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

  1. Hardy Diagnostics. Campylobacter [Internet]. 2016. Available from: https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/Campylobacter.htm#:~:text=In%20general%2C%20Campylobacter%20spp.%20appear%20as%20gray%2C%20flat%2C,glistening%2C%20with%20little%20spreading.%20Campylobacter%20spp.%20are%20non-hemolytic.
  2. Perez-Perez GI, Blaser MJ. Campylobacter and Helicobacter. In: Baron S, editor. Medical Microbiology. Galveston (TX): University of Texas Medical Branch at Galveston Copyright © 1996, The University of Texas Medical Branch at Galveston.; 1996.
  3. Centers for Disease Control and Prevention. Campylobacter (Campylobacteriosis) For Health Professionals [Internet]. 2019 [updated December 23, 2019]. Available from: https://www.cdc.gov/campylobacter/technical.html.
  4. Ehrenpreis ED. Campylobacter infection [Internet]. Epocrates2022 [updated January 22, 2022]. Available from: https://online.epocrates.com/v2/print/disease/1175?subSectionId=11#:~:text=Bacteria%20of%20the%20genus%20Campylobacter%20cause%20a%20variety,%5B%203%5D%20There%20are%20many%20species%20of%20Campylobacter.
  5. Centers for Disease Control and Prevention. Campylobacter (Campylobacteriosis) Symptoms [Internet]. 2019. Available from: https://www.cdc.gov/campylobacter/symptoms.html.
  6. Reddy KR, Farnum JB, Thomas E. Acute hepatitis associated with campylobacter colitis. J Clin Gastroenterol. 1983;5(3):259-62. Epub 1983/06/01. doi: 10.1097/00004836-198306000-00013. PubMed PMID: 6863882.
  7. Humphrey KS. Campylobacter infection and hepatocellular injury. Lancet. 1993;341(8836):49. Epub 1993/01/02. doi: 10.1016/0140-6736(93)92521-t. PubMed PMID: 8093289.
  8. Vermeij CG, van Dissel JT, Veenendaal RA, Lamers CB, van Hoek B. Campylobacter jejuni peritonitis in a patient with liver cirrhosis. Eur J Gastroenterol Hepatol. 1996;8(12):1219-21. Epub 1996/12/01. doi: 10.1097/00042737-199612000-00016. PubMed PMID: 8980944.
  9. Korman TM, Varley CC, Spelman DW. Acute hepatitis associated with Campylobacter jejuni bacteraemia. Eur J Clin Microbiol Infect Dis. 1997;16(9):678-81. Epub 1997/11/14. doi: 10.1007/bf01708559. PubMed PMID: 9352262.
  10. Yoon JG, Lee SN, Hyun HJ, Choi MJ, Jeon JH, Jung E, et al. Campylobacter jejuni Bacteremia in a Liver Cirrhosis Patient and Review of Literature: A Case Study. Infect Chemother. 2017;49(3):230-5. Epub 2017/06/14. doi: 10.3947/ic.2017.49.3.230. PubMed PMID: 28608661; PubMed Central PMCID: PMCPMC5620392

-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.

Hematology Case Study: An Unusual case of Leukemic Reticuloendotheliosis (aka Hairy Cell Leukemia)

Leukemic Reticuloendotheliosis (LRE) is a term that was first used in 1923 but is a name that most of us would not recognize today. In 1958, Bournocle et al. published a paper that characterized LRE as a separate clinical disorder and described the clinical course, pathologic features, treatment options and prognosis. The study also described an unusual morphology of the malignant cells seen in this condition. The malignant cells were noted to be small mononuclear cells with projections around the circumference of the cytoplasm. Another decade went by before these cells were given the nickname “hairy cells”. At the time, though LRE was considered a fatal disease, splenectomy appeared to be a beneficial treatment, thus pointing to a lymphocytic disorder. Later, in 1976, an article was published that suggested that these hairy cells were monocytic rather than lymphocytic in origin. The true lineage of these hairy cells was unknown until the development of newer immunophenotypic methodologies in the mid to late 1970s. Today, hairy cell leukemia (HCL) is considered a rare, chronic B cell leukemia that comprises 2% of lymphoid leukemias and responds well to therapy.

Patients may be entirely asymptomatic at diagnosis, and the finding of hairy cells on the peripheral smear from a routine CBC prompts further investigation. Patients do not usually require treatment at diagnosis, and many patients live a normal lifespan. Originally, diagnosis was based on clinical and laboratory result correlation: CBC results, observation of the characteristic hairy cells, and splenomegaly. One of the first tests used for  diagnosis of HCL was tartrate-resistant acid phosphatase activity (TRAP stain). Today, standard practice is immunophenotyping by flow cytometry. HCL is characterized by the expression of B-cell antigens CD19, CD20, and CD22 in addition to bright CD11c expression with CD103, CD25, CD123 and Annexin A1 (ANXA1) co-expression. Annexin A1 is the most specific immunohistochemical marker for HCL. In 2011, the BRAF-V600E mutation was identified as the genetic causal event of HCL, allowing even more advances in the diagnosis and therapy for HCL.

As the disease progresses, most patients experience increasing cytopenia, including monocytopenia, and persistent splenomegaly. Treatment is usually started when a patient meets certain guidelines, which include a severe cytopenia or pancytopenia, malignant lymphocytosis, increased susceptibility to infection or symptomatic splenomegaly. Historically, the only available treatment was splenectomy. In the 1980’s, interferon therapy was introduced and was able to induce partial responses in some patients. In the 1990’s the purine analogs, cladribine or pentostatin, became available as the preferred first line treatment for HCL. Treatment response is good and offers prolonged remission rates. For patients who experience relapse, rituximab may be used in combination with a purine analog. Most recently, anti-CD22 immunotoxins and molecular targeted therapy with BRAF inhibitors have been introduced for cases that do not respond to other therapies.

Additional discoveries into the biology of the disease have identified new subtypes of HCL. It is important to distinguish between classic HCL and Hairy Cell leukemia variant (HCLv) because they are treated differently. HCLv may be more aggressive and does not respond well to purine analogs alone. HCLv is often diagnosed at older age than classic HCL In HCLv the WBC is often elevated, with lymphocytosis, and there is a lack of monocytopenia. The hairy cells seen on a peripheral blood smear may be more abundant than in classic HCL. These HCLv cells also often have a distinct nucleolus not seen in HCL cells. As well, these cells can have a morphology that appears to be somewhere between prolymphocytes and hairy cells. Unlike HCL, HCLv cells are negative for CD25 and BRAF-V600E. HCLv represents only about 10% of HCL cases. Because of its rarity, and the gray areas surrounding differential diagnosis between HCL and HCLv, studying these rare cases can help lead to a better understanding and management of both HCL and HCLv patients.

About 1200 new cases of HCL are diagnosed each year in the US. HCL is 4-5 times more common in males, with a median age at diagnosis of 55-60. This is an unusual case because the patient is female, was older at diagnosis, with no cytopenia or splenomegaly noted. This patient is a 79-year-old female who, one year ago, was referred to a Hematology Oncology practice with a several year history of a mildly elevated WBC with increased lymphocytes, without absolute lymphocytosis. She was referred after a peripheral smear exhibited prolymphocytes and the “hairy’ appearing lymphocytes shown below in Image 1.

