an important question “What can laboratories do to solve these discrepancies?”

The first step in detection of discrepancies requires educating and teaching the lab staff to be vigilant in looking for odd susceptibility patterns (from results within a drug class and also the overall AST profile). Next, check if there was pure isolation of the organism on the purity plate; if not, each individual isolate should be subbed, identified and re-tested on both genotypic and phenotypic platforms. Of note, subbing the bacteria under selective antibiotic pressure (e.g. growing the isolate on agar plate with an antibiotic disk) can increase the potential of detecting resistance. Alternative methods (e.g. CarbaNP, mCIM, etc) could be considered if one is looking into specific resistant mechanisms. Due diligence in checking for clerical, transcription errors and contamination on equipment, especially when there is a consistent pattern of detection for a specific molecular target, is highly recommended. As such, a lab should maintain constant communication with the test manufacturer in case there are issues with batches or lots of reagents.1,2

While these rapid, genotypic panels tend to include the more common AMR mechanisms, there are still other mechanisms of resistance not on the panels. For gram negatives, AMR mechanisms such as AmpC beta-lactamases, porin mutations, efflux pumps and rare carbapenemases such as GES, IMI, and SME types are typically not included.3 Additionally, although the gene blaCTX-M is used as a marker for Extended Spectrum Beta-Lactamases (ESBL), different variants of ESBLs confer different cephalosporin (e.g. 3rd and 4th generation) phenotypes.4 A heteroresistant subpopulation, decreased or lack of expression of an AMR gene may also be potential explanations.

If a discrepancy is not resolved, it is suggested to report the isolate as resistant. If both the discrepant genotypic and phenotypic results are reported, one should consider recommending an infectious diseases consult or to contact the antimicrobial stewardship team.1 Additional information and suggested laboratory workflow can be found in Appendix H of the M100 guidelines from the Clinical Laboratory and Standards Institute.2 While molecular AMR approaches have many advantages such as a shorter turnaround time, phenotypic susceptibility testing can still offer valuable clinical information.5

  1. CLSI. Performance Standards for Antimicrobial Susceptibility Test. CLSI supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute; 2022, Edition 32
  2. Yee R, Dien Bard J, Simner PJ. The Genotype-to-Phenotype Dilemma: How Should Laboratories Approach Discordant Susceptibility Results? J Clin Microbiol. 2021 May 19;59(6):e00138-20.
  3. Tamma PD, Sharara SL, Pana ZD, Amoah J, Fisher SL, Tekle T, Doi Y, Simner PJ. 2019. Molecular epidemiology of ceftriaxone non-susceptible Enterobacterales isolates in an academic medical center in the United States. Open Forum Infect Dis 6:ofz353.
  4. Paterson DL, Bonomo RA. 2005. Extended-spectrum beta-lactamases: a clinical update. Clin Microbiol Rev 18:657–686.
  5. Dien Bard J, Lee F. 2018. Why can’t we just use PCR? The role of genotypic versus phenotypic testing for antimicrobial resistance testing. Clin Microbiol Newsl 40:87–95. 10.1016/j.clinmicnews.2018.05.003. 

Rami Abdulbaki, MD is a Pathology Resident (PGY-3) at The George Washington University Hospital. His academic interest includes hematopathology and molecular 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.

Microbiology Case Study: How to “Pin” a Diagnosis

Case History

A 7 year old female presented to the emergency department with left sided abdominal pain and a temperature of 103 degrees Fahrenheit. Labs drawn showed mild leukocytosis with a CT scan suggestive of acute appendicitis. The patient underwent uncomplicated appendectomy with no complication. Gross examination of the appendix revealed an unremarkable, non-perforated serosa and a fecalith within the lumen. Representative tissue sections submitted for microscopic analysis per grossing policy. The findings below led to the submission of the entire appendix to be evaluated.

Figure 1. Low power image of an appendix demonstrating mild acute inflammation, lymphoid hyperplasia and congestion.