Image 1. Hairy Cells seen on peripheral blood smear.

Peripheral blood was sent for myeloid/lymphoid disorders and acute leukemia analysis by flow cytometry. Remarkable in this case were the results of the flow cytometry studies. Flow cytometry performed on the peripheral blood revealed 2 distinct morphological populations of lymphocytes. The majority of lymphs appeared to be small, with scant cytoplasm, round nucleus, clumped chromatin, and inconspicuous nucleoli. These cells were identified as a monoclonal kappa restricted B cell population exhibiting co-expression of CD23 and CD5, consistent with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). A second population of lymphs were larger, with more abundant granular cytoplasm and hairy projections, large nuclei, condensed chromatin, and inconspicuous nucleoli. This second population displayed CD20 expression and was positive for CD11c, CD103 and FMC-7. CD25, CD5 and CD23 were negative.

The immunophenotyping of this second population of cells suggests a diagnosis HCL; or is it suggestive of HCLv? The patient was older at diagnosis, leukocytosis and lymphocytosis are present, and monocytopenia is absent. Hairy cells were over 8% of the differential, though lacking the distinct nucleoli of HCLv. Prolymphocytes were noted. CD25 was negative in this patient and is usually exhibited in HCL.

An immunological scoring system for HCL has been proposed with one point given to each of markers for CD11c, CD103, CD123 and CD25. One point is given if the marker is expressed and no point when it is not expressed. A score of 3 or 4 is observed in 98% of cases of HCL and is usually 0-1 in other HCL-like disorders. This patient’s cells showed expression of CD11c and CD103, was CD25 negative and CD123 was not evaluated so would score at least a 2, which puts her somewhere in an inconclusive score. Additionally, a bone marrow biopsy has not been done and there therefore results for TRAP or annexin A1 immunostaining, or BRAF-V600E mutations are not available.

With a diagnosis of a B-lymphocytosis consistent with CLL/SLL and a simultaneous HCL, or HCLv, this patient is an interesting case. Several articles and reviews in literature of other patients with CLL and HCL give further insight into the biology of HCL. Literature suggests that concurrent HCL and CLL may indicate a common origin. Patients with HCL may subsequently develop CLL, which can mimic a relapse of HCL. Therapy requires treating each case individually and watchful waiting in asymptomatic cases. Rituximab with or without purine analogs have been useful to treat both disorders simultaneously.

Table 1. CBC results from a patient in 2022 and 2022.

This patient at 1 year following diagnoses has developed a mildly increasing lymphocytosis and is being monitored. Both her CLL/SLL and HCL still appear to be in the indolent, “wait and see” stage. The patient has declined further workups at this time.

References

  1. Bain, Barbara J. Blood Cells: A Practical Guide. 5th ed. Wiley Blackwell, 2015. Print.
  2. Chang-Hun Park, Hyun-Young Kim, M.D.et al. Efficacy of Annexin A1 Immunostaining in Bone Marrow for the Diagnosis of Hairy Cell Leukemia. Laboratory Medicine Online 2019; 9(4): 236-241
  3. Falini B, Tiacci E. New treatment options in hairy cell leukemia with focus on BRAF inhibitors. Hematol Oncol. 2019; 37(Suppl. 1): 30– 7..Maitre, E.; Cornet, E.; Troussard, X. Hairy cell leukemia: 2020 update on diagnosis, risk stratification, and treatment. Am. J. Hematol. 2019, 94, 1413–1422.
  4. Obiorah IE, Francischetti IMB, Wang HW, Ahn IE, Wang W, Raffeld M, Kreitman RJ, Wiestner A, Calvo KR. Concurrent chronic lymphocytic leukemia/small lymphocytic lymphoma and hairy cell leukemia: clinical, pathologic and molecular features. Leuk Lymphoma. 2020 Dec;61(13):3177-3187.
  5. Scheinberg M, Brenner AI, Sullivan AL, Cathcart ES, Katayama I. The heterogeneity of leukemic reticuloendotheliosis, “hairy cell leukemia”. Evidence for its monocytic origin. Cancer. 1976 Mar;37(3):1302-7
  6. Shao, Haipeng et al. “Distinguishing hairy cell leukemia variant from hairy cell leukemia: development and validation of diagnostic criteria.” Leukemia research vol. 37,4 (2013)
  7. Verma V, Giri S, Bhatt VR, Amador-Ortiz C, Armitage JO. Synchronous or Metachronous Hairy Cell Leukemia and Chronic Lymphocytic Leukemia: A Case Series and Literature Review. Front Oncol. 2017 Jan 9;6:270.
  8. X. Troussard, M.R. Grever. The revised guidelines for the diagnosis and management of hairy cell leukaemia and the hairy cell leukaemia variant. r J Haematol, 193 (1) (2021), pp. 11-14
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 69 Year Old Man with Chronic Cutaneous Disease

Case Description

A 69 year old man with hepatitis B and chronic cutaneous Rosai-Dorfman disease presented to the dermatology clinic for regular follow-up. He was being treated with subcutaneous injection of methotrexate every other week and intralesional Kenalog (ILK) injections for individual lesions. The patient presented with a new complaint of a painful nodule on his left thumb where he was stuck with a splinter two months prior. He denied fever, chills, weight loss, or other systemic symptomology. Upon physical examination, an erythematous nodule on the lateral left thumb with central pallor and crusting consistent with a foreign body was observed (Figure 1). Surgical excision was recommended.

Figure 1. Photograph of a nodular lesion on the lateral aspect of the left thumb with surrounding erythema and central pallor, which was described as painful, and had been present for two months following traumatic splinter implantation.

Following excisional biopsy, histopathology revealed a relatively circumscribed lesion with suppurative granulomatous dermatitis and numerous pigmented hyphae observed on hematoxylin and eosin stained slides (H&E; [Figures 2-3]). A diagnosis of phaeohyphomycosis was made; the patient’s methotrexate was held and an infectious disease (ID) consult was placed. Precautionary blood cultures were drawn which remained negative following five days of incubation. The patient was started on a course of empiric oral doxycycline for two weeks which he completed. At presentation for ID follow up, the patient felt well and denied constitutional symptoms or recurrence of the thumb lesion. Physical exam revealed no associated sporotrichoid lesions (lymphocutaneous spread of infection) or palpable lymphadenopathy. ID recommended a 3-month course of oral itraconazole as secondary prophylaxis, which was completed without adverse effects or recurrence of symptoms.

Figure 2. Hematoxylin and Eosin (H&E) stained excisional biopsy demonstrating a relatively circumscribed lesion with suppurative granulomatous inflammation (100x magnification). Thick arrows highlight lavender staining epithelioid-histiocytes that comprise the bulk of the granuloma and arrowheads point to the admixed aggregates of dark pink and purple staining neutrophils, giving this granuloma its suppurative nature.
Figure 3. High-power magnification photomicrograph of the lesion with a close-up view of the suppurative nature of this granulomatous inflammation with small arrows highlighting the numerous cross-sections of hyphae that demonstrate melanized pigment observed in this case of phaeohyphomycosis (H&E stain; 400x magnification).

Case Discussion

Phaeohyphomycosis describes a constellation of clinical syndromes caused by infection with a broad group of “dematiaceous” or “melanized” molds and some pigmented yeasts.1 Many of these organisms are ubiquitous in the environment though some are more selective in their habitat, restricting the likelihood of infection to specific geography or select patient populations.2,3 Despite significant microbiological diversity, a unifying characteristic of dematiaceous molds is the production of the pigment melanin. Melanin is theorized to serve as a virulence factor, as loss of melanization often results in attenuation.3,4  In contrast to other diseases caused by dematiaceous molds with more defined etiologies and presentations (e.g., eumycetoma, chromoblastomycosis), manifestations of phaeohyphomycosis are highly variable and can include keratitis, cutaneous disease, pulmonary infection, central nervous system penetration and/or disseminated disease.

Laboratory diagnosis of phaeohyphomycosis is reliant on histopathological evaluation, as surgical debridement is often necessary for management. In this setting, darkly pigmented, septate hyphae invading tissue in a nonspecific background of inflammation may be observed.1 H&E staining is generally sufficient to confirm diagnosis; however, special stains that can highlight fungi, namely Grocott-Gomori methenamine silver (GMS) or periodic acid-Schiff (PAS) stains, can outline the presence of hyphal elements. Additionally, melanin production can be highlighted using Fontana-Masson staining. Careful evaluation and interpretation of fungal cultures, when collected, are important as results can be complex given the ubiquitous nature of many etiological agents, particularly from non-sterile anatomical sites. Additionally, there are no alternate methods routinely available to aid in diagnosis, outside of culture, to specifically identify etiologic agents of phaeohyphomycosis.3 Importantly, optimal antifungal therapy for these infections remains unclear due to a lack of randomized control trials and relative infrequency of presentation.

Superficial infections, such as the one described in this case, are generally considered to be consequences of local trauma, and exhibit minimal tissue invasion. However, in the setting of the immunocompromised host or immunosuppression, disseminated infection can occur.3 The prognosis of invasive phaeohyphomycosis is poor, exhibiting a mortality is as high as 10% for deep local infections and 50% for disseminated disease.1 This patient’s advanced age and chronic immunosuppression were cause for great concern. Fortunately, the biopsy demonstrated granuloma formation effectively localizing the infection to the subcutaneous tissue of the thumb. The patient has remained free of further disease to date, suggestive of a curative surgical resection.

References

  1. Arcobello, JT, Revankar, SG. Phaeohyphomycosis. Respiratory and Critical Care Medicine. 2020. DOI: 10.1055/s-0039-3400957
  2. Wong EH, Revankar SG. Dematiaceous Molds. Infectious Disease Clinics of North America. 2016. 10.1016/j.idc.2015.10.007
  3. Revankar, SG., Baddley, JW., Chen, S.C-A., Kauffman, CA., Slavin, M., Vazquez, JA, Seas, C., Morris, MI., Nguyen, MH et. al. A Mycoses Study Group International Prospective Study of Phaeohyphomycosis: and Analysis of 99 Proven/Probable Cases. Open Forum Infectious Diseases. 2017. DOI: 10.1093/ofid/ofx200
  4. Sharkey PK, Graybill JR, Rinaldi MG, Stevens DA, Tucker RM, Peterie JD, Hoeprich PD, Greer DL, Frenkel L, Counts GW, et al. Itraconazole treatment of phaeohyphomycosis. Journal of the American Academy of Dermatology. 1990. doi: 10.1016/0190-9622(90)70259-k

-Kevin Burningham is a 4th year medical student at UT Southwestern Medical School.

-Dominick Cavuoti is a Professor at UT Southwestern Medical Center who specializes in Cytopathology, Infectious Disease pathology and is a medical director of the Microbiology laboratory at Parkland Health and Hospital System.

-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 Elderly Women Presents with Altered Mental Status, Fever, and Gastrointestinal Symptoms

Case Description

An elderly woman presented to the emergency department with sudden onset diarrhea, fever and altered mental status for 1 day. On admission, she had fever with chills, tachycardia, tachypnea, and diminishing mental status. Her white blood cell (WBC) count was slightly elevated from baseline with neutrophilia and thrombocytopenia. Blood and CSF were sent for bacterial culture. Stool was sent for C. difficile PCR and occult blood, which came back negative.

Organisms were recovered from the aerobic blood culture bottle. Gram stain showed gram positive short rods. A multiplex PCR was run directly from the blood, which detected Listeria monocytogenes. The care team was notified immediately. The organism grew on blood agar and aerobic Columbia Naladixic Acid Agar (CNA agar) with characteristic beta hemolysis. The identification was re-confirmed by MALDI-TOF MS as Listeria monocytogenes.

Image 1. Listeria colonies with beta-hemolysis on blood agar

Discussion

Listeria monocytogenes is an intracellular Gram positive rod that is pathogenic to humans. The organism is a non-spore forming facultative anaerobe which thrives at low temperature and can survive at low pH and high salt concentration, making it an organism of concern in ready to eat- refrigerated food products, including soft cheese, deli meat, and packaged salads. There have been multiple outbreaks of Listeria infection related to certain these food products. The most recent Listeria outbreaks, as per the CDC’s report December 2021, has been linked to Dole packaged salads.

The primary route of infection with Listeria is oral ingestion. The amount of inoculum ingested is responsible for the degree of severity of infection. Most people get noninvasive gastroenteritis, which is self-limited, and recovery typically occurs within a week of infection with supportive treatment. Invasive infection is mostly seen in immunocompromised people, people with hematologic malignancies, chronic illnesses like diabetes, pregnant individuals, and extremes of ages. Infected pregnant woman can transmit the infection to fetus vertically, which can lead to fetal demise. Because of this, pregnant women are advised to avoid foods where Listeria is often found, such as deli meat. The infection rate in US currently is 24 cases per thousand and around 800 cases are reported annually, this number does not include the cases that are unreported which is most likely comprised of the noninvasive gastroenteritis cases.1,2,5

Invasive disease can range from severe form of gastroenteritis to meningoencephalitis. The fetal infection with Listeria is known to result in the most severe form of outcomes ranging from granuloma infantisepticum to fetal demise in utero. Bacteria enter the gastrointestinal tract through the intestinal lining. Upon entry, the bacteria travel intracellularly through the lamina propria into the vascular system and get disseminated throughout the body including the brain and placenta in pregnant woman.1,2,5


Bacterial culture remains the gold standard, of which blood culture is the most sensitive test when it comes to invasive diseases. Blood culture takes about 24 hours to grow the organism. Listeria can be cultured in media containing horse, sheep or rabbit blood. Listeria produces smooth, round, translucent colonies with a narrow zone of beta- hemolysis. Listeria can be morphologically difficult to differentiate from other Gram positive rods. In these cases, simple biochemical tests can help identify the organism. For example, catalase test, esculin test, oxidase test that are typically positive in case of Listeria, while H2S and indole are not produced by the organism and urea and gelatin are not hydrolyzed. Additionally, hanging drop method can help demonstrate the characteristic tumbling motility of the organism. With motility agar, Listeria demonstrates an umbrella motility pattern. Direct identification of organism from a positive blood culture, CSF or tissue specimen by using PCR assays should be performed, when available, and is particularly useful in patient who have undergone antimicrobial therapy. There is microarray based nucleic acid test available that can identify Listeria from blood culture within 3 hours. MALDI-TOF mass spectrometry is an efficient assay for rapid identification of L. monocytogenes once an organism is recovered in culture.2.6

For the treatment of severe listeriosis in at risk populations, ampicillin is commonly used and aminoglycosides can be added for synergy. Trimethoprim/sulfamethoxazole (TMP/SMX) can be given in case of Beta-lactam allergy. Listeria is intrinsically resistant to cephalosporins and therefore these agents should not be used for therapy. In case of suspected treatment failures, antimicrobial susceptibility testing can be performed as per the available CLSI guidelines, which provides susceptibility breakpoints for penicillin, ampicillin, TMP/SMX and meropenem.2,6 Our patient received ampicillin along with gentamicin for her symptoms and recovered well.