Figure 2. High power image, Cross-section of an adult female E. vermicularis from the same specimen shown in Figure 1. Adherent to the appendiceal surface. Note the presence of the alae (blue arrow), and the presence of almond shaped eggs (red arrow).

Discussion

The nematode Enterobius vermicularis, widely known as the human pinworm, is one of the most common parasitic worm infections today in the United States, infecting approximately 40 million people. The patient population is often children who are infected via fecal-oral transmission, with autoinfection being common. Humans are the only known host of this nematode. Once E. vermicularis embryonated oocytes are ingested, the larvae hatch and inhabit the gastrointestinal system. At night, the larvae migrate down to the anus, lay their eggs, and the cycle recurs.

The clinical presentation can be asymptomatic or can present with perianal pruritus at night, which can be explained via the life cycle of the parasite as stated above. The method of choice for diagnosing E. Vermicularis is microscopic examination of the eggs via cellulose tape slide test. A piece of scotch tape collects the eggs near the perianal area of the patient, which is then used for analysis and identification of the eggs. Microscopically, E. Vermicularis can be identified by the spines or ‘alas’ on the surface with oval shaped, thick capsuled oocytes within, seen in figure 2. To improve the sensitivity of the scotch tape test, it is best to do this test in the early morning, when there is an increased chance of sampling the eggs.

Rarely, is this worm associated with any severe symptoms but patients can present with abdominal pain, suggesting intestinal obstruction, extra intestinal manifestations like vulvovaginitis, or appendicitis. The relationship between E. Vermicularis and appendicitis is up for debate as to whether there is a causative relationship or if it is an incidental finding seen within appendicitis. Regardless of the relationship, once a diagnosis of Enterobius vermicularis is made, treatment with an anthelmintic needs to be given to the patient, such as Albendazole or Pyrantel Pamoate. In addition, treatment for everyone in the household needs to be considered in confirmed cases of infection.

Routine surgical specimens, such as appendices, can perhaps be overlooked once acute inflammation is noted. It is important to be able to identify organisms, such as pinworms, on such specimens to get the patient the appropriate treatment.

References

  1. https://www.cdc.gov/dpdx/enterobiasis/index.html.
  2. https://www.sciencedirect.com/science/article/pii/S204908012030412X
  3. https://www.uptodate.com/contents/enterobiasis-pinworm-and-trichuriasis-whipworm?search=enterobius%20vermicularis&source=search_result&selectedTitle=1~32&usage_type=default&display_rank=1#H12

-Alexandra Medeiros, MD, is a first year anatomic and clinical pathology resident at Medical College of Georgia at Augusta University. Her academic interests include Forensic pathology, and surgical pathology.

-Hasan Samra, MD, is the Director of Clinical Microbiology at Augusta University and an Assistant Professor at the Medical College of Georgia.

Microbiology Case Study: A 44 Year Old Male Finds a Tick on His Leg

Case History A 44 year old male pulled this (image 1) off of his leg after dragging brush out of a tree line in Vermont.

Image 1. Ixodes scapularis under a microscope. Characteristic features such as eight black legs, dorsal shield, and dark red color can be appreciated.

Ixodes scapularis

Ixodes scapularis, also known as the blacklegged tick or deer tick, is commonly found in the eastern and northern Midwest regions of the United States as well as southeastern Canada. This species of tick is approximately 3 mm in length. Morphologically, females have a black head and a dorsal shield with a dark red abdomen, while males are entirely black or dark brown. Both sexes have eight black legs and a characteristic anal opening, appearing within a horseshoe-shaped ridge on the ventral lower abdomen. Unlike other tick species, Ixodes scapularis does not have ridges on the edge of the lower abdomen. Ixodes scapularis can live up to 2 years in the wild and die after reproduction.1