References

  1. Radoshevich, L., Cossart, P. Listeria monocytogenes: towards a complete picture of its physiology and pathogenesis. Nat Rev Microbiol 16, 32–46 (2018). https://doi.org/10.1038/nrmicro.2017.126
  2. https://www.cdc.gov/listeria/index.html
  3. Bierne, H. & Cossart, P. When bacteria target the nucleus: the emerging family of nucleomodulins. Cell. Microbiol. 14, 622–633 
  4. Boujemaa-Paterski, R. et al. Listeria protein ActA mimics WASP family proteins: it activates filament barbed end branching by Arp2/3 complex. Biochemistry 40, 11390–11404 (2001)
  5. Lancet Infect Dis. 2017;17(5):510. Epub 2017 Jan 28
  6. Nele Wellinghausen
  7. , 2019. Listeria and Erysipelothrix, Manual of Clinical Microbiology, 12th Edition. ASM Press, Washington, DC. doi: 10.1128/9781683670438.MCM.ch28

-Kritika Prasai, MD. is a PGY-1 Anatomic and Clinical Pathology resident at University of Chicago (NorthShore). 

-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: Worm Seen in Toddler’s Stool

Case History

A worm specimen—as shown in Image 1—was found in a stool sample from a 21 month old, otherwise healthy female.

Image 1. Specimen collect from patient’s stool.

Discussion

The worm specimen in question is Ascaris lumbricoides, the largest of the nematode parasites. Females typically measure 20-35 cm long with straight tails, while males are smaller at 15-31 cm with curved tail.1 A characteristic feature in adults of both sexes are the three “lips” at the anterior end of the body, as shown in Image 2.

Image 2. Close up of the anterior end of an adult A. lumbricoides. Three “lips” are highlighted with a black arrow.

Humans are the definitive host for these roundworm parasites. Infection with these soil-transmitted helminths is quite common, with an estimated 807 million to 1.2 billion people affected.2,3 Children are infected much more frequently than adults.4 Nearly all A. lumbricoides cases occur in tropical and subtropical areas of Asia, sub-Saharan Africa, and the Americas. This infection is rare or absent in developed countries, but sporadic cases may occur in rural regions.3

Individuals affected with adult Ascariasis worms usually show no acute symptoms. However, since these worms are commonly situated in the small intestines, the clinical presentation of a heavy worm burden in children might include stunted growth via malnutrition. In both adults and children, a high worm burden may result in abdominal pain and intestinal obstruction leading to potential perforations. Migrating worms may lead to symptomatic occlusion of the biliary tract, appendicitis, or nasopharyngeal expulsion.3

In the clinical setting and for diagnosis, A. lumbricoides eggs should be found in the feces, juvenile worms in the sputum, and in some cases adults in the feces. For deworming, the recommended treatment are anti-helminthic medications such as albendazole and mebendazole.3 These medications kill the adults, but not the migrating larvae thus repeat treatment might be needed.

References

  1. Centers for Disease Control and Prevention. DPDx – Laboratory Identification of Parasites of Public Health Concern. Internet [updated July 19, 2019]. Available from: https://www.cdc.gov/dpdx/ascariasis/index.html.
  2. Jourdan PM, Lamberton PHL, Fenwick A, Addiss DG. Soil-transmitted helminth infections. Lancet. 2018;391(10117):252-65. Epub 2017/09/09. doi: 10.1016/s0140-6736(17)31930-x. PubMed PMID: 28882382.
  3. Centers for Disease Control and Prevention. Parasites – Ascariasis. Internet [updated November 23, 2020]. Available from: https://www.cdc.gov/parasites/ascariasis/index.html.
  4. Veesenmeyer AF. Important Nematodes in Children. Pediatr Clin North Am. 2022;69(1):129-39. Epub 2021/11/20. doi: 10.1016/j.pcl.2021.08.005. PubMed PMID: 34794670.

-Amelia Lamberty is a Masters Student in the Department of Pathology and Laboratory 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

Microbiology Case Study: A Middle-Aged Female with Fever, Chills, Night Sweats, and Syncope

Case History

A middle-aged female presented to the emergency department after experiencing a fall and loss of consciousness due to syncope. Upon presentation, the patient endorsed an almost four-week history of fevers, chills, abdominal discomfort, night sweats, and dizziness. She also reported poor oral intake and recent unintended weight loss since the onset of her symptoms. When asked, she noted she had returned from a month-long trip to Italy and Ghana two months prior to presentation. She initially presented to an outside hospital with generalized weakness, body aches, and a fever where she was treated with antibiotics for a urinary tract infection. She then presented to a different outside hospital with similar symptoms. There, she confirmed she had not taken malaria prophylaxis and was bitten by mosquitos on her recent trip. Blood was taken for a peripheral blood smear review but no Plasmodium sp. were observed.

At her current presentation, the patient denied a history of seizures but continued to endorse recurrent fevers, malaise, nausea, and vomiting. She was mildly tachycardic, afebrile, and bloodwork revealed normocytic anemia (hemoglobin 10.3), and elevated creatinine. Given the uncertainty surrounding her syncopal episode, the patient was admitted for further workup. After admission, she spiked a fever up to 103°F and the infectious disease service was consulted. As part of her workup, blood was again drawn for Giemsa-stained peripheral blood smears which were read in the microbiology laboratory.

Laboratory Identification

Upon receipt of the patient’s blood, Giemsa-stained thick and thin smears and an immunochromatographic assay for the detection of malarial antigens (BinaxNOW® Malaria, Abbott Laboratories, Abbott Park, IL) were performed. The BinaxNOW® assay was positive for the detection of pan-malarial antigen (T2), but not the histidine-rich protein II antigen specific to P. falciparum (T1). These findings were suggestive of infection with a non-falciparum Plasmodium species (Image 1). Analysis of the Giemsa-stained thin smear revealed several Plasmodium parasites at various stages of development. Importantly, parasites (and particularly ring forms) were only rarely encountered (Image 2, Image 3A). “Basket” (Image 3B) and “Band” (Image 3C) trophozoite forms were observed, as well as schizonts with 6-12 merozoites typical rosette patterns around central pigment (Image 3D). In the context of a positive antigen test, the patient was definitively diagnosed with a Plasmodium malariae infection based on morphology with a calculated parasitemia of less than 0.1%.

Image 1. BinaxNOW® Malaria assay.  This patient’s assay was positive for the common malarial antigen (T2), but the histidine-rich protein II antigen (T1) specific to P. falciparum was not detected.  These results suggest an infection with a non-falciparum Plasmodium species.
Image 2. Developing ring-form trophozoites of P. malariae.  Ring form trophozoites of P. malariae are less-frequently encountered in peripheral smears compared to other Plasmodium species that infect humans.  A) P. malariae rings usually have a single chromatin dot and are generally thicker than that of P. falciparum.  B) As rings develop, the cytoplasm can extend across the cell or can appear with vacuolation leading to “band” or “basket” forms, respectively.
Image 3Gimesa-stained thin smear of erythrocytes infected with P. malariae A)  CellaVision® field with rare infected erythrocytes notated by black arrowheads.  B) “Basket” form trophozoite of P. malariae.  C) “Band” trophozoite of P. malariae.  D) Schizont of P. malariae with 6-12 merozoites surrounding central pigment in a characteristic “rosette”.

Discussion

Plasmodium malariae is one of the five species of Plasmodium (along with P. falciparum, P. vivax, P. ovale and P. knowlesi) which cause human malaria. Infection begins when sporozoites are injected from the salivary glands of the female Anopheles mosquito into the host upon taking a blood meal. Sporozoites migrate to the liver where they infect hepatocytes and develop into schizonts which eventually rupture, releasing infectious merozoites. These merozoites enter the circulation and infect erythrocytes, subsequently developing into immature ring form trophozoites (Image 2A). Ring form trophozoites develop into either mature trophozoites or become gametocytes which can be taken up by another mosquito upon feeding (Image 2B). Mature P. malariae trophozoites adopt unique morphologies not seen with other Plasmodium species including “band” (Image 3B) and “basket” (Image 3C) forms. Mature trophozoites then develop into schizonts (Image 3D) which rupture, releasing 6-12 merozoites which perpetuate the erythrocytic cycle of infection. P. malariae elaborates fewer merozoites than other Plasmodium species which are often arranged in a “rosette” pattern around centrally localized pigment in the schizont (Image 3D).

The P. malariae infectious cycle has several unique hallmarks compared to that of other Plasmodium species. Unlike P. vivax and P. ovale, the P. malariae lifecycle does not include a latent hypnozoite form, and thus is devoid of classical relapse. P. malariae also preferentially infects older erythrocytes, as opposed to P. vivax which prefers younger cells. Additionally, the infected erythrocyte does not enlarge or fimbriate when infected with P. malariae as opposed to P. vivax and P. ovale, respectively. Patterns of erythrocyte infection and lysis lead to elevated parasite burden, characteristic cyclic fevers and anemia. However, the time needed for development from ring trophozoite to rupturing schizont is different among malarial parasites: P. knowlesi exhibits the most rapid development (24-hours), followed by P. falciparum, P. ovale, and P. vivax (48-hours), and then P. malariae (72-hours).  

P. malariae has a global distribution overlapping with P. falciparum. While P. falciparum is the primary species causing reported infection in Ghana, P. malariae infection is encountered less frequently. Associated parasitemia are characteristically lower in P. malariae infections compared to other species due to fewer merozoites produced during infection, an extended 72-hour developmental cycle, and the preference for the infection of older erythrocytes. This can complicate microscopic diagnosis as well as lead to more indolent symptomology. Indeed, patients can often remain asymptomatic for months to years after leaving endemic areas. In this patient’s case, definitive diagnosis was made months following her travel to an endemic region. The patient completed a 5-day course of artemether/lumefantrine with complete resolution of symptoms prior to discharge.

-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: A Preteen Boy with Cold and Cough

Case History

A preteen boy presented to primary care office with a complaint of flu-like symptoms for the past five weeks. His symptoms improved after 2-3 weeks but noted acute worsening of symptoms in the last two weeks, including sore throat, head congestion, and cough. The physical exam was unremarkable except for nasal congestion, mucosal edema, and some drainage. A chest X-ray was taken, which was normal. Results were negative for a Streptococcal infection, SARS-CoV-2, Bordetella pertussis, and influenza. Bordetella parapertussis was detected by PCR (Image 1).

Image 1. Bordetella pertussis and Bordetella parapertussis PCR. Green = IS481, target gene for B. pertussis. Red = IS1001, target gene for B. parapertussis. Purple = Internal control (IC)

Discussion

Bordetella is a small, non-fermentative, gram negative coccobacilli. The genus Bordetella has 15 species, and B. pertussisB. parapertussis are most commonly found in human infections causing pertussis. B. parapertussis usually cause milder disease, but reports of outbreaks of B. parapertussis have increased in recent literature. The epidemic cycles for pertussis occur at 3–4 years intervals2 and pertussis vaccination does not prevent B. parapertussis infection. B. parapertussis generally occurs in a younger age group than disease caused by B. pertussis.4 Cherry et al. indicated that B. parapertussis infections contribute significantly to the disease burden, which was previously thought to be vaccine failure in children.2

Pertussis is primarily a toxin-mediated disease; the bacteria attach to the cilia of the respiratory epithelial cells and produce toxins that paralyze the cilia and cause inflammation of the respiratory tract, which interferes with the clearing of pulmonary secretions.1 B. pertussis and B. parapertussis are almost identical at the DNA level and produce many similar virulence factors like as filamentous hemagglutinin (FHA), pertactin, tracheal cytotoxin, dermonecrotic toxin, and adenylate cyclase-hemolysin. An essential difference between the two is that B. parapertussis does not secrete pertussis toxin.3,5-9 Despite the high degree of homology shown by the amino acid sequences of the main antigens, the two species differ in respect to several protective epitopes.10

Pertussis (whooping cough) can cause serious illness in babies. Symptoms of pertussis usually develop within 5-10 days of exposure. Early non-specific symptoms, including runny nose, low-grade fever, and occasional cough, can last for 1 to 2 weeks. After 1 to 2 weeks, as the disease progresses, paroxysms occur, which are many, rapid coughs followed by a high-pitched “whoop” sound. Vomiting or exhaustion develops at this stage. Recovery from pertussis is slow, the cough becomes milder and less common, but coughing fits can return with other respiratory infections for many months after the pertussis infection started. The “whoop” is often absent or mild in less severe disease. The illness is generally milder in teens and adults, especially those who have gotten the pertussis vaccine. The cough can be minimal or absent in babies, but they might get apnea, which is most dangerous.1

Bordetella is a fastidious organism as it requires special media, prolonged incubation, timely transport, and rapid plating for recovery of the organism. Regan low and Bordet Gengou are the special media used for culture of B. parapertussis and B. pertussis. Unlike B. pertussis, B. parapertussis can grow on blood and chocolate agar. Colonies may appear like mercury drop and produce beta hemolysis on prolonged incubation. Culture has the highest recovery if a nasopharyngeal swab is collected within two weeks of symptom onset. Sensitivity can be as high as 56% in early disease and decrease over time, while specificity is 100%.1 Serological assay are not clinically validated and do not help differentiate between recent or remote infection or vaccination. PCR is the most sensitive methodology and should be performed from a nasopharyngeal swab taken within three weeks of symptom onset; after the fourth week of cough, the amount of bacterial DNA rapidly diminishes, which increases the risk of obtaining falsely-negative results. PCR-detectable B. pertussis DNA in some pertussis vaccines and the contamination of the clinic environment by those vaccines increases the risk of false-positive PCR. As per CDC guidelines, PCR in asymptomatic persons, asymptomatic close contacts of a confirmed case, and after five days of antibiotic use is unlikely to benefit and is generally not recommended because of the risk of false positivity. In our lab, we use the DiaSorin Simplexa Bordetella direct assay system – RT PCR which targets IS481 and IS1001 for pertussis PCR (other PCR may use different targets). B. pertussis contains ∼238 copies of IS481 and no copies of IS1001, multiple copies of IS481 are responsible for the high sensitivity of PCR and increased risk of false-positive. B. parapertussis has ∼22 copies of IS1001 and no copies of IS481; false-positive identification of IS1001 seems unlikely, as IS1001 is not present in vaccines and its copy numbers are low.2