Life Cycle, Transmission, and Infection

Ixodes scapularis is a three-host tick with a different host at each stage of development. Their life cycle lasts approximately 2 years, where they undergo 4 distinct developmental/life stages: egg, six-legged larva, eight-legged nymph, and adult. After hatching from the egg, it should have a blood meal at every developmental stage to survive. Ixodes scapularis is known to parasitize and feed from mammals, birds, reptiles, and amphibians, and its best-known host is the white-tailed deer. This species is unable to fly or jump so it usually waits for a host while resting in the tips of grass or shrubs. Depending on the developmental stage, preparation for feeding can take between 10 minutes to 2 hours.2 Once the tick finds a feeding spot on the host, it grasps onto the skin and cuts into the surface inserting its feeding tube, which can have barbs and can secrete a cement-like surface for better attachment. Moreover, the tick can also secrete small amounts of saliva with anesthetic properties to remain undetected during the blood meal. If attached to a sheltered spot, the tick can remain unnoticed for long periods. Ixodes scapularis will attach to its host and suck on the blood for a few days. The lengthy feeding process makes them good at transmitting infection. If the host has a bloodborne infection (e.g., Lyme disease), the tick may ingest the pathogen and become infected. If the tick feeds on a human later, that human can become infected with the same pathogen if it is a prolonged blood meal. However, if the tick is removed quickly (~ 24 hours), the risk of acquiring disease is reduced.2 The longer the tick is attached, the greater the risk of becoming infected. The risk of human infection is greater during the spring and summer.

Ixodes scapularis as a Disease Vector

Babesiosis

The causative agent of babesiosis are Basebesia microti and other Babesia species. These parasites preferentially infect red blood cells. In the United States, most cases are caused by Babesia microti.3 Babesiosis is most frequently reported in the upper midwestern and northeastern regions of the United States, where Babesia microti is endemic. Although this parasite is generally transmitted by Ixodes scapularis, Babesia parasites can also be transmitted via blood transfusions and, in some cases, congenitally. Babesiosis can range from asymptomatic to life-threatening. Some of the common signs and symptoms include fever, chills, sweats, general malaise or fatigue, myalgia, arthralgia, headaches, anorexia, nausea, and dark urine. Less common symptoms include cough, sore throat, emotional lability, depression, photophobia, conjunctival infection.3 Not all infected persons are symptomatic or febrile. Clinical presentation usually manifests within several weeks after exposure, but may develop or recur months after infection. The incubation period for Babesia species parasites is approximately 1-9+ weeks. Laboratory findings associated with babesiosis include decreased hematocrit due to hemolytic anemia, thrombocytopenia, elevated serum creatinine and blood urea nitrogen values, and mildly elevated hepatic transaminase values.3 To diagnose babesiosis in the laboratory, identification of intraerythrocytic Babesia parasites by light-microscopic examination of a blood smear, positive Babesia (or Babesia microti) PCR analysis, or isolation of Babesia parasites from a whole blood specimen by animal inoculation in a reference lab are recommended procedures. Additionally, demonstration of a Babesia-specific antibody titer by indirect fluorescent antibody testing for IgG can be used as supportive laboratory criteria—although it is not enough evidence to support a diagnosis of an active infection.3 Treatment for babesia usually lasts 7-10 days with a combination of two drugs: atovaquone plus azithromycin or clindamycin plus quinine, with the latter being the standard of care for severely ill patients.

Anaplasmosis

Anaplasmosis, formerly known as Human Granulocytic Ehrlichiosis, is caused by Anaplasma phagocytophilum. Anaplasmosis is commonly reported in the upper Midwest and northeastern regions of the United States. The incubation period for Anaplasma phagocytophilum is 5-14 days.3 Some of the common signs and symptoms of anaplasmosis include fever, chills, rigors, severe headaches, malaise, myalgia, gastrointestinal symptoms such as nausea, vomiting, diarrhea, and anorexia, and, in some cases, rash. The general laboratory findings for anaplasmosis during the first week of clinical disease include mild anemia, thrombocytopenia, leukopenia, and mild to moderate elevations in hepatic transaminases.3 Under the microscope, the visualization of morulae in the cytoplasm of granulocytes during examination of blood smears is indicative of diagnosis. However, to definitely determine diagnosis in the laboratory, detection of DNA by PCR of whole blood is recommended during the first week of illness. Additionally, demonstration of a four-fold change in IgG specific antibody titer by indirect immunofluorescence antibody assay in paired serum samples is recommended. The first serum sample should be taken during the first week of illness and the second serum sample should be taken 2-4 weeks after. Moreover, immunohistochemical staining of the organism from the skin, tissue, or bone marrow biopsies is also recommended for diagnosis.3 Anaplasmosis is treated with doxycycline. Treatment should be started once there is a clinical suspicion of disease, as delaying treatment may result in severe illness or in death.