The recommended antimicrobial agents for treatment or chemoprophylaxis is azithromycin. Antibiotic susceptibility data indicate that the same antibiotics recommended for treating and preventing B. pertussis might help treat and prevent B. parapertussis.11,12 CDC recommends vaccinating young children, preteens, pregnant women, and adults, but pertussis vaccine immunity is short-lived and wanes after 7- 10 years. Immunized children become susceptible after that and can transmit B. pertussis to their very young infant siblings or get B. parapertussis as the vaccine does not protect against it. The average age of patients with B. parapertussis is much younger than those with B. pertussis, and some literature suggest B. parapertussis should be considered when developing new pertussis vaccines.13

References

  1. https://www.cdc.gov/pertussis/index.htmlJames D. Cherry, Brent L. Seaton, Patterns of Bordetella parapertussis Respiratory Illnesses: 2008–2010, Clinical Infectious Diseases, Volume 54, Issue 4, 15 February 2012, Pages 534–537, https://doi-org.proxy.uchicago.edu/10.1093/cid/cir860
  2. Arico B, Rappuoli R. Bordetella parapertussis and Bordetella bronchiseptica contain transcriptionally silent pertussis toxin genes. J Bacteriol.1987;169:2847-2853.
  3. https://www.mayocliniclabs.com/test-catalog/overview/80910#Clinical-and-Interpretive
  4. Blom J., Hansen G. A., and Poulsen F. M.Morphology of cells and hemagglutinogens of Bordetella species: resolution of substructural units in fimbriae of Bordetella pertussis.Infect. Immun.421983308317 CrossrefPubMed.
  5. Cookson B. T. and Goldman W. E.Tracheal cytotoxin: a conserved virulence determinant of all Bordetella species.J. Cell. Biochem.11B1987124
  6. Endoh M., Takezawa T., and Nakase Y.Adenylate cyclase activity of Bordetella organisms. Its production in liquid medium.Microbiol. Immunol.24198095104 PubMed.
  7. Li L. J., Dougan P., Novotny P., and Charles I. G.P70 pertactin, an outer membrane protein from Bordetella parapertussis: cloning, nucleotide sequence and surface expression in Escherichia coli.Mol. Microbiol.51991409417 PubMed.
  8. Mooi F. R., van der Heide H. G. J., TerAvest A. R., Welinder K. G., Livey I., van der Zeisj B. M. A., and Gaastra W.Characterization of fimbrial subunits from Bordetella species.Microb. Pathog.3198718 PubMed.
  9. He Q, Viljanen MK, Arvilommi H, Aittanen B, Mertsola J. Whooping Cough Caused by Bordetella pertussis and Bordetella parapertussis in an Immunized Population. JAMA. 1998;280(7):635–637. doi:10.1001/jama.280.7.635
  10. Hoppe JE, Bryskier A. In vitro susceptibilities of Bordetella pertussis and Bordetella parapertussis to two ketolides (HMR 3004 and HMR 3647), four macrolides (azithromycin, clarithromycin, erythromycin A, and roxithromycin), and two ansamycins (rifampin and rifapentine). Antimicrob Agents Chemother. 1998 Apr;42(4):965-6. doi: 10.1128/AAC.42.4.965. PMID: 9559823; PMCID: PMC105582.
  11. Mortensen JE, Rodgers GL. In vitro activity of gemifloxacin and other antimicrobial agents against isolates of Bordetella pertussis and Bordetella parapertussis. J Antimicrob Chemother. 2000 Apr;45 Suppl 1:47-9. doi: 10.1093/jac/45.suppl_3.47. PMID: 10824032
  12. Karalius VP, Rucinski SL, Mandrekar JN, Patel R. Bordetella parapertussis outbreak in Southeastern Minnesota and the United States, 2014. Medicine (Baltimore). 2017 May;96(20):e6730. doi: 10.1097/MD.0000000000006730. PMID: 28514288; PMCID: PMC5440125.
  13. Karalius VP, Rucinski SL, Mandrekar JN, Patel R. Bordetella parapertussis outbreak in Southeastern Minnesota and the United States, 2014. Medicine (Baltimore). 2017 May;96(20):e6730. doi: 10.1097/MD.0000000000006730. PMID: 28514288; PMCID: PMC5440125.

-Payu Raval, MD is a 1st year anatomic and clinical pathology resident at University of Chicago (NorthShore). Her academic interests include hematology, molecular, and surgical pathology.

-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.

While We Are Young

Preface: I wish we lived in a world where cancer did not exist. While I do not foresee that being a reality in my lifetime, I hope I can witness a milestone in oncology when the development of safe and effective therapies surpasses the rate of metastasis. I hope to see the day when instead of being five steps behind cancer, we gain our footing and come out at least one step ahead. We can’t be that far away, right? When I see a patient who is younger than me or around my age, I can’t help but ask myself, “why they have to go through this? Why do they have to be diagnosed with cancer?” Now, they have to choose whether or not to undergo fertility sparing treatment in addition to making a shared decision regarding cytotoxic therapy and radiation. They’re just beginning their life, their career, their family; they’re finally building their story, and it’s interrupted by cancer. Rather than a partner or friend, I see many younger patients present to our clinic with a parent. Imagine how that feels to be a parent – knowing your child was just diagnosed with a terminal disease. How does one cope with that? No parent should have to endure the pain and emotional suffering of losing their child to cancer, let alone anything. We’re meant to outlive our parents, we’re meant to enjoy a full and healthy life, and this case tore right through me.


The patient, a 28 year old male, first presented to an out-of-state health system after managing intermittent right hip discomfort with NSAIDs and ice for three years. When the pain worsened, he went to an urgent care toward the end of 2018, but his X-rays were negative. After his pain became unbearable six months later, he had an MRI which demonstrated a proximal femur lesion with hip and sacral involvement. The femoral lesion biopsied via FNA, and the patient underwent 6 cycles of induction chemotherapy prior to resection. The patient’s PET scan showed improvement, and the 10.5 cm hip mass was resected demonstrating fibrosis and no residual disease. Additional chemotherapy was prescribed for consolidation followed by radiation to the sacral lesions. Seven months after no evidence of disease, a surveillance PET scan demonstrated abundant bone lesions in the arms, ribs, and pelvis. A new relapse chemotherapy regimen was prescribed for refractory disease, but within cycle 2, new calvarial lesions were identified. By the beginning of 2021, the patient underwent stem cell harvesting followed by myeloablative/salvage chemotherapy and an autologous stem cell transplant. Soon after he transferred his care to our institution a few months later, the patient underwent a successful stem cell transplant and remained stable for three months post-transplant. The patient began experiencing severe pain which prompted earlier surveillance imaging, and both PET scan and MRI demonstrated extensive disease progression. To improve comfort and confirm etiology, a right thoracentesis was performed, and 1,200 mL’s of bloody fluid was sent to our cytology lab for analysis. The findings are presented below.

At our institution, we use one or two drops of fresh fluid to make a cytospin for triage purposes. We examine the air-dried, Diff-Quik-stained cytospin to determine whether or not the cells are likely to contaminate our routine preparations, and if the cells are overtly malignant, the remaining cytopreparations need to be stained in our known-positive fluid set-up. In this case, the specimen was deemed routine despite the few clusters of cells that do not resemble the predominating mesothelial cells. The tightly clustered group in Image 1 has small, round nuclei; fine chromatin; scant, blue cytoplasm; and atypical mitoses.

Image 1. Pleural fluid, right. DQ-stained cytospin.

The pap-stained smears and liquid-based SurePath preparation were then screened and the cells of interest highlighted below.

Images 2-4. Pleural fluid, right. 2-3, Pap-stained smears; 4, SurePath liquid-based prep.

Similar to the Diff-Quik preparation, the small, round cells of interest are forming tight clusters with each cell approximately twice the size of a normal lymphocyte (Image 3). The nuclei are fairly uniform with fine chromatin, relatively small, inconspicuous nucleoli, and scant cytoplasm (Image 2 & 3). The clustering, while not characteristic of this type of tumor, is most likely artifact due to reactivity of being suspended in fluid. What is not pictured in this specimen is the classic Homer-Wright pseudorosettes.

The cell block sections are consistent with the previous cytopreparations, demonstrating a two-cell population. Small, round blue cells (some forming distorted rosettes) in a background of benign-appearing mesothelial cells.

Images 5-6: pleural fluid, right, H&E cell block sections. 5, 100x; 6, 400x.

This pleural fluid was diagnosed as positive for malignant cells: consistent with the patient’s known Ewing sarcoma. While immunostains were not necessary for this stage IV diagnosis, the cell block sections are expected to stain positive for CD99 (membranous), vimentin, and FLI1 (as an EWSR1 [22q12] break apart rearrangement (83%) was detected in this patient). A few days after this fluid was signed out, the patient passed away, and his family is now undergoing genetic testing.

-Taryn Waraksa, 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 36 Year Old Male Traveler with Fever

Case Description

A 36 year old male presented to the emergency department with complaints of fevers, chills, night sweats, nausea, diarrhea, weakness, and decreased appetite for 6 days. He often travels between India and Dallas, and five months prior to presentation returned from two years abroad. While overseas, he developed similar symptoms, but due to COVID-19 restrictions, he was unable to see a provider at that time. His family doctor prescribed a course of medication for presumed malaria, which he completed but could not recall the name of the medication. He endorsed being ill for two weeks at that time and improved with medication to complete resolution of his symptoms. Prior to presentation, he also endorsed 3-4 episodes of non-bloody diarrhea per day and remembered a period of self-resolving chills a month prior. His fever and rigors were cyclic, occurring every other day, worsening up to presentation.

Given his travel history and symptomology, blood was drawn in the emergency department for analysis including a malaria smear. CBC and CMP were significant for elevated bilirubin (Total bilirubin 1.6 mg/dL, Direct bilirubin 0.4 mg/dL), leukopenia (3.60 x 10(9)/L), macrocytosis (92.5 femtoliters), thrombocytopenia (86 x 10(9)/L), and elevated CRP (6.6 mg/dL). His blood differential was significant for neutrophilia (91%), lymphocytopenia (7%), and monocytopenia (1%). The malaria smear was positive, and the patient was given a dose of artemether/lumefantrine in the emergency department. Plasmodium vivax was identified at a parasitemia of 0.5% (Figure 1), and the infectious disease service recommended admission for further workup including testing for G6PD deficiency prior to starting primaquine. He was not G6PD deficient, and an ultrasound of the spleen was unremarkable. The patient was treated with chloroquine for the erythrocytic and primaquine for the exo-erythrocytic stages of P. vivax malaria.

Figure 1. Photomicrograph of Plasmodium vivax ring forms observed in this patient’s Giemsa-stained peripheral blood smear, which are counted to determine the level of parasitemia in a patient’s bloodstream (500x oil immersion).

Discussion

Malaria is an infection caused by protozoan parasites of the genus Plasmodium. These organisms are transmitted by female Anopheles mosquitos upon taking a blood-meal. Human malaria is caused by five defined Plasmodium species: P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi.1 While not endemic to the United States, there is significant disease burden worldwide. In 2019, an estimated 230 million cases of malaria were reported causing approximately 409,000 deaths.2

The two lifecycles of Plasmodium sp. in the human host are classically defined as “erythrocytic” and “exo-erythrocytic”, involving red blood cells and hepatocytes, respectively. Plasmodium sporozoites are inoculated into the human host from the salivary glands of the mosquito upon feeding. From there, the sporozoites travel to the liver where they infect hepatocytes, mature into schizonts and ultimately merozoites. The infected hepatocyte then ruptures, releasing merozoites which enter the circulation and infect erythrocytes, initiating the erythrocytic cycle. This is a unifying trait of all Plasmodium sp. causing human malaria. Importantly, P. vivax and P. ovale form hypnozoites (dormant forms) in the liver, which can reactivate (oftentimes months to years later) following bloodstream clearance, resulting in relapse. It is therefore important that Plasmodium sp. infections be accurately speciated, as management of liver stage parasites differs from that of those in the bloodstream. By contrast, P. malariae and P. falciparum do not form hypnozoites and thus do not chronically infect the liver.

Plasmodium speciationis accomplished by evaluating thin and thick blood spears,4 allowing for assessment of parasite morphology and determination of parasitemia to guide patient management. In cases of P. vivax, the red blood cells are often enlarged (1.5 to 2 times the size of uninfected erythrocytes). Ring forms in all stages of development can be observed in P. vivax infection. These ring forms subsequently mature into trophozoites or gametocytes. P. vivax trophozoites exhibit a large, amoeboid cytoplasm, large chromatin dots, and fine yellow-brown pigment. Trophozoites subsequently develop into schizonts in the infected erythrocytes, subsequently rupturing leading to autoinfection. P. vivax schizonts are large with coalesced pigment and harbor 12 or more merozoites3 (Figure 2). P. vivax gametocytes are large and round to oval shaped and have scattered brown pigment, hemozoin, that may fill the erythrocyte (Figure 3). Gametocytes will migrate to the capillaries which are taken up by a mosquito upon taking a blood-meal, completing the Plasmodium lifecycle.

Figure 2. Photomicrograph of Plasmodium vivax merozoites in a schizont (1000x oil immersion) from this patient.
Figure 3. Photomicrograph of Plasmodium vivax gametocyte with malaria pigment (500x oil immersion) from this patient.

Here we present a case of relapsed P. vivax infection. Blood stage P. vivax parasites are susceptible to chloroquine, but dormant hypnozoites in the liver are resistant to its effects. Hypnozoites can be treated with primaquine, and thus routine management of either P. ovale or P. vivax usually consists of a combination of both antimalarial drugs. It is important to note that primaquine is contraindicated in cases of G6PD deficiency and pregnancy due to hemolytic complications,2 which is why this patient was tested prior to initiating therapy.