Lyme Disease

The causative agents for Lyme disease include Borrelia burgdorferi and Borrelia mayonii. Lyme disease is most frequently reported in the Upper Midwestern and northeastern regions of the United States with some cases being reported in northern California, Oregon, and Washington. Data from 2015 shows that 95% of Lyme disease cases were reported in the following 14 states: Connecticut, Delaware, Maine, Maryland, Massachusetts, Minnesota, New Hampshire, New Jersey, New York, Pennsylvania, Rhode Island, Vermont, Virginia, and Wisconsin.3 The incubation period for Borrelia parasites is usually 3-30 days.3 Some of the early (3-30 days after a tick bite) signs and symptoms of Lyme disease include fever, chills, headache, fatigue, muscle and joint aches, and swollen lymph nodes may occur with an absence of rash. Erythema migrans is a characteristic rash of Lyme disease and it occurs in 70%-80% of infected people.4 This rash starts at the site of a tick bite after an average of 3-30 days (average is 7 days) and it gradually expands over several days reaching up to 30 cm across.4 As it enlarges, it can result in the characteristic “bulls-eye” appearance; it may feel warm to the touch and it is rarely itchy or painful. Some of the later (days to months after a tick bite) signs and symptoms include severe headache and neck stiffness, additional erythema migrans rashes in other areas of the body, facial palsy, arthritis with severe joint pain and swelling—especially in the knees, intermittent pain in the tendons, muscles, joints, and bones. It may also lead to heart palpitations or Lyme carditis, episodes of dizziness or shortness of breath, inflammation of the brain and spinal cord, nerve pain, and shooting pains, numbness, or tingling of the hands and feet.4 Laboratory diagnosis for Lyme disease includes the demonstration of IgM or IgG antibodies in serum and a two-step testing protocol is highly recommended.5 Moreover, isolation of an organism from a clinical specimen is also recommended. Treatment for Lyme disease includes antibiotics such as doxycycline, cefuroxime axetil, or amoxicillin.

When assessing a patient for any tick-borne diseases, the clinical presentation should be considered alongside the likelihood that the patient has been exposed to an infected Ixodes scapularis tick, or any other tick. Moreover, if a tick is found, engorgement of the tick should be considered when assessing for the possibility of disease transmission.

References

  1. Thevanayagam S. Ixodes scapularis [Internet]. 2012. Available from: https://animaldiversity.org/accounts/Ixodes_scapularis/.
  2. Centers for Disease Control and Prevention. Lifecycle of Blacklegged Ticks [Internet]. 2011 [updated November 15, 2011]. Available from: https://www.cdc.gov/lyme/transmission/blacklegged.html.
  3. Centers for Disease Control and Prevention. Tickborne Diseases of the United States: A Reference Manual for Healthcare Providers [Internet]2018. Available from: https://www.cdc.gov/ticks/tickbornediseases/TickborneDiseases-P.pdf.
  4. Centers for Disease Control and Prevention. Lyme Disease – Signs and Symtoms [Internet]. 2021. Available from: https://www.cdc.gov/lyme/signs_symptoms/index.html.
  5. Mead P, Petersen J, Hinckley A. Updated CDC Recommendation for Serologic Diagnosis of Lyme Disease. MMWR Morb Mortal Wkly Rep. 2019;68(32):703. Epub 2019/08/16. doi: 10.15585/mmwr.mm6832a4. PubMed PMID: 31415492; PubMed Central PMCID: PMCPMC6818702 potential conflicts of interest. No potential conflicts of interest were disclosed.

Amelia Lamberty is a Master’s student in the Pathology Master’s Program.

-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 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.

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.