P. vivax has a worldwide distribution but has higher prevalence in colder climates as compared to other malaria species. P. vivax is most commonly encountered in Latin America and Southeast Asia. In addition to colder climate adaptation, P. vivax is interesting in that the parasite uses Duffy red cell antigens to enter erythrocytes and in populations with low frequency of Duffy on the surface of RBCs those groups are generally resistant to P. vivax infection. However, there have been rare cases of P. vivax in Africans who are Duffy-null.5

References

  1. Gladwin, M., Mahan, C. S., & Trattler, B. (2021). Malaria. In Clinical microbiology made ridiculously simple (pp. 343–346). essay, MedMaster, Inc.
  2. Menkin-Smith L, Winders WT. Plasmodium Vivax Malaria. [Updated 2021 Jul 23]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK538333/
  3. Procop, G. W., Koneman, E. W., & Winn, W. C. (2017). Malaria. In Koneman’s color Atlas and textbook of diagnostic microbiology (pp. 1467–1470). essay, Lippincott Williams & Wilkins.
  4. Laboratory diagnosis of malaria: Plasmodium vivax. Laboratory Identification of Parasites of Public Health Concern. (n.d.). Retrieved September 14, 2021, from https://www.cdc.gov/dpdx/resources/pdf/benchAids/malaria/Pvivax_benchaidV2.pdf.
  5. Gunalan, K., Niangaly, A., Thera, M. A., Doumbo, O. K., & Miller, L. H. (2018). Plasmodium vivax infections of duffy-negative erythrocytes: Historically undetected or a recent adaptation? Trends in Parasitology, 34(5), 420–429. https://doi.org/10.1016/j.pt.2018.02.006

-Elisa Lin is a fourth-year medical student at UT Southwestern Medical Center in Dallas, Texas. She is interested in AP/CP track residencies.

-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: A Middle-Aged Man with Generalized Weakness

Case History

A middle age male with a past medical history of liver cirrhosis presented to the emergency department with one day of fever, chills, generalized weakness, and nausea. Complete blood count with differential showed leukopenia and neutropenia. Infectious work up was initiated including collection of 2 sets of blood cultures and imaging studies. A computed tomography (CT) scan of the abdomen revealed an irregularly shaped hypodense lesion in the right hepatic lobe concerning for abscess (Image 1). Ultrasound guided aspiration for the hepatic lesion yielded cloudy yellow bilious fluid, which was sent to the microbiology lab for aerobic and anaerobic cultures.

Image 1. CT scan of abdomen showing irregularly shaped hypodense lesion (yellow circle).

Two sets of blood cultures turned positive and Gram stain showed slender Gram positive rods in chains (Image 2). The aspirated fluid culture also showed 4+ Gram positive rods. Small gray colonies appeared on blood agar, chocolate agar, and Columbia Naladixic Acid (CNA) agar from both specimen types (Image 3). Lactobacillus rhamnosus was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Minimal inhibitory concentration (MIC) was determined by broth microdilution assay and the organism was susceptible to penicillin while resistant to vancomycin. With appropriate antibiotics and abscess drainage, the patient’s condition improved and he was discharged to home.

Image 2. Gram stain of blood culture demonstrating gram positive rods in chains.
Image 3. Small gray colonies in blood agar from blood culture.

Discussion

Lactobacillus species are facultatively anaerobic, gram positive, non-spore forming rods that can have varying Gram stain morphology including short plump rods or long slender rods in chains or palisides.1 Lactobacillus species are bacterial inhabitant of the human mouth, gastrointestinal tract, and female genital tract. Isolation from clinical specimens could be considered by many to have questionable clinical significance.2 Lactobacillus species are often present in probiotics and fermented dairy products, including yogurt, and have been reported to provide benefits in gastrointestinal health,3–5 which led to increase in consumption of these products by many people, including our patient.

Cases of liver abscess and bacteremia caused by Lactobacillus species have been rarely reported in the literature and risk factors for the infection were immunosuppression, uncontrolled diabetes, hepatopancreaticobiliary disease, bacterial translocation, and use of probiotics or heavy dairy product consumption.6,7 The causative strains included L. rhamnosus, L. acidophilus, and L. paracasei.7

Pathophysiology of liver abscess and bacteremia due to Lactobacillus species is not well understood but it is postulated that several mechanisms may contribute to the pathogenicity of lactobacilli. Some strains are able to bind to intestinal mucosa, which may aid in translocation of the organism into the bloodstream. Also some strains can adhere to extracellular matrix proteins, aggregate platelets, and produce glycosidases and proteases.7 Furthermore, some strains are more resistant to intracellular killing by macrophages and nitric oxide.8

It is worth noting that many species of Lactobacillus are intrinsically resistant to vancomycin. However, they are usually susceptible to penicillin and ampicillin, as it was seen in our patient, and antimicrobial susceptibility testing can be performed by determining MIC of antimicrobials.9

References

  1. Goldstein EJC, Tyrrell KL, Citron DM. Lactobacillus Species: Taxonomic Complexity and Controversial Susceptibilities. Clin Infect Dis. 2015;60(suppl_2):S98-S107. doi:10.1093/CID/CIV072
  2. Chan JFW, Lau SKP, Woo PCY, et al. Lactobacillus rhamnosus hepatic abscess associated with Mirizzi syndrome: a case report and review of the literature. Diagn Microbiol Infect Dis. 2010;66(1):94-97. doi:10.1016/J.DIAGMICROBIO.2009.08.009
  3. Kligler B, Cohrssen A. Probiotics. Am Fam Physician. 2008;78(9):1073-1078. Accessed November 17, 2021. http://www.aafp.org/afp.
  4. Anukam KC, Osazuwa EO, Osadolor HB, Bruce AW, Reid G. Yogurt containing probiotic Lactobacillus rhamnosus GR-1 and L. reuteri RC-14 helps resolve moderate diarrhea and increases CD4 count in HIV/AIDS patients. J Clin Gastroenterol. 2008;42(3):239-243. doi:10.1097/MCG.0B013E31802C7465
  5. Adolfsson O, Meydani SN, Russell RM. Yogurt and gut function. Am J Clin Nutr. 2004;80(2):245-256. doi:10.1093/AJCN/80.2.245
  6. Omar AM, Ahmadi N, Ombada M, et al. Breaking Bad: a case of Lactobacillus bacteremia and liver abscess. J Community Hosp Intern Med Perspect. 2019;9(3):235. doi:10.1080/20009666.2019.1607704
  7. Sherid M, Samo S, Sulaiman S, Husein H, Sifuentes H, Sridhar S. Liver abscess and bacteremia caused by lactobacillus: Role of probiotics? Case report and review of the literature. BMC Gastroenterol. 2016;16(1):1-6. doi:10.1186/S12876-016-0552-Y/TABLES/1
  8. Asahara T, Takahashi M, Nomoto K, et al. Assessment of Safety of Lactobacillus Strains Based on Resistance to Host Innate Defense Mechanisms. Clin Diagn Lab Immunol. 2003;10(1):169. doi:10.1128/CDLI.10.1.169-173.2003
  9. CLSI. M45. Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria ; Proposed Guideline. Vol 35.; 2015. Accessed November 17, 2021. http://www.clsi.org.

Do Young Kim, MD is a medical microbiology fellow at University of Chicago (NorthShore). His academic interests include clinical microbiology and infectious diseases, epidemiology, and public health.

-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